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The generation of T cell anergy is associated with upregulation of ubiquitin E3 ligases including Casitas B-lineage lymphoma (Cbl-b), Itch, gene related to anergy in lymphocyte, and deltex1 (DTX1). These E3 ligases attenuate T cell activation by targeting to signaling molecules. For example, Cbl-b and Itch promote the degradation of protein kinase Cθ (PKCθ) and phospholipase C-γ1 (PLC-γ1) in anergic Th1 cells. How these anergy-associated E3 ligases coordinate during T cell anergy remains largely unknown. In the current study, we found that PKCθ and PLC-γ1 are also downregulated by DTX1. DTX1 interacted with PKCθ and PLC-γ1 and stimulated the degradation of PKCθ and PLC-γ1. T cell anergy-induced proteolysis of PKCθ was prevented in Dtx1(-/-) T cells, supporting the essential role of DTX1 in PKCθ downregulation. Similar to Cbl-b and Itch, DTX1 promoted monoubiquitination of PKCθ. Proteasome inhibitor did not inhibit DTX1-directed PKCθ degradation, but instead DTX1 directed the relocalization of PKCθ into the lysosomal pathway. In addition, DTX1 interacted with Cbl-b and increased the protein levels of Cbl-b. We further demonstrated the possibility that, through the downregulation of PKCθ, DTX1 prevented PKCθ-induced Cbl-b degradation and increased Cbl-b protein stability. Our results suggest the coordination between E3 ligases during T cell anergy; DTX1 acts with Cbl-b to assure a more extensive silencing of PKCθ, whereas DTX1-mediated PKCθ degradation further stabilizes Cbl-b., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Leukotrienes generated by 5-lipoxygenase (5-LOX)-catalyzed reaction are key regulators of inflammation. In ionophore-stimulated (A23187; 1-2.5 μM) human blood neutrophils or differentiated HL-60 cells, vitamin E forms differentially inhibited leukotriene B(4) (LTB(4)) with an IC(50) of 5-20 μM for γ-tocopherol, δ-tocopherol (δT), and γ-tocotrienol, but a much higher IC(50) for α-tocopherol. 13'-Carboxychromanol, a long-chain metabolite of δT, suppressed neutrophil- and HL-60 cell-generated LTB(4) with an IC(50) of 4-7 μM and potently inhibited human recombinant 5-LOX activity with an IC(50) of 0.5-1 μM. In contrast, vitamin E forms had no effect on human 5-LOX activity but impaired ionophore-induced intracellular calcium increase and calcium influx as well as the subsequent signaling including ERK1/2 phosphorylation and 5-LOX translocation from cytosol to the nucleus, a key event for 5-LOX activation. Further investigation showed that δT suppressed cytosolic Ca(2+) increase and/or LTB(4) formation triggered by ionophores, sphingosine 1-phosphate, and lysophosphatidic acid but not by fMLP or thapsigargin, whereas 13'-carboxychromanol decreased cellular production of LTB(4) regardless of different stimuli, consistent with its strong inhibition of the 5-LOX activity. These observations suggest that δT does not likely affect fMLP receptor-mediated signaling or store depletion-induced calcium entry. Instead, we found that δT prevented ionophore-caused cytoplasmic membrane disruption, which may account for its blocking of calcium influx. These activities by vitamin E forms and long-chain carboxychromanol provide potential molecular bases for the differential anti-inflammatory effects of vitamin E forms in vivo.

Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

We recently reported the synthesis and anti-inflammatory properties of a novel long chain polyunsaturated fatty acid (PUFA) with an oxygen atom in the beta-position, beta-oxa-21:3 n-3 (Z,Z,Z)-(octadeca-9,12,15-trienyloxy) acetic acid). Our data, from studies aimed at elucidating the mechanism of its action, show that pretreatment of human neutrophils with the beta-oxa-PUFA substantially depresses the production of leukotriene B(4) (LTB(4)) in response to calcium ionophore, A23187, comparable to standard leukotriene inhibitors such as zileuton and nordihydroguaiaretic acid. Interestingly, the n-6 equivalent, beta-oxa 21:3 n-6, is also a strong inhibitor of LTB(4) production. In contrast, naturally occurring PUFA only slightly reduce, for eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids, or increase, for arachidonic acid (20:4n-6), the formation of LTB(4). The parent beta-oxa-21:3n-3 molecule, rather than its derivatives (methyl ester, saturated, monohydroperoxy, or monohydroxy forms), is exclusively responsible for attenuation of LTB(4) formation. beta-Oxa-21:3n-3 inhibits the conversion of [(3)H]20:4n-6 to [(3)H]5-hydroxyeicosatetraenoic acid and [(3)H]LTB(4) by neutrophils in the presence of calcium ionophore and also suppresses the activity of purified 5-lipoxygenase, but not cyclooxygenase 1 and 2. Beta-oxa-21:3n-3 is taken up by neutrophils and incorporated into phospholipids and neutral lipids. In the presence of calcium ionophore, the leukocytes convert a marginal amount of beta-oxa-21:3n-3 to a 16-monohydroxy-beta-oxa-21:3n-3 derivative. After administration to rodents by gavage or i.p. injection, beta-oxa-21:3n-3 is found to be incorporated into the lipids of various tissues. Thus, beta-oxa-21:3n-3 has the potential to be used in the treatment of inflammatory diseases, which are mediated by products of the lipoxygenase pathway.

Human recombinant histamine-releasing factor (HrHRF) preincubation enhances the secretion of histamine, IL-4, and IL-13 from FcepsilonRI-stimulated human basophils. In GM-CSF-primed human eosinophils, HrHRF increases IL-8 production. Our recent experiments were designed to evaluate the effects of HrHRF on human T cell cytokine production. Purified T cells were preincubated with GST-tagged HrHRF, followed by stimulation with PMA and A23187 overnight. A partial inhibition of IL-2 and IL-13 production (30 and 75%, respectively) was detected compared with that in cells treated with PMA/A23187 alone. However, the production of IFN-gamma was similar in PMA/A23187 stimulated cells with or without HrHRF. The inhibition of cytokine protein production was dose dependent and specific to the HrHRF portion of GST-HrHRF. The inhibition was not due to endotoxin, since preincubation with polymyxin B and HrHRF gave similar results to that with HrHRF alone. The same pattern and specificity of cytokine regulation were replicated in the Jurkat T cell line as for primary T cells. The PMA/A23187-stimulated activity of a proximal promoter IL-13, IL-4, or IL-2 luciferase construct transfected into Jurkat cells was partially inhibited (60, 32, or 70%, respectively) upon GST-HrHRF preincubation, suggesting that HrHRF functions to inhibit cytokine production in Jurkat cells by preventing gene transcription. The inhibition of IL-2 promoter activation was specific to the HrHRF portion of GST-HrHRF. We conclude that HrHRF, in addition to functioning as a histamine-releasing factor, can differentially modulate the secretion of cytokines from human basophils, eosinophils, T cells, and murine B cells, suggesting that it may induce a complex array of responses at sites of allergic inflammation.

We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC(4) synthase expression in mononuclear phagocytes. Conditioning of the monocyte-like cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC(4) release. The putative LPS receptor, Toll-like receptor 4, was expressed in THP-1 cells. LPS down-regulated LTC(4) synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated cells with LPS resulted in no change in the rate of LTC(4) synthase mRNA decay. LPS treatment of THP-1 cells, transiently transfected with a LTC(4) synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-alpha and inhibition of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of cells with a Toll-like receptor 4-blocking Ab and an inhibitor of NF-kappaB activation resulted in inhibition of the LPS effect, while activation of NF-kappaB and p50/p65 overexpression down-regulated the LTC(4) synthase gene. LPS down-regulates cysteinyl LT release and LTC(4) synthase gene expression in mononuclear phagocytes by an NF-kappaB-mediated mechanism.

PGs produced from arachidonic acid by the action of cyclooxygenase enzymes play a pivotal role in the regulation of both inflammatory and immune responses. Because leukotriene B4 (LTB4), a product of 5-lipoxygenase (5-LO) pathway, can exert numerous immunoregulatory and proinflammatory activities, we examined the effects of PGs on LTB4 release from dendritic cells (DC) and from peritoneal macrophages. In concentration-dependent manner, PGE1 and PGE2 inhibited the production of LTB4 from DC, but not from peritoneal macrophage, with an IC50 of 0.04 microM. The same effect was observed with MK-886, a 5-LO-activating protein (FLAP)-specific inhibitor. The decreased release of LTB4 was associated with an enhanced level of IL-10. Furthermore, the inhibition of LTB4 synthesis by PGs was significantly reversed by anti-IL-10, suggesting the involvement of an IL-10-dependent mechanism. Hence, we examined the effects of exogenous IL-10 on the 5-LO pathway. We demonstrate that IL-10 suppresses the production of LTB4 from DC by inhibiting FLAP protein expression without any effect on 5-LO and cytosolic phospholipase A2. Taken together, our results suggest links between DC cyclooxygenase and 5-LO pathways during the inflammatory response, and FLAP is a key target for the PG-induced IL-10-suppressive effects.

