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Estrogen has known anti-inflammatory effects, but the mechanism whereby 17β-estradiol (E2) protects oviduct sheep epithelial cells from inflammation remains unknown. In this study, we detected the E2 synthetase and E2 nuclear and membrane receptors in sheep oviducts, primarily in epithelial cells. Using lipopolysaccharide (LPS)-stimulated sheep oviduct epithelial cells as an in vitro inflammation model, we demonstrated that E2 attenuates the expression of inflammatory factors in a concentration-response manner. E2 also inhibited the LPS-stimulated phosphorylation of p38 MAPK and NF-κB p65 but did not reduce the phosphorylation of JNK and ERK 1/2. This attenuation was partially antagonized by an intracellular estrogen antagonist that was involved in genomic regulation and enhanced by a G protein-coupled estrogen receptor agonist that was involved in nongenomic cellular modulation. These results suggest that E2 has an inhibitory effect on LPS-induced oviduct epithelial cell inflammation in sheep, which is mediated by the downstream regulatory effects of estrogen receptors.

Macrophages (Mφ) undergo activation to pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes in response to pathophysiologic stimuli and dysregulation of the M1-M2 balance is often associated with diseases. Therefore, studying mechanisms of macrophage polarization may reveal new drug targets. Human Mφ polarization is generally studied in primary monocyte-derived Mφ (PBMC Mφ) and THP-1-derived Mφ (THP-1 Mφ). We compared the polarization profile of THP-1 Mφ with that of PBMC Mφ to assess the alternative use of THP-1 for polarization studies. Cellular morphology, the expression profiles of 18 genes and 4 cell surface proteins, and phagocytosis capacity for apoptotic cells and S. aureus bioparticles were compared between these Mφ, activated towards M1, M2a, or M2c subsets by stimulation with LPS/IFNγ, IL-4, or IL-10, respectively, for 6 h, 24 h and 48 h. The Mφ types are unique in morphology and basal expression of polarization marker genes, particularly CCL22, in a pre-polarized state, and were differentially sensitive to polarization stimuli. Generally, M1 markers were instantly induced and gradually decreased, while M2 markers were markedly expressed at a later time. Expression profiles of M1 markers were similar between the polarized Mφ types, but M2a cell surface markers demonstrated an IL-4-dependent upregulation only in PBMC Mφ. Polarized THP-1 Mφ but not PBMC Mφ showed distinctive phagocytic capacity for apoptotic cells and bacterial antigens, respectively. In conclusion, our data suggest that THP-1 may be useful for performing studies involving phagocytosis and M1 polarization, rather than M2 polarization.

In this study, a complete molecular characterization of tumor necrosis factor receptor 1 (TNFR1) which was identified from the constructed cDNA library of striped murrel Channa striatus (Cs) is reported. The CsTNFR1 encoded a type I membrane receptor protein that contains 399 amino acids including three cysteine-rich domains (CRDs) at CRD1(41-46), CRD2(79-118) and CRD3(120-159) in the extracellular region and a putative TNF receptor-associated factor (TRAF) site at 245-253 and a death domain between 297 and 388 in the cytoplasmic region which is essential for induction of apoptosis. The predicted molecular mass of CsTNFR1 is 45kDa and its corresponding theoretical isoelectric point (pI) is 6.3. CsTNFR1 shared maximum identity with TNFR1 from olive flounder Paralichthys olivaceus (82%). Real-time PCR showed that CsTNFR1 gene was expressed most abundantly (P

Graphical abstract, Highlights • The IgE antibody plays a central role in allergic disease. • Binding of IgE to the B-cell receptor CD23 regulates IgE synthesis. • Seven crystal forms of the IgE-binding head domain of CD23 have been analyzed and compared. • The thirty-five independent structures reveal conformational plasticity in two IgE-binding loops., IgE antibodies play a central role in allergic disease. They recognize allergens via their Fab regions, whilst their effector functions are controlled through interactions of the Fc region with two principal cell surface receptors, FcɛRI and CD23. Crosslinking of FcɛRI-bound IgE on mast cells and basophils by allergen initiates an immediate inflammatory response, while the interaction of IgE with CD23 on B-cells regulates IgE production. We have determined the structures of the C-type lectin “head” domain of CD23 from seven crystal forms. The thirty-five independent structures reveal extensive conformational plasticity in two loops that are critical for IgE binding.

Mast cells (MCs) can have either detrimental or beneficial effects on malignant processes but the underlying mechanisms are poorly understood. Here we addressed this issue by examining the interaction between Lewis Lung Carcinoma (LLC) cells and MCs. In vivo, LLC tumors caused a profound accumulation of MCs, suggesting that LLC tumors have the capacity to attract MCs. Indeed, transwell migration assays showed that LLC-conditioned medium had chemotactic activity towards MCs, which was blocked by an antibody towards stem cell factor. In order to gain insight into the molecular mechanisms operative in tumor–MC interactions, the effect of LLC on the MC gene expression pattern was examined. As judged by gene array analysis, conditioned medium from LLC cells caused significant upregulation of numerous cell surface receptors and a pro-angiogenic Runx2/VEGF/Dusp5 axis in MCs, the latter in line with a role for MCs in promoting tumor angiogenesis. Among the genes showing the highest extent of upregulation was Tnfrsf9, encoding the anti-tumorigenic protein 4-1BB, suggesting that also anti-tumorigenic factors are induced. Quantitative RT-PCR analysis showed that 4-1BB was upregulated in a transient manner, and it was also shown that tumor cells induce 4-1BB in human MCs. Immunohistochemical analysis showed that LLC-conditioned medium induced 4-1BB also at the protein level. Together, this study provides novel insight into the molecular events associated with MC–tumor interactions and suggests that tumor cells induce both pro- and anti-tumorigenic responses in MCs.

Factors in physiological fluids that regulate the chemotactic activity of complement activation peptides C5a and C5a des Arg are not well understood. The vitamin D binding protein (DBP) has been shown to significantly enhance chemotaxis to C5a/C5a des Arg. More recently, platelet-derived thrombospondin-1 (TSP-1) has been shown to facilitate the augmentation of C5a-induced chemotaxis by DBP. The objective of this study was to better characterize these chemotactic cofactors and investigate the role that cell surface TSP-1 receptors CD36 and CD47 may play in this process. The chemotactic activity in C-activated normal serum, citrated plasma, DBP-depleted serum or C5 depleted serum was determined for both normal human neutrophils and U937 cell line transfected with the C5a receptor (U937-C5aR). In addition, levels of C5a des Arg, DBP and TSP-1 in these fluids were measured by RIA or ELISA. Results show that there is a clear hierarchy with C5a being the essential primary signal (DBP or TSP-1 will not function in the absence of C5a), DBP the necessary cofactor and TSP-1 a dependent tertiary factor, since it cannot function to enhance chemotaxis to C5a without DBP. Measurement of the C5a-induced intracellular calcium flux confirmed the same hierarchy observed with chemotaxis. Moreover, analysis of bronchoalveolar lavage fluid (BALF) from patients with the adult respiratory distress syndrome (ARDS) demonstrated that C5a-dependent chemotactic activity is significantly decreased after anti-DBP treatment. Finally, results show that TSP-1 utilizes cell surface receptors CD36 and CD47 to augment chemotaxis, but DBP does not bind to TSP-1, CD36 or CD47. The results clearly demonstrate that C5a/C5a des Arg needs both DBP and TSP-1 for maximal chemotactic activity and suggest that the regulation of C5a chemotactic activity in physiological fluids is more complex than previously thought.

Proteolytic processing of transmembrane receptors and ligands can have a dramatic impact on cell signaling processes and subsequent cellular responses, including activation and differentiation. A member of the disintegrin and metalloproteinase family, ADAM10, has emerged as a prominent regulator of numerous receptors and ligands, including Notch and CD23. Here, we review studies resulting from the recent generation of ADAM10 conditional knockout mice which revealed a critical role for ADAM10 in Notch-dependent lymphocyte development. Additionally, we discuss results of numerous in vitro and ex vivo studies indicating that ADAM10 regulates the production of multiple secreted factors that contribute to autoimmune reactions.