Mucosal mast cells (MMC) or their precursors migrate through the intestinal lamina propria to reside intraepithelially, where expression of mouse mast cell protease-1 indicates the mature phenotype. Alterations in expression of integrins that govern cell adhesion to the extracellular matrix may regulate this process. As the key cytokine mediating differentiation of mouse mast cell protease-1-expressing MMC homologues in vitro, TGF-beta1 was considered a likely candidate for regulation of the integrins that facilitate intraepithelial migration of MMC. Therefore, we examined adhesion of bone marrow-derived mast cells cultured with and without TGF-beta1 to laminin-1, fibronectin, and vitronectin along with expression of integrins likely to regulate this adhesion. Adhesion of PMA-stimulated cultured mast cells to laminin-1 increased from 5.3 +/- 3.6% (mean +/- SEM) in the absence of TGF-beta1 to 58.7 +/- 4.0% (p < 0.05) when cultured mast cells had differentiated into MMC homologues in the presence of TGF-beta1. Increased adhesion of MMC homologues to laminin-1 was also stimulated by FcepsilonRI cross-linking and the calcium ionophore A23187. Expression of the laminin-binding integrin alpha(7) by MMC homologues grown in the presence of TGF-beta1 was demonstrated by RT-PCR and flow cytometry, and preincubation of MMC homologues with the alpha(7)-neutralizing Ab 6A11 inhibited adhesion to laminin-1 by 98% (p < 0.05), demonstrating a novel role for this molecule in adhesion of a hemopoietic cell to laminin-1.

Soluble GM-CSF receptor alpha subunit (sGMRalpha) is a soluble isoform of the GMRalpha that is believed to arise exclusively through alternative splicing of the GMRalpha gene product. The sGMRalpha mRNA is expressed in a variety of tissues, but it is not clear which cells are capable of secreting the protein. We show here that normal human monocytes, but not lymphocytes, constitutively secrete sGMRalpha. Stimulation of monocytes with GM-CSF, LPS, PMA, or A23187 rapidly up-regulates the secretion of sGMRalpha in a dose-dependent manner, demonstrating that secretion is also regulated. To determine whether sGMRalpha arose exclusively through alternative splicing of the GMRalpha gene product, or whether it could also be generated through ectodomain shedding of GMRalpha, we engineered a murine pro-B cell line (Ba/F3) to express exclusively the cDNA for cell surface GMRalpha (Ba/F3.GMRalpha). The Ba/F3.GMRalpha cell line, but not the parental Ba/F3 cell line, constitutively shed a sGMRalpha-like protein that bound specifically to GM-CSF, was equivalent in size to recombinant alternatively spliced sGMRalpha (60 kDa), and was recognized specifically by a mAb raised against the ectodomain of GMRalpha. Furthermore, a broad-spectrum metalloprotease inhibitor (BB94) reduced constitutive and PMA-, A23187-, and LPS-induced secretion of sGMRalpha by monocytes, suggesting that shedding of GMRalpha by monocytes may be mediated in part through the activity of metalloproteases. Taken together, these observations demonstrate that sGMRalpha is constitutively secreted by monocytes, that GM-CSF and inflammatory mediators up-regulate sGMRalpha secretion, and that sGMRalpha arises not only through alternative splicing but also through ectodomain shedding of cell surface GMRalpha.

Human macrophages (Mphi) respond to Mycobacterium tuberculosis (Mtb) infection by undergoing apoptosis, a cornerstone of effective antimycobacterial host defense. Virulent mycobacteria override this reaction by inducing necrosis leading to uncontrolled Mtb replication. Accordingly, Mphi death induced by inoculation with Mtb had the characteristics of apoptosis and necrosis and correlated with moderate increase of mitochondrial permeability transition (MPT), mitochondrial cytochrome c release, and caspase-9 and -3 activation. We hypothesized that changes in intramitochondrial Ca(2+) concentration ([Ca(2+)](m)) determine whether Mphi undergo either apoptosis or necrosis. Therefore, we induced mechanism(s) leading to predominant apoptosis or necrosis by modulating [Ca(2+)](m) and examined their physiological consequences. Adding calcium ionophore A23187 to Mphi inoculated with Mtb further increased calcium flux into the cells which is thought to lead to increased [Ca(2+)](m), blocked necrosis, stabilized MPT, decreased mitochondrial cytochrome c release, lowered caspase activation, and accompanied effective antimycobacterial activity. In contrast, Mphi infected with Mtb in presence of the mitochondrial calcium uniporter inhibitor ruthenium red showed increased mitochondrial swelling and cytochrome c release and decreased MPT and antimycobacterial activity. Thus, in Mtb-infected Mphi, high levels of mitochondrial membrane integrity, low levels of caspase activation, and diminished mitochondrial cytochrome c release are hallmarks of apoptosis and effective antimycobacterial activity. In contrast, breakdown of mitochondrial membrane integrity and increased caspase activation are characteristic of necrosis and uncontrolled Mtb replication.

To understand the biochemical events that occur in the airways after rhinovirus (RV) infection, we developed for the first time a model in which the cell lines from human mast cells (HMC-1) and basophils (KU812) can be infected with RV14, a major group RV. Viral infection was confirmed by demonstrating that viral titers in culture supernatants, and RV RNA increased with time. RV14 infection alone and a combination of PMA plus calcium ionophore A23187, did not increase histamine production by these cells, although IgE plus anti-IgE increased the histamine production. However, histamine content in the supernatants increased in response to PMA plus A23187, or IgE plus anti-IgE after RV14 infection. PMA plus A23187 or IgE plus anti-IgE induced the production of IL-8 and GM-CSF in supernatants of HMC-1 cells and IL-4 and IL-6 in supernatants of KU812 cells. RV14 infection further increased the production of the cytokines, whereas RV14 infection alone did not alter the production of the cytokines by these cells. An Ab to ICAM-1 inhibited RV14 infection of the cells and decreased the production of cytokines and histamine after RV14 infection. RV14 infection enhanced the increases in intracellular calcium concentration and activation of NF-kappaB by PMA plus A23187 in the cells. These findings suggest that RV14 infection may prime the cytokine and histamine production from mast cells and basophils and may cause airway inflammation in asthma.

The transcription factor NF-kappaB is a critical regulator of T cell function that becomes strongly activated in response to coengagement of TCR and CD28. Although events immediately proximal to NF-kappaB activation are well understood, uncertainty remains over which upstream signaling pathways engaged by TCR and CD28 lead to NF-kappaB activation. By using Jurkat T cell lines that are deficient or replete for either the protein tyrosine kinase ZAP-70 or the cytosolic adapter molecule SLP-76, the role of these proteins in modulating NF-kappaB activation was examined. NF-kappaB was not activated in response to coengagement of TCR and CD28 in either the ZAP-70- or SLP-76-negative cells, whereas stimuli that bypass these receptors (PMA plus A23187, or TNF-alpha) activated NF-kappaB normally. Protein kinase C (PKC) theta activation, which is required for NF-kappaB activation, also was defective in these cells. Reexpression of ZAP-70 restored PKCtheta and NF-kappaB activation in response to TCR and CD28 coengagement. p95(vav) (Vav)-1 tyrosine phosphorylation was largely unperturbed in the ZAP-70-negative cells; however, receptor-stimulated SLP-76/Vav-1 coassociation was greatly reduced. Wild-type SLP-76 fully restored PKCtheta and NF-kappaB activation in the SLP-76-negative cells, whereas 3YF-SLP-76, which lacks the sites of tyrosine phosphorylation required for Vav-1 binding, only partially rescued signaling. These data illustrate the importance of the ZAP-70/SLP-76 signaling pathway in CD3/CD28-stimulated activation of PKC theta and NF-kappaB, and suggest that Vav-1 association with SLP-76 may be important in this pathway.

To facilitate the study of signaling pathways involved in myeloid dendritic cell (DC) differentiation, we have developed a serum-free culture system in which human CD14+ peripheral blood monocytes differentiate rapidly in response to bacterial LPS, TNF-alpha, or calcium ionophore (CI). Within 48-96 h, depending on the inducing agent, the cells acquire many immunophenotypical, morphological, functional, and molecular properties of DC. However, there are significant differences in the signaling pathways used by these agents, because 1) LPS-induced, but not CI-induced, DC differentiation required TNF-alpha production; and 2) cyclosporin A inhibited differentiation induced by CI, but not that induced by LPS. Nevertheless, all three inducing agents activated members of the NF-kappaB family of transcription factors, including RelB, suggesting that despite differences in upstream elements, the signaling pathways all involve NF-kappaB. In this report we also demonstrate and offer an explanation for two observed forms of the RelB protein and show that RelB can be induced in myeloid cells, either directly or indirectly, through a calcium-dependent and cyclosporin A-sensitive pathway.

Although proteinase 3 (PR3) is known to have the potential to promote inflammation and injure tissues, the biologic forms and function of PR3 in polymorphonuclear neutrophils (PMN) from healthy donors have received little attention. In this paper, we show that PMN contain 3.24 +/- SD 0.24 pg of PR3 per cell, and that the mean concentration of PR3 in azurophil granules of PMN is 13.4 mM. Low levels of PR3 are detectable on the cell surface of unstimulated PMN. Exposure of PMN to cytokines or chemoattractants alone induces modest (1.5- to 2.5-fold) increases in cell surface-bound PR3. In contrast, brief priming of PMN with cytokines, followed by activation with a chemoattractant, induces rapid and persistent, 5- to 6-fold increases in cell surface expression of PR3, while causing minimal free release of PR3. Membrane-bound PR3 on PMN is catalytically active against Boc-Alanine-Alanine-Norvaline-thiobenzyl ester and fibronectin, but in marked contrast to soluble PR3, membrane-bound PR3 is resistant to inhibition by physiologic proteinase inhibitors. PR3 appears to bind to the cell surface of PMN via a charge-dependent mechanism because exposure of fixed, activated PMN to solutions having increasing ionic strength results in elution of PR3, HLE, and CG, and there is a direct relationship between their order of elution and their isoelectric points. These data indicate that rapidly inducible PR3 expressed on the cell surface of PMN is an important bioactive form of the proteinase. If PR3 expression on the cell surface of PMN is dysregulated, it is well equipped to amplify tissue injury directly, and also indirectly via the generation of autoantibodies.

T cell activation initiates signals that control gene expression of molecules important for T cell function. The focal adhesion kinase Pyk2 has been implicated in T cell signaling. To further analyze the involvement of Pyk2 in T cell processes, we examined the effect of T cell stimulation on the expression of Pyk2. We found that TCR ligation or PMA increased Pyk2 expression in Jurkat T cells and in normal T cells. In contrast, TCR ligation and PMA failed to induce any detectable increase in the expression of the other member of the focal adhesion kinase family, Fak, in Jurkat T cells and induced only a weak increase in Fak expression in normal T cells. The serine/threonine kinases, protein kinase C and mitogen-activated protein/extracellular signal-related kinase kinase (MEK), regulated Pyk2 expression, as inhibitors of these kinases blocked stimulus-induced Pyk2 expression. Cyclosporin A, FK506, and KN-62 did not block Pyk2 expression; thus, calcineurin and Ca2+/calmodulin-activated kinases are not critical for augmenting Pyk2 expression. TCR ligation increased Pyk2 mRNA, and the transcriptional inhibitor actinomycin D blocked Pyk2 expression. Strikingly, Ca2+ ionophores, at concentrations that in combination with other stimuli induced IL-2 expression, blocked TCR- and PMA-induced up-regulation of Pyk2 expression. Thus, the increase in Ca2+ has opposing effects on IL-2 and Pyk2 expression. Cyclosporin A and FK506, but not KN-62, blocked Ca2+ ionophore-mediated inhibition of Pyk2 expression, implicating calcineurin in down-regulating Pyk2 expression. These results show that TCR-triggered intracellular signals increase Pyk2 expression and shed light on the molecular mechanisms that regulate Pyk2 expression in T cells.

Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst. Two distinct PLD isoforms, designated PLD1 and PLD2, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear. In our present study we investigated how PLD2 activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD2, and in PLD2 -transfected COS-7 cells. Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lines, suggesting that Ca2+ might be implicated in the regulation of the PLD2 activity. In addition to the A23187-induced PLD2 activation, A23187 also increases PLA2-mediated arachidonic acid release, and the A23187-stimulated PLD2 and PLA2 activities could be blocked by pretreatment of the cells with cytosolic calcium-dependent PLA2 (cPLA2) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induced PLD2 and PLA2 activities could be inhibited by cotransfection with antisense cPLA2 oligonucleotide. These results suggest a role for cPLA2 in the regulation of PLD2 activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD2 activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD2 activity is up-regulated by Ca2+ influx and that cPLA2 may play a key role in the Ca2+-dependent regulation of PLD2 through generation of lysophosphatidylcholine.

Numerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-kappa B contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-kappa B protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-kappa B binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-kappa B activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-kappa B binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-kappa B activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-kappa B activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients.

Cysteinyl-leukotrienes are potent bronchoconstrictor mediators synthesized by the 5-lipoxygenase (5-LO) pathway. Eosinophilopoietic cytokines such as IL-5 enhance cysteinyl-leukotriene synthesis in eosinophils in vitro, mimicking changes in eosinophils from asthmatic patients, but the mechanism is unknown. We hypothesized that IL-5 induces the expression of 5-LO and/or its activating protein FLAP in eosinophils, and that this might be modulated by anti-inflammatory corticosteroids. Compared with control cultures, IL-5 increased the proportion of normal blood eosinophils immunostaining for FLAP (65 +/- 4 vs 34 +/- 4%; p < 0.0001), enhanced immunoblot levels of FLAP by 51 +/- 14% (p = 0.03), and quadrupled ionophore-stimulated leukotriene C4 synthesis from 5.7 to 20.8 ng/106 cells (p < 0.02). IL-5 effects persisted for 24 h and were abolished by cycloheximide and actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (p < 0.0001). Neither IL-5 nor dexamethasone altered 5-LO expression, but IL-5 significantly increased 5-LO immunofluorescence localizing to eosinophil nuclei. Compared with normal subjects, allergic asthmatic patients had a greater proportion of circulating FLAP+ eosinophils (46 +/- 6 vs 27 +/- 3%; p < 0.03) and a smaller IL-5-induced increase in FLAP immunoreactivity (p < 0.05). Thus, IL-5 increases FLAP expression and translocates 5-LO to the nucleus in normal blood eosinophils in vitro. This is associated with an enhanced capacity for cysteinyl-leukotriene synthesis and mimics in vivo increases in FLAP expression in eosinophils from allergic asthmatics.

Mast cells secrete a variety of biologically active substances that mediate inflammatory responses. Synaptotagmin(s) (Syts) are a gene family of proteins that are implicated in the control of Ca2+-dependent exocytosis. In the present study, we investigated the possible occurrence and functional involvement of Syt in the control of mast cell exocytosis. Here, we demonstrate that both connective tissue type and mucosal-like mast cells express Syt-immunoreactive proteins, and that these proteins are localized almost exclusively to their secretory granules. Furthermore, expression of Syt I, the neuronal Ca2+ sensor, in rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells, resulted in prominent potentiation and acceleration of Ca2+-dependent exocytosis. Therefore, these findings implicate Syt as a Ca2+ sensor that mediates regulated secretion in mast cells to calcium ionophore.

IL-8 is an important neutrophil and eosinophil chemoattractant in asthma. A recent report has suggested that bradykinin (BK), an asthmatic mediator, induces the release of IL-8 in nonairway cells. We have recently reported that BK causes cyclooxygenase (COX)-2 induction and PGE2 release in human airway smooth muscle (ASM) cells. In this study, we tested the ability of BK to induce IL-8 from these cells and explored the role of COX products and COX-2 induction in this process. Confluent serum-deprived human ASM cells were studied. IL-8 was assayed by specific ELISA. Unstimulated cells released low levels of IL-8. BK enhanced IL-8 release in a concentration- and time-dependent fashion (maximum 50-fold increase over basal). The nonselective COX inhibitor indomethacin and the selective COX-2 inhibitor NS-398 strongly inhibited BK-stimulated PGE2 and IL-8 production. The COX substrate arachidonic acid also caused PGE2 and IL-8 production, and its effect was inhibited by nonselective COX inhibitors but unaffected by NS-398. Both the BK- and arachidonic acid-induced IL-8 production was inhibited by the protein synthesis inhibitors cycloheximide and actinomycin D and by the steroid dexamethasone. Furthermore, exogenous PGE2 and calcium ionophore A23187 also stimulated IL-8 release. BK-induced IL-8 release was mimicked by the BK B2 receptor agonist (Tyr(Me)8)-BK and was potently inhibited by the selective B2 receptor antagonist HOE-140. These results suggest that human ASM can be a source of IL-8 and also that endogenous prostanoids, involving both COX-1 and COX-2, have a novel role in mediating BK-induced IL-8 production.

Activation of cytosolic phospholipase A2 (cPLA2) by TNF has been shown to be an important component of the signaling pathway leading to cell death. The role of cPLA2 in the cytotoxic action of TNF was investigated in a panel of human leukemic cell lines. TNF could activate cPLA2 only in U937 and HL60 TNF-sensitive leukemic cells, but not in KG1a, CEM, and CEM/VLB100 cells that are relatively resistant to TNF. Pretreatment with 4-bromophenacyl bromide, a cPLA2 inhibitor, rendered U937 and HL60 cell lines resistant to the cytotoxic effect of TNF. Immunoblot and reverse-transcriptase PCR demonstrated that cPLA2 expression was detectable at both transcriptional and translational levels in all leukemic cell lines studied, although CEM and CEM/VLB100 cells expressed cPLA2 mRNA and protein at lower levels. The protein synthesis inhibitor, cycloheximide, increased TNF-induced cPLA2 activity and cytotoxicity in both CEM and CEM/VLB100 cell lines. Low levels of cPLA2 activity in the KG1a cell line could be activated by the cPLA2 activator mellitin, or the calcium ionophore A23187. The data suggest that cPLA2 activity is involved in TNF-induced cytotoxicity in leukemic cells. Resistance to TNF-induced cytotoxicity may involve either protein inhibitors that act upstream of cPLA2 in the TNF-signaling pathway or constitutive defects of cPLA2 itself, possibly involving calcium utilization.