Pattern recognition receptors (PRR) recognize invariant bacterial, viral, protozoan and certain synthetic ligands. PRR may be expressed as outer membrane (or endosomal) or cytosolic proteins and function to signal cell activation processes during inflammation responses. In the present study, a novel membrane receptor, NCC cationic antimicrobial protein-1 (NCAMP-1), is described that is expressed on nonspecific cytotoxic cell (NCC) membranes and is found in granule extracts from these cells. In recombinant form, full-length (amino acids 1-203) and truncated N (NT; amino acids 1-60) and C (CT; amino acids 116-203) terminal forms of NCAMP-1 had antibacterial activity against bovine, avian and lab strain Escherichia coli. Recombinant NCAMP-1-NT also killed the gram-negative fish pathogen Edwardsiella ictaluri. Maximal bacterial killing of a representative avian E. coli, APEC 3721, occurred at 60min post-treatment with 2microg/ml of rNCAMP-1-NT. Killing occurred by NCAMP-1-NT-induced alterations in the permeability of the bacterial cell wall. Polyclonal antibody anti-NCAMP-1 specifically neutralized the antimicrobial activity of recombinant NCAMP-1-NT against E. coli APEC 3751. Expression of NCAMP-1 as a NCC membrane protein was analyzed by flow cytometry using anti-NCAMP-1 monoclonal antibody 9C9. Merged images from immunofluorescence microscopy showed that NCAMP-1 and the NCC receptor protein (NCCRP-1) are co-expressed on NCC membranes. NCAMP-1 was identified in acetic acid granule extracts of NCC by Western blot analysis using polyclonal anti-NCAMP-1 and killing of E. coli by these extracts was specifically inhibited by this polyclonal. These data suggested that NCAMP-1 is a membrane protein and may participate in antibacterial innate immunity by granule exocytosis during inflammatory responses in teleosts.

Toll-like receptors (TLRs) play an essential role in the detection and elimination of invading microbes. They are type-1 transmembrane receptors, containing extracellular leucine rich repeats and an intracellular Toll/interleukin-1 receptor (TIR) domain. Upon stimulation, these receptors interact with specific TIR domain-containing adaptor proteins. Five such adaptors have been discovered to date, MyD88, Mal (MyD88 adaptor-like)/TIRAP (TIR domain-containing adaptor protein), Trif (TIR-domain-containing adaptor inducing interferon-β), TRAM (Trif-related adaptor molecule) and SARM (SAM and ARM-containing protein). Different TLRs use different combinations of these adaptors, leading to the activation of common and unique pathways involved in the elimination of the invading microbe.

Aggregation of cell surface receptors is a ubiquitous means of initiating signal transduction in many cellular systems. In this manuscript, we describe a combined theoretical and experimental approach based on multiparameter flow cytometry for measuring the time course of ligand induced aggregation of IgE–FceRI on RBL cells. By fluorescently labeling both the ligand and surface IgE (sIgE), we have developed an assay that permits us to simultaneously measure both occupancy of sIgE combining sites and association of antigen with the cell surface. This allows for a direct calculation of the degree of receptor aggregation present on the cell. By employing new mixing technologies developed for flow cytometry, we are able to look at aggregation in the sub second time domain. To extend our work, we have synthesized a new set of chemically well defined ligands (of valences 1–3) to use as probes in our studies. We show that the magnitude of the cellular response is dramatically increased as the valence of our ligand is raised from two to three.