We examined herein the functional linkage of enzymes regulating the initial, intermediate, and terminal steps of PG biosynthesis to provide PGs in rat peritoneal macrophages stimulated with LPS and/or A23187. Quiescent cells stimulated with A23187 produced thromboxane B2 (TXB2) in marked preference to PGE2 within 30 to 60 min (constitutive immediate response), which was mediated by preexisting cytosolic phospholipase A2 (cPLA2), cyclooxygenase-1 (COX-1), and TX synthase. Cells treated with LPS predominantly produced PGE2 during culture for 3 to 24 h (delayed response), where cPLA2 and secretory PLA2 functioned cooperatively with inducible COX-2, which was, in turn, coupled with inducible PGE2 synthase. Cells primed for 12 h with LPS and stimulated for 30 min with A23187 produced PGE2 in marked preference to TXB2 (induced immediate response), in which three inducible enzymes, cPLA2, COX-2, and PGE2 synthase, were functionally linked. Preferred coupling of the two inducible enzymes, COX-2 and PGE2 synthase, was further confirmed by the ability of LPS-treated cells to convert exogenous arachidonic acid to PGE2 optimally at a time when both enzymes were simultaneously induced. These results suggest that distinct PG biosynthetic enzymes display segregated functional coupling following different transmembrane stimulation events even when enzymes that catalyze similar reactions in vitro coexist in the same cells.

Arachidonic acid (AA) released from membrane phospholipids by phospholipase A2 (PLA2) is important as a substrate for eicosanoid formation and as a second messenger for superoxide anion (O2-) generation in neutrophils. Different isoforms of PLA2 in neutrophils might mobilize AA for different functions. To test this possibility, we sought to characterize the PLA2s that are activated by the neutrophil stimuli, Aroclor 1242, a mixture of polychlorinated biphenyls, and A23187, a calcium ionophore. Both Aroclor 1242 and A23187 caused release of [3H]AA; however, O2- production was seen only in response to Aroclor 1242. Eicosanoids accounted for >85% of the radioactivity recovered in the supernatant of A23187-stimulated cells but <20% of the radioactivity recovered from cells exposed to Aroclor 1242. Omission or chelation of calcium abolished A23187-induced AA release, but did not alter AA release in Aroclor 1242-stimulated neutrophils. AA release and O2- production in response to Aroclor 1242 were inhibited by bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2. BEL, however, did not alter Calcium-independent activity was inhibited >80% by BEL, whereas calcium-dependent activity was inhibited <5%. Furthermore, calcium-independent, but not calcium-dependent, PLA2 activity was significantly enhanced by Aroclor 1242. These data suggest that Aroclor 1242 and A23187 activate distinct isoforms of PLA2 that are linked to different functions: Aroclor 1242 activates a calcium-independent PLA2 that releases AA for the generation of O2-, and A23187 activates a calcium-dependent PLA2 that mobilizes AA for eicosanoid production.

Human peripheral blood contains a small subpopulation of immature dendritic cells (iDC) distinguished from circulating monocytes by their low expression of CD14. We utilized leukapheresis and countercurrent centrifugal elutriation to obtain myeloid origin mononuclear cell (MOMC) fractions of monocytes and iDC for study. These subpopulations were ultrastructurally and immunophenotypically similar before culture. After a 20- to 96-h culture either alone, with recombinant human granulocyte-monocyte CSF, or with endotoxin, greater up-regulation of costimulatory molecule expression was observed among iDC than among monocytes, and only iDC expressed the activation molecule CD83. Treatment with rhIL-4 caused many MOMC to develop morphologic properties of dendritic cells within 96 h, but costimulatory molecule up-regulation and CD14 down-regulation were heterogeneous, and CD83 expression was infrequent. In contrast, calcium ionophore (CI) treatment induced rapid and consistent effects in MOMC from both healthy volunteers and cancer patients, including down-regulated CD14 expression, acquisition of dendritic cell morphologic properties, up-regulated MHC and costimulatory molecule expression, and de novo CD83 expression. Many such effects occurred within 20 h of treatment. CI treatment activated purified CD14+ monocytes and also enhanced the spontaneous activation of purified CD14-/dim iDC in culture. Unfractionated MOMC, purified monocytes, and purified iDC displayed equivalently enhanced T cell-sensitizing efficiency following CI treatment. CD4+ T cell sensitization to keyhole limpet hemocyanin and CD8+ T cell sensitization to MART-1 melanoma-associated peptide were achieved in a single culture stimulation. Therefore, circulating monocytes and iDC can be induced by CI to manifest properties of activated DC, providing large numbers of efficient, nontransformed autologous APC for T cell sensitization strategies.

Rat basophilic leukemia mast cells (RBL-2H3) secrete histamine when activated by Ag. This secretion correlates with increased phosphorylation of myosin light chain by protein kinase C (PKC). Calcium ionophores (A23187) also elicit secretion, which is enhanced by PMA. To analyze the roles of Ca2+ and PKC in the secretory process, A23187-induced myosin light chain phosphorylation was examined in the presence and absence of PMA. A23187-induced secretion correlated best with myosin light chain phosphorylation by PKC, not with phosphorylation by myosin light chain kinase (MLCK). A23187 induced the translocation to membranes of the alpha, beta, delta, and epsilon isozymes of PKC. PMA not only increased the phosphorylation of myosin light chains at PKC-specific sites (Ser1 and Ser2) but also at sites attributed to MLCK (Ser19 and Thr18-Ser19). A23187 plus PMA induced higher levels of secretion concomitantly with increased myosin light chain phosphorylation at the PKC-specific sites. However, there was little correlation between the translocation of specific PKC isozymes and the phosphorylation of myosin light chains by PKC. Activation induced a novel triphosphorylated form of myosin light chain with a higher level of phosphorylation at the diphosphorylated MLCK sites. Quantitation of A23187 plus PMA-induced myosin light chain phosphorylation revealed that phosphorylation at PKC sites increased from zero to 0.35 mol/mol, was little changed at the monophosphorylated MLCK site (0.30 mol/mol), and increased from zero to 0.06 mol/mol at the diphosphorylated MLCK sites. Therefore, Ca2+-induced secretion correlates best with myosin light chain phosphorylation by PKC, and diphosphorylation by MLCK is unlikely to contribute to secretion.

Macrophage inflammatory protein-1alpha (MIP-1alpha) has previously been shown to be produced by mononuclear cells, eosinophils, and neutrophils. Its production by basophils has not been investigated. The objective of this study was to investigate the production of MIP-1alpha by basophils. Peripheral blood basophils were separated by Percoll gradient centrifugation, cultured overnight, and processed for double immunocytochemistry using Abs against MIP-1alpha and FcepsilonRIalpha (alpha subunit of IgE receptor type 1). We demonstrated that basophils expressed immunoreactive MIP-1alpha upon stimulation with anti-IgE. Less than 5% of the basophils stained for MIP-1alpha without stimulation. The secretion of MIP-1alpha by basophils was studied by ELISA. In these experiments, basophils were further enriched to 65 to 99% (median, 86%) by a negative selection method. Basophils released MIP-1alpha when stimulated by Abs against IgE and FCepsilonRIalpha as well as IL-3 and the calcium ionophore, A23187. In parallel experiments, PBMC, eosinophils, and neutrophils did not produce MIP-1alpha in response to anti-IgE, but they did so in response to A23187. No MIP-1alpha release was detected in platelet preparations. Preincubation with IL-3 (15 min or 18 h) augmented anti-IgE-included basophil MIP-1alpha production. The secretion of MIP-1alpha by basophils was detectable shortly after stimulation and gradually increased over 24 h. Since MIP-1alpha has potent inflammatory and histamine-releasing activities, its production by basophils may indicate a positive feedback mechanism for allergic inflammation.

Ceramide, a product of sphingomyelin hydrolysis by sphingomyelinase, elicits various cellular functions and has recently been regarded as a second messenger. To investigate the role of ceramide in rat basophilic leukemia (RBL-2H3) cells, the effects of a cell-permeable analogue, N-acetylsphingosine (C2-ceramide), on Ag-mediated cellular responses were examined. C2-Ceramide inhibited Ag- or PMA-induced activation of phospholipase D (PLD), whereas Ca2+ ionophore A23187-induced PLD activation was not affected. C2-Ceramide failed to inhibit PLD activity in two different in vitro assay systems. Since PLD activity is known to be regulated by several factors, the effects of C2-ceramide on these factors were examined. We have previously reported the possible involvement of protein tyrosine kinase in Ag-mediated PLD activation. However, C2-ceramide had no effect on Ag-induced protein tyrosine phosphorylation, including mitogen-activated protein (MAP) kinases. In fura-2-loaded RBL-2H3 cells, C2-ceramide suppressed Ag-induced Ca2+ influx, leaving initial Ca2+ increase and inositol phosphate production unaffected. Western blot analysis revealed that Ag caused translocation of protein kinase C (PKC) alpha, beta 1, beta 2, delta and epsilon isozymes from cytosol to membrane fraction. Translocation of alpha, beta 1, and beta 2 isozymes was specifically prevented by C2-ceramide. Moreover, C2-ceramide suppressed Ag-induced serotonin release. In the absence of extracellular Ca2+, Ag-induced PLD activation and release reaction were greatly reduced. The inhibitory profile was nearly the same as that obtained in C2-ceramide-treated cells. These results suggest that C2-ceramide inhibits Ag-induced PLD activation and serotonin release, probably through the blockage of Ca2+ influx and translocation of Ca(2+)-dependent PKC isozymes in RBL-2H3 cells.

In HIV-1-infected asymptomatic carriers, the vast majority of infected cells in PBMCs are believed to be latently or nonproductively infected. We have isolated a subclone (MOLT-20-2) from an infected T cell line that expressed HIV-1 Ags at a very low level. However, viral Ag expression was markedly up-regulated by stimulation with either TNF-alpha, A23187, or PMA, indicating that the subclone might provide a suitable model of HIV-1 latency. Our previous studies have shown that the carboxyl-terminal region of the extracellular form of HIV-1 Nef played an important role in the interaction of infected cells with uninfected T cells, and could induce the cytostatic state. This suggested that Nef might contribute to intracellular signal transduction through an interaction with latently infected cells. We show in this study that stimulation of MOLT-20-2 with soluble Nef leads to HIV-1 activation from latency in a dose-dependent manner. Moreover, using a total of 14 overlapping Nef-related synthetic peptides, stimulatory activity was mapped to a discrete peptide (amino acid residues 132-147) that had the potential to activate latent HIV-1. This novel Nef function was confirmed by activation of virus production from the PBMCs of asymptomatic carriers. In addition, Nef-dependent HIV-1 activation from latency was also observed in another independently derived, latently infected cell line, U1, though not in cell line ACH-2. These results extend the significance of the Nef activity in vivo to the regulation of productive HIV-1 infection from latency, and define the regions of the protein involved.

Imbalances in anti-inflammatory and proinflammatory cytokines may be responsible for initiation or progression of diverse pathologic states including autoimmune and infectious diseases. IL-4 production of proinflammatory cytokines and IL-12 promotes differentiation and activation of IFN-gamma-producing T cells, but does a counter-regulatory effect of proinflammatory cytokines on IL-4 production exist? This study evaluates the effect of proinflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-12, and TNF-alpha) on IL-4 production in primary human T cell cultures. PBMCs from healthy individuals were tested for IL-4 production in response to PHA and various cytokines. IL-4 was measured by proliferation of the IL-4-sensitive T cell line (CT.h4S) or ELISA. IL-1 alpha and IL-1 beta inhibited IL-4 production by 20 to 80% in > 92% of healthy individuals (p = 0.0001, paired t-test). IL-12 had an inhibitory effect on PBMC IL-4 production as previously described, but neither IL-6 nor TNF-alpha inhibited IL-4 production. IL-1 had no effect on PHA-induced PBMC or purified T cell proliferation or IL-2 production. IL-4 production by purified T cells stimulated by PHA or the combination of PMA with calcium ionophore (A23187) was inhibited by IL-1, and reconstitution with peripheral blood-derived adherent macrophages had no effect. IL-12 did not inhibit IL-4 production in stimulated purified T cells. Steady state IL-4 mRNA levels were determined by semiquantitative competitive reverse transcribed PCR (RT-PCR). Marked inhibition of IL-4 mRNA levels were seen at 5 h after exposure to IL-1. This interaction between IL-1 and IL-4 may be an important physiologic regulator of the balance between proinflammatory cytokines from activated macrophages and anti-inflammatory cytokines from T cells.

Activation of MMC-34 cells, a murine mast cell line, or bone marrow-derived mast cells by aggregation of IgE cell surface receptors or addition of calcium ionophore stimulates prostaglandin (PG) D2 synthesis and secretion. An initial rapid burst of PGD2 synthesis, complete within 30 min, is followed by a slower subsequent production of PGD2 that reaches a maximum 4 to 8 h after activation in MMC-34 cells. PG synthase 1 (PGS-1) message and protein are expressed constitutively in MMC-34 cells and are not modulated by exposure to calcium ionophore or aggregation of IgE receptors. In contrast, activation of MMC-34 or bone marrow-derived mast cells induces expression of the PG synthase 2 (PGS-2) gene. PGS-2 induction following mast cell activation is blocked by dexamethasone. The initial PGD2 burst in activated MMC-34 cells is prevented by aspirin pretreatment, suggesting that constitutive PGS-1 present in mast cells before activation is responsible for the early PGD2 production in response to activation. In contrast, the later phase of PGD2 production is blocked by dexamethasone, cycloheximide, or NS-398, a PGS-2-specific nonsteroidal anti-inflammatory drug that inhibits PGS-2 enzyme activity but not PGS-1 activity. These data demonstrate that mast cell activation results 1) in the induction of PGS-2 gene expression, and 2) in both PGS-1-dependent PGD2 synthesis and PGD2 synthesis that is dependent on the activation-induced synthesis and activity of PGS-2.

IL-8 is a potent neutrophil chemoattractant that has been detected in high concentrations at acutely inflamed sites in vivo. Many cell types, including peripheral blood neutrophils, produce IL-8 that can be released by a variety of pro-inflammatory stimuli. However, the functional importance of neutrophil IL-8 during exudation is not yet known. We now report that neutrophils, harvested from skin lesions on the forearms of normal human volunteers (exudative neutrophils), expressed 100-fold higher levels of cell-associated IL-8 and spontaneously released up to 50-fold more IL-8 than freshly isolated peripheral blood neutrophils from the same donor. Furthermore, cell-associated IL-8 in peripheral blood neutrophils increased 20-fold during incubation at 37 degrees C in vitro and was increased over 200-fold after treatment with the Ca2+ ionophore A23187. More than 35% of the cell-associated IL-8 could be released by stimulation with either Ca2+ ionophore A23187 or phorbol myristate acetate. IL-8 was localized by sucrose gradient centrifugation to a subcellular fraction of heterogeneous, light membranous organelles. The accumulation of IL-8 within these organelles is inhibited by cycloheximide but not actinomycin D, suggesting that IL-8 accumulation is under translational, rather than transcriptional control. These studies indicate that peripheral blood neutrophils are capable of synthesis of large amounts of IL-8. Subsequent release of IL-8 during exudation may regulate neutrophil migration into sites of inflammation.

To investigate whether ADP-ribosylation of proteins by cholera toxin could influence B cell activation, B cells were incubated with the A subunit of cholera toxin. Ionomycin acted synergistically to induce B cell proliferation with the A subunit of cholera toxin but not with cAMP-enhancing agents or with the B subunit of cholera toxin, suggesting that the synergistic effect of the A subunit was mediated via ADP-ribosylation and not via cAMP elevations or ganglioside GM1 binding. Indeed, inhibitors of ADP-ribosylation blocked the synergistic effect. Unlike anti-Ig, B cell proliferation stimulated by LPS or by the combination of the A subunit and ionomycin was observed in protein kinase C (PKC)-depleted B cells. However, neither the A subunit nor ionomycin enhanced B cell proliferation stimulated by low dose LPS, suggesting that the A subunit plus ionomycin stimulated an activation pathway distinct from the LPS-stimulated pathway. Additionally, unlike LPS, the A subunit plus ionomycin did not stimulate B cells in vitro to secrete Ig. IL-4 acted synergistically with the A subunit to induce B cell proliferation to the same extent as it did with anti-Ig; unlike the anti-Ig plus IL-4 synergy, however, the A subunit plus IL-4-mediated synergy persisted in PKC-depleted B cells. Taken together, our data suggest that cholera toxin A subunit-catalyzed ADP-ribosylation modifies a non-Gs protein involved in the activation of B cells, either through a novel pathway or at a point distal to the activation of PKC along the anti-Ig-stimulated pathway.

The CD28 molecule expressed on the surface of T cells plays a pivotal role in transducing costimulatory signals necessary for cell activation. CD28 coligation enhances tyrosine phosphorylation and phosphoinositol 3-kinase association in responsive cells. CD28 cross-linking has also been reported to activate inositol phospholipid turnover and to cause release of intracellular calcium. Here we examine the effects of CD28 cross-linking on early activation of protein kinase C (PKC). We have reported recently that either PMA or CD28 cross-linking synergizes with signals delivered by superantigen and cytokines to induce the proliferation of APC-depleted T cells. Unlike PMA, CD28 cross-linking alone failed to induce an increase in membrane-associated PKC activity. However, PKC activation was seen in resting T cells when CD28 was cross-linked in the presence of superantigen plus APC-derived supernatant, which by themselves had no effect on PKC activity. Inhibition of PKC activity using calphostin C blocked the response of pure T cells to superantigen in the presence of either autologous APC, PMA, or CD28 cross-linking. This effect was specific; it was only seen when calphostin C was added within the first hour of stimulation. Assays of [Ca2+]i levels showed that CD28 cross-linking augmented and prolonged the rise in [Ca2+]i induced in T cells by superantigen and APC-derived cytokines. In the presence of superantigen, the proliferative response of T cells costimulated by CD28 cross-linking was cyclosporin A-sensitive, whereas in the presence of PMA, CD28 cross-linking conferred resistance to cyclosporin A. Both the phosphorylation of phospholipase C gamma 1 at tyrosine and the rise in [Ca2+]i induced by CD28 cross-linking in preactivated T cells were blocked by herbimycin A. Herbimycin A treatment also blocked the ability of CD28 cross-linking to induce a rise in [Ca2+]i in resting T cells. We conclude that CD28 costimulatory signals augment superantigen-induced TCR signals by converging onto common TCR effector pathways involving the activation of phospholipase C gamma 1 and PKC and by generating a cyclosporin A-sensitive pathway.

The role of increases in intracellular calcium levels on tyrosine phosphorylation in human B lymphocytes was studied. Stimulation of normal, resting B lymphocytes or B lymphoblastoid cells with the calcium ionophores ionomycin or A23187 induced the tyrosine phosphorylation and the enzymatic activation of microtubule-associated protein-2 kinase (MAP2-K). Treatment of these cells with PMA induced tyrosine phosphorylation of a protein with the identical mobility, as well as the enzymatic activation of MAP2-K. Stimulation of these cells with ionomycin also resulted in increased ribosomal S6 kinase activity. Activation of MAP2-K in B lymphocytes by calcium ionophore was rapid (detectable within 1 min), transient (returning to background levels by 45 min), and dependent on extracellular calcium. These results demonstrate that transmembrane calcium flux induced by calcium ionophore results in the tyrosine phosphorylation and enzymatic activation of MAP2-K in human B cells.

We identified a chymotrypsin-like activity in the granules of IL-2 lymphokine-activated killer (LAK) cells and a NK cell line (YT) that reacted preferentially with the oligopeptide substrate succinyl-Phe-Leu-Phe-thiobenzyl ester (Suc-Phe-Leu-Phe-SBzl). The enzyme was isolated by detergent extraction of sedimented cytotoxic granules and then by a sequence of sieve, hydrophobic, and anion exchange chromatography. On SDS-PAGE, the protein migrated at 42 kDa in nonreduced form and became two bands (31 and 19 kDa, respectively) after reduction. Amino-terminal sequencing of the reduced protein bands revealed 100% homology with cathepsin A-like protective protein (CAPP), a lysosomal enzyme that expresses serine carboxypeptidase and deamidase activities. The carboxypeptidase activity of lymphocyte CAPP was verified by showing that the protease preferred hydrophobic amino acids in the penultimate position of the C terminus (i.e., cleaved arginine from dansyl-Phe-Leu-Arg). The presence of lymphocyte CAPP in secretory lysosomes was demonstrated by showing that Suc-Phe-Leu-Phe-SBzl activity co-migrated with tryptase and Asp-ase activities on Percoll density gradients and that 95% of the Suc-Phe-Leu-Phe-SBzl activity in granule fractions of cavitated YT cells could be immunoprecipitated with an anti-CAPP antiserum. In addition, calcium ionophore-stimulated YT cells were shown to secrete immunoprecipitable CAPP. As proposed for platelets, lymphocyte CAPP may be secreted to function extracellularly by inactivating bioactive peptides.

mRNA expression of the immediate-early gene NGFI-B was investigated in T cells during the G0/G1 transition as well as throughout the G1 phase. After stimulation of T lymphocytes with Con A or phorbol 12,13 dibutyrate (PDBu), NGFI-B gene expression showed an induction of at least sevenfold within 3 h of stimulation. Twenty-four h later, however, the level of NGFI-B transcripts had fallen to almost basal levels. Activation of the Ca2+ signaling pathway also produced an induction of this gene, although to a lesser extent than the one obtained after protein kinase C activation. Similar transient kinetics of NGFI-B mRNA were also observed after PDBu stimulation of G1 lymphoblasts. However, the induction of NGFI-B by IL-2 is dependent on the presence of cycloheximide. Con A-induced activation of NGFI-B gene expression was not overcome by cyclosporin A or by 8Br-cAMP, but was partially prevented by dexamethasone. In lymphoblasts, okadaic acid caused the induction of NGFI-B gene expression, indicating a role for the serine/threonine protein phosphatases PP1 and PP2A in the regulation of this gene in resting cells. Okadaic acid-induced NGFI-B transcripts were significantly more stable than PDBu-induced NGFI-B mRNA. Thus, the level of NGFI-B transcripts in T cells might be determined by the balance between the activities of several serine/threonine protein kinases and phosphatases. Together, these findings indicate that the transient induction of NGFI-B transcripts is associated with normal lymphocyte activation. Because the mRNA for NGFI-B codes for a zinc-finger DNA-binding protein, these results suggest that NGFI-B participates in transcriptional regulation during T cell activation.

IL-4 protein and mRNA have recently been detected in pure basophil cultures after stimulation. It has also been established in mixed leukocyte cultures obtained by elutriation and challenged with anti-IgE that the basophil is the only cell that makes detectable IL-4. We have used cells prepared using Percoll gradients (5 to 30% basophils) to study the relationship between histamine release and IL-4 synthesis. In cultures challenged with anti-IgE after a 15-min pretreatment with IL-3, detectable IL-4 secretion ranged from 40 to 630 pg/10(6) basophils in 18 of 22 donors, with no significant correlation (r = 0.21, p = 0.34) with basophil purity. IL-4 synthesis was dissociated from histamine release, with optimal production consistently occurring at 10 ng/ml anti-IgE, whereas histamine levels peaked at 50 to 100 ng/ml of stimulus. Stimulation with anti-IgE alone was sufficient for IL-4 secretion but protein levels were enhanced two- to threefold in low responders by increasing the calcium concentration to 5 mM with no significant changes in histamine release. The IgE-independent secretagogues, F-met peptide (1 microM) and C5a (25 ng/ml), demonstrated limited ability to generate detectable IL-4, despite promoting vigorous histamine release. Additional studies showed no significant difference between IL-4 secretion in atopic and nonatopic donors. Finally, IL-4 levels generated with specific Ags were comparable to those levels produced in response to anti-IgE. We predict that basophil IL-4 generation will have a proinflammatory effect, but it appears that the signal transduction mechanisms for its synthesis and release differ from those for histamine release and thus may require different methods of pharmacologic control.

An eosinophilic substrain of HL-60 cells (HL-60#7) predominantly synthesized cysteinyl leukotrienes after stimulation with the calcium ionophore A23187. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) specifically attenuated cysteinyl leukotriene production without affecting the biosynthesis of non-cysteinyl leukotrienes. The inhibition of cysteinyl leukotriene biosynthesis was prevented only by specific PKC inhibitors (staurosporine and bisindolylmaleimide) but not by inhibitors of tyrosine kinases (genistein, tyrphostin 47, and herbimycin A), protein kinase A (KT5720), or the oxidative burst (apocynin). Similar results were obtained when LTC4 synthase enzymatic activity was measured directly in the presence of saturating concentrations of exogenously added substrates. Therefore, the inhibitory effects of PKC activation on cysteinyl leukotriene formation in intact cells was attributable to effects on the LTC4 synthase enzyme. The mechanism of inhibition of LTC4 synthase by PKC activation was determined by kinetic analysis to be noncompetitive in both eosinophil-like HL-60#7 cells and monocytic THP-1 cells. Contrary to the effect of PKC activation on cysteinyl leukotriene biosynthesis, the formation of prostaglandin E2 and thromboxane B2 was elevated twofold to threefold after PMA treatment, which was prevented by the PKC inhibitor, staurosporine. We propose a regulatory model in which PKC activation shifts the profile of eicosanoid mediators produced by eosinophils from cysteinyl leukotrienes to prostanoids.

TCA3 is a pro-inflammatory cytokine expressed in mast cells after activation. To examine how this gene is regulated, TCA3 mRNA levels were examined and nuclear run-on experiments and promoter analysis were performed. Northern blot analysis showed that TCA3 message appeared in mast cells within 2 h after induction, with a t1/2 of 30 min. Nuclear run-on experiments revealed that the appearance of TCA3 mRNA occurred in large part because of an increase in the level of de novo transcription. Analysis of the promoter region demonstrated that inducible gene expression was directed by a region extending between 1.324 kb and 0.082 kb upstream from the transcription start site. There was a 60- to 80-fold induction with TCA3 CAT constructs extending between 1.324 kb and 0.324 kb upstream from the transcription start site after treatment with PMA/A23187 and a 30- to 40-fold induction with Fc epsilon RI cross-linking. There was a seven- to eightfold induction with the region extending between 0.136 kb and 0.082 kb upstream from the transcription start site. TCA3 CAT constructs containing regions encompassing either the 0.042 kb or 2.0 kb sequence from the transcription start site were not able to direct CAT-protein synthesis. The TCA3 5' flanking sequence contained negative regulatory activity. Electrophoretic mobility shift assays revealed that protein in nuclear extracts of activated mast cells bound to an NF-kappa B element of the TCA3 gene. These findings demonstrate that the TCA3 gene is regulated transcriptionally in mast cells, with minimal promoter sequences contained within the 0.082 kb upstream region of the TCA3 gene; a putative enhancer NF-kappa B element is contained between 0.324 kb and 0.136 kb and a putative inhibitory element is contained between 1.324 kb and 2.0 kb upstream from the transcription start site.

Mast cells produce a number of cytokines including IL-6. In view of the large amounts of de novo synthesis induced by the activation of rat peritoneal mast cells and previous observations of expression of this cytokine by human lung mast cells, we have studied the regulation of IL-6 production. We examined the hypothesis that mast cell IL-6 production is not related to previous histamine release. Highly purified rat peritoneal mast cells were activated with anti-IgE, calcium ionophore A23187, or LPS. Histamine was used as a marker of preformed mediator release and IL-6 production was assessed by using the B9 hybridoma growth factor bioassay. Anti-IgE activation of rat peritoneal mast cells induced IL-6 production and histamine release. In contrast, LPS activation induced substantial, serum-dependent, IL-6 production without a significant level of histamine release. No preformed IL-6 was detected in the cells. Calcium ionophore induced histamine release from mast cells to a greater extent than did anti-IgE, but no A23187-induced IL-6 production was observed. A23187-treated cells retained high viability and produced a significant amount of TNF-alpha. To further examine the concordance of IL-6 production and histamine release we used mast cell stabilizing drugs. Dexamethasone and nedocromil significantly inhibited IL-6 production in response to anti-IgE. Our results demonstrate that there is not a direct relationship between mast cell degranulation and IL-6 production. Our observations are important for understanding the role of mast cells in inflammation and for developing strategies to modulate mast cell function in disease.

We have investigated mechanisms that regulate the generation of IL-8 by human neutrophils on contact with zymosan particles in vitro. Zymosan stimulated IL-8 production, which increased with increasing particle numbers and was abolished by the protein synthesis inhibitor cycloheximide. IL-8 was detectable in culture supernatant at 8 h reaching a maximum at 24 h. In all further experiments IL-8 was measured at 24 h. mAbs to neutrophil CD18 (60.3 and 6.5E) caused a marked suppression of IL-8 generation, but only if added up to 2 h after zymosan stimulation. An anti-CD11b mAb (KIM 225) substantially inhibited zymosan-induced IL-8 release. We investigated whether other mediators generated during phagocytosis modulate IL-8 production. Two selective platelet-activating factor (PAF) receptor antagonists, WEB 2086 and UK 74505, produced a profound suppression of IL-8 generation, when added within 30 min to 1 h of zymosan stimulation. An IL-1R antagonist, a leukotriene B4 antagonist, and an anti-TNF-alpha Ab had no effect on IL-8 generation. FMLP, PAF, and a stable PAF agonist did not stimulate significant IL-8 production, however, a calcium ionophore (A23187) did induce IL-8 release and this was suppressed by UK 74505. We conclude that zymosan-induced IL-8 generation involves stimulation of the neutrophil via a CD11b/CD18 receptor resulting in beta 2-integrin mediated activation of signal transduction mechanisms that leads to cytokine synthesis. Furthermore, endogenously generated PAF, or a PAF, or a PAF-like molecule, appears to have an autocrine function in regulating this pathway of IL-8 production at an early stage after the interaction between the neutrophil and the particles.

We analyzed the effects of rIL-10 on IL-5 production by human resting T cells isolated from peripheral blood. Resting T cells of healthy individuals required activation for 48 h with either anti-CD3 mAb cross-linked on B7/CD32-transfected mouse fibroblasts or PMA in conjunction with anti-CD28 mAb for optimal IL-5 secretion. In each condition, IL-5 secretion measured by ELISA was inhibited in a dose-dependent manner by rIL-10, whereas IFN-gamma production was not suppressed. The inhibitory effect of rIL-10 on IL-5 synthesis induced by PMA and anti-CD28 mAb was also observed at the mRNA level. In contrast with its action on T cells costimulated by B7/CD28 signaling, rIL-10 did not block IL-5 secretion in response to PMA and A23187 calcium ionophore. The inhibition of IL-5 production by rIL-10 was not due to IL-2 deprivation because it was not modified by the addition of exogenous rIL-2. Moreover, cyclosporin A, which inhibited IL-2 more efficiently than rIL-10 in response to anti-CD3 mAb and B7/CD32 transfected fibroblasts, did not reduce and even enhanced IL-5 production. Finally, we analyzed the influence of endogenously produced IL-10 on IL-5 secretion by T cells stimulated by PMA and anti-CD28 mAb. Addition of a neutralizing anti-IL-10 mAb increased IL-5 release in this system, indicating that endogenous IL-10 controls IL-5 production. We conclude that both rIL-10 and endogenous IL-10 inhibit IL-5 production by T cells costimulated by B7/CD28 signaling.

CD43, an anionic rod-like mucin molecule on white blood cells, is thought to provide a barrier that prevents interactions of other surface molecules and acts as negative regulator of cell function. As a correlate, CD43 is expected to be altered or down-regulated when blood cells are functionally activated. This study examines CD43 of blood neutrophils before and after treatment with known activating agents. Flow cytometry indicated that PMA and A23187, and to a much lesser extent, FMLP and IL-8, decrease neutrophil expression of CD43. Two separate mechanisms were identified for CD43 down-regulation. Both are proteolytic processes. PMA-induced down-regulation is a rapid process involving proteolysis at a minimum of two sites, one within the N-terminal distal region recognized by mAbs and the other at a membrane-proximal site. The PMA-induced protease, cd43' ase, is characterized by insensitivity to DFP, TLCK, leupeptin, pepstatin, and 1,10 phenanthroline (< 5 mM). PMA-induced CD43 down-regulation is extensive but never complete, terminating at approximately 10 min after down-regulating 65 to 85% of molecules, and thereby converting neutrophils from dense to sparse CD43 expression. The second CD43 down-regulation mechanism, although likely a regulated event in vivo, occurred slowly in this study in neutrophils incubated without additives; the process is not affected by PMA, involves the action of a DFP-sensitive protease, releases N-terminal mAb-reactive fragments of 52 kDa or 40 kDa and can be mimicked by exogenous neutrophil elastase. The complexity and apparent tight regulation described here for the two down-regulatory mechanisms are consistent with an important role for CD43 in preventing or dampening cell surface interactions of blood neutrophils.

Cefdinir, a new oral 2-amino-5-thiazolyl cephalosporin, inhibited the luminol-amplified chemiluminescence (LACL) response of human neutrophils stimulated by PMA but not opsonized zymosan, in a concentration-dependent but not time-dependent manner. The LACL response to opsonized zymosan in cytochalasin B-treated neutrophils was, however, inhibited by cefdinir. Various cephalosporins, regardless of the presence of a 2-amino-5-thiazolyl moiety, did not significantly alter the neutrophil LACL response triggered by PMA and zymosan. The LACL response induced by the calcium ionophore A23187 and FMLP was also impaired by cefdinir, and this impairment was increased in cytochalasin B-treated neutrophils. Superoxide anion generation by neutrophils, measured in terms of lucigenin-amplified chemiluminescence and cytochrome c reduction, was not altered. Spontaneous and FMLP-induced neutrophil degranulation, assessed by lysozyme and beta-glucuronidase release, were not modified by cefdinir. Furthermore, cefdinir inhibited LACL generation in cell-free systems consisting of H2O2, NaI, and either horseradish peroxidase or a myeloperoxidase-containing neutrophil extract. Orthodianisidine oxidation in these two acellular systems was inhibited by cefdinir. Cefdinir did not alter neutrophil bacterial killing at concentrations that inhibited myeloperoxidase-containing neutrophil extract-dependent reactions induced by soluble stimuli. Taken together, these data strongly suggest that cefdinir directly inhibits the activity of myeloperoxidase-containing neutrophil extract released into the extracellular medium during neutrophil stimulation by soluble mediators, but has no effect on that released into the phagolysosome during phagocytosis. This unusual property of a member of the beta-lactam family could be of interest in modulating the exaggerated inflammatory process often associated with infectious diseases.

IL-11 and IL-6 are fibroblast-derived cytokines with overlapping biologic properties. To determine whether IL-11 and IL-6 are similarly regulated, we characterized the effects of rIL-1 and TGF-beta (beta 1 and beta 2) on human lung fibroblast IL-11 production and compared this regulation with that of IL-6. Unstimulated fibroblasts did not produce significant amounts of IL-11, whereas rIL-1 alpha and TGF-beta were dose-dependent stimulators of IL-11 protein production, mRNA accumulation, and gene transcription. rIL-1 alpha and TGF-beta also interacted in a synergistic fashion to further increase IL-11 protein production and mRNA accumulation. The effects of rIL-1 and TGF-beta individually were not altered by the cyclic nucleotide-dependent protein kinase inhibitor HA1004, protein kinase C (PKC) inhibition with staurosporine, or chronic phorbol ester preincubation, or the calmodulin antagonists W7 and TFP. The effects of rIL-1 alpha and TGF-beta in combination were also unaltered by HA1004, staurosporine, and chronic phorbol ester exposure. A23187, however, did induce IL-11 mRNA accumulation and W7 and TFP did reverse the synergistic stimulation caused by rIL-1 and TGF-beta in combination. In contrast with the regulation of IL-11, TGF-beta did not effectively stimulate IL-6 mRNA accumulation, rIL-1 alpha was a more potent stimulator of IL-6 than IL-11 production, and rIL-1-induced IL-6 mRNA accumulation was augmented by W7 and TFP. These studies demonstrate that: 1) rIL-1, TGF-beta, and agents that increase intracellular calcium stimulate lung fibroblast IL-11; 2) the IL-11 stimulatory effects of rIL-1 and TGF-beta are, at least partially, transcriptionally mediated and are the result of signal transduction pathways that are largely PKC, cyclic nucleotide, and calmodulin independent; and 3) rIL-1 and TGF-beta interact in a synergistic fashion to further increase fibroblast IL-11 production and that this synergy is mediated by a largely PKC- and cyclic nucleotide-independent and calmodulin-dependent activation pathway. Importantly, they also demonstrate that rIL-1 and TGF-beta stimulate lung fibroblast IL-6 and IL-11 production via distinct and differentially regulatable activation pathways.

Arachidonic acid (AA) incorporation into and release from cellular glycerolipids are thought to be crucial events for the regulation of eicosanoid biosynthesis in inflammatory cells. The goal of our study was to determine the distribution of AA in the lipid pools of human lung macrophages (HLM) isolated from human lung parenchyma and to define the changes in AA pools occurring during cell activation. Mass spectrometry analysis indicated that the major pools of AA in HLM were located in phosphatidylethanolamine (PE), phosphatidylcholine (PC), and triglycerides (TG), and, to a lesser extent, in phosphatidylinositol/phosphatidylserine (PI/PS) and in a phospholipid similar to bis-(monoacylglyceryl)-phosphate (BMP). Exogenous AA was initially incorporated into TG and subsequently distributed within phospholipids. After 24 h of labeling, the distribution of exogenous AA in glycerolipid classes closely mimicked that of endogenous AA. Under these equilibrium conditions, HLM released 18.7 and 20.2% of cellular AA when stimulated with TPA or A23187, respectively. Free AA was the major product released by TPA- or A23187-stimulated HLM. However, A23187 but not TPA also induced the formation of leukotriene B4 and 5-HETE. AA was released from PC > PI/PS > or = PE > BMP. In contrast to phospholipids, the amount of AA in TG increased 15 to 90 min after cell activation and returned to prestimulation levels after 180 min. These data indicate that AA is stored in several glycerolipid pools of HLM. Among these pools, BMP is unique of macrophages and TG are particularly enriched in AA in HLM. During the early stage of cell activation, phospholipids act as a source of AA, whereas TG function as a reacylation pool for AA released from phospholipids. These data indicate that TG and phospholipid pools have a differential role in the control of free AA levels generated during HLM activation.

One immune function of astrocytes is IL-6 production. Synthesis of IL-6 within the central nervous system (CNS) can produce several different responses, acting on glia, neurons, and lymphocytes infiltrating brain tissue, and some of these effects are associated with CNS autoimmune disease. IL-6 gene expression in astrocytes is regulated by cytokines, infectious agents, neuropeptides, and neurotransmitters, and most of these stimuli interact synergistically. To examine the integration of these diverse factors in the control of IL-6 production, we have studied the involvement of underlying signal transduction processes using neonatal rat astrocytes. We have focused on signal transduction related to the stimulation of IL-6 gene expression by IL-1 beta and TNF-alpha. Our results indicate that stimuli related to protein kinase C (PKC), such as PMA and calcium ionophore A23187, increase IL-6 expression, whereas pharmacologic inhibitors of PKC inhibit IL-6 induction by IL-1 beta and TNF-alpha. Furthermore, both IL-1 beta and TNF-alpha stimulate PKC activity in astrocytes. Stimulators of the cAMP pathway, such as cholera toxin, forskolin, and dibutyryl cAMP, also induced astrocyte IL-6 gene expression. However, inhibition of the cAMP pathway effector, protein kinase A, did not reduce the induction of astrocyte IL-6 gene expression in response to IL-1 beta or TNF-alpha, and an ELISA for cAMP detected only very small increases in cAMP synthesis in response to these cytokines. These data suggest that although cAMP does activate astrocyte IL-6 gene expression, it is the PKC pathway that plays a primary role in the stimulation of astrocyte IL-6 gene expression by IL-1 beta and TNF-alpha.

Ag-induced cross-linking of IgE bound to its high affinity receptor (Fc epsilon RI) at the surface of basophils or mast cells triggers a number of biochemical events culminating in the release of several inflammatory mediators. In rat basophilic leukemia (RBL-2H3) cells expressing the G protein-coupled m1 muscarinic receptor, Ag/IgE-induced cross-linking of Fc epsilon RI, calcium ionophore A23187, and carbachol through M1 receptors stimulated tyrosine phosphorylation of several proteins, including two of 42 and 44 kDa. Proteins of identical molecular masses were recognized by anti-MAP-kinase antibodies, and these immunoreactive proteins exhibited in part a slightly increased molecular mass on SDS polyacrylamide gels after incubation of cells with secretory stimuli. All stimuli led to the activation of MAP kinase, which co-purified on Mono Q chromatography with 42- and 44-kDa proteins, which were tyrosine phosphorylated in response to secretory stimuli and reacted with anti-(MAP kinase) antibodies. Finally, 42- and 44-kDa proteins immunoprecipitated by anti-MAP-kinase antibodies and anti-phosphotyrosine antibodies were recognized by anti-phosphotyrosine and anti-MAP-kinase antibodies, respectively. Primarily threonine and tyrosine residues were found to be phosphorylated in 42- and 44-kDa proteins immunoprecipitated from [32P]phosphate-labeled cells that had been treated with secretory stimuli. The dose dependence of secretagogue-induced MAP kinase activation correlated with that of increases in serotonin release from activated cells, and the maximum of MAP kinase activation coincided with the maximum rate of secretion. Down-regulation or inhibition of protein kinase C as well as incubation of cells with the tyrosine kinase inhibitor genistein markedly inhibited MAP kinase activation in parallel with serotonin release. Taken together, these findings demonstrate that 42- and 44-kDa MAP kinases are activated in response to secretory stimuli and provide some evidence for a functional link between MAP kinase activation and signaling events leading to mediator release in RBL cells.

We investigated whether cyclosporin A (CsA) in vivo exerts anti-inflammatory effects by inhibiting IgE- and non-IgE-dependent mediator release from human basophils. Six healthy volunteers were given oral CsA (5 mg/kg twice daily) or placebo for 5 days. Plasma CsA and basophil releasability in response to anti-IgE, FMLP, and A23187 were monitored 1 day before treatment, on alternate days during the treatment course, and 1 and 8 days after cessation of treatment. A constant plasma level of approximately 250 ng/ml CsA was obtained during CsA treatment. Basophil releasability in response to anti-IgE, FMLP, and A23187 was diminished by 20 to 60% throughout the course of CsA treatment. Placebo had no effect on basophil releasability. There was a significant correlation between plasma CsA and the decrease of histamine release induced by anti-IgE (rs = -0.66; p < 0.0005), FMLP (rs = -0.59; p < 0.001) and A23187 (rs = -0.68; p < 0.0001). In a second study, eight normal volunteers were given a single oral dose of CsA (7 mg/kg) or placebo, and plasma CsA and basophil releasability were monitored at different times thereafter. A rapid and significant reduction of histamine release induced by anti-IgE, FMLP, and A23187 paralleled a sharp increase of CsA plasma levels, which peaked at 5 h and lasted approximately 13 h. This study indicates that oral administration of CsA in normal subjects causes a rapid and significant inhibition of histamine release from basophils. This is the first evidence that in vivo administration of CsA can modulate the release of proinflammatory mediators from basophils obtained ex vivo.

Recruitment of neutrophils is a common feature in diseases that are associated with mast cell activation. The mechanisms that mediate neutrophil activation are not well understood. IL-8 is a recently described potent chemotactic factor that might be pathogenetically involved in this process. We therefore studied the human mast cell line HMCI and human skin mast cells for their ability to produce IL-8 using various stimuli. IL-8-mRNA was expressed in a stimulus- and time-dependent fashion as detected by Northern blot analysis with an IL-8-specific cDNA probe. The molecular mass of HMCI-derived IL-8 was determined to be about 8 kDa by immunoblot analysis. Immunoreactive and biologically active IL-8 protein was measured in the cell culture supernatants of HMCI cells by an ELISA and a chemotaxis assay, respectively. On immunoelectron microscopy of stimulated skin mast cells, IL-8 was found along cytoplasmatic membranes and in intracellular granules. Our data indicate that mast cells may contribute to neutrophil recruitment by secretion of IL-8.