A series of compounds were tested as inhibitors of receptor-binding and uptake of fluorescein-derivatized antigens in primary peritoneal and J774 murine macrophage. Results were analysed with regard to the inhibitory potency of the tested ligands and revealed that the putative cell surface receptor preferentially recognized and bound aromatic structures containing phenyl rings. L-phenylalanine was found to be the most potent inhibitor among the ligands tested. Significant inhibition of FITC10BSA binding to macrophage was observed even at 10(-9) M concentration of specific monovalent ligands tested indicating that these ligands were bound by the putative macrophage receptor with high apparent affinity. Structural comparisons of the various inhibitors employed, demonstrated that accessible, unconjugated phenyl rings were bound by the putative receptor with high apparent affinity whereas non-phenyl derivatives were bound with either low apparent affinity or via non-specific interactions. Therefore, the fluorescein hapten appeared to utilize a receptor with specificity for an essential aromatic amino acid for gaining entry into the endocytic pathway of murine macrophage. Finally, the binding of the hapten was enhanced when polyvalent fluorescein-derivatized antigens were used as a result of receptor crosslinkage on the cell surface.

Aggregation of cell surface receptors plays an important role in signal transduction in many receptor systems. In the T cell receptor (TCR), as in many other cell surface receptors, this aggregation results in insolubility in certain nonionic detergents. We have characterized this insolubility for TCR, and we show it is not preexisting in HPB-ALL cells but increases with increasing TCR aggregation. It is not likely to be due to a direct interaction with cellular cytoskeletal elements, as it is not affected by inhibitors of actin or tubulin polymerization. It may be due to interaction with detergent-resistant membrane domains that have been found in various cell types and contain tyrosine kinases, the earliest known participants in TCR signal transduction. This aggregation-dependent insolubility occurs as rapidly as the anti-TCR antibody binds, so the kinetics are consistent with an involvement in signal transduction. It is not, however, dependent on signal transduction, as inhibitors of tyrosine kinases do not inhibit the insolubility. Insolubility is also enhanced by preaggregation of CD4, an important T cell surface molecule which also associates with the tyrosine kinase p56lck.

CD28, which is a member of the immunoglobulin superfamily of molecules (IgSF), is a homodimer of two polypeptides containing a single V-like domain with short transmembrane and cytoplasmic regions. It serves as a co-signalling molecule for T cell activation through binding to its cognate counter-receptors CD80 and B70, expressed on antigen presenting cells. In the current study, we investigated the regions of CD28 which are involved in its interactions with CD80 and B70, using site directed mutagenesis, CD28 mAb epitope mapping, receptor based adhesion assays and direct binding of Ig-fusion proteins to cell surface receptors. Truncation or substitution of a stretch of a proline rich "hallmark" sequence, "MYPPPY", abrogates binding to CD80 or B70, while retaining CD28 mAb epitopes and cell surface expression. On an Ig-fold model of the CD28 V-domain, this fully conserved motif localizes to a CDR3-like region. Mutations introduced into other loops, including the CDRI-like and CDR2-like regions, had very little effect on CD80 or B70 binding. Mutations introduced within the predicted beta-strand regions caused loss of receptor expression. Conservative substitution of both the flanking tyrosine residues within the "MYPPPY" motif with phenylalanine, caused loss of binding to B70 but not to CD80. These results show that, although the same overall region on CD28 may be involved in the interactions with CD80 and B70, subtle but important differences distinguish recognition by the two molecules. These finding, along with previous observations on the differential pattern of expression and tissue distribution of CD80 and B70, support the contention that these molecules play distinct roles in the regulation of immune responses in vivo.

Cell surface receptors mediate many cellular responses in health and disease. Recent progress in our understanding of how ligand binding to the extracellular domains of receptors triggers intracellular signaling has underlined the role of ligand-promoted receptor clustering following by oligomerization of the cytoplasmic signaling domains. The clustering suggests the requirement of ligand multivalency and is especially important for triggering receptors involved in innate and adaptive immune responses. However, although numerous studies have established that multivalent, but not monovalent, ligands induce receptor-mediated signal transduction, considerable uncertainty still remains. Here, I hypothesize that “monovalent” ligands that have been reported to trigger immune receptors in vitro are not necessarily truly monovalent. This is illustrated by focusing on studies of signal transduction by toll-like receptor-4 and T cell receptor. By generalizing this concept to a variety of lipid and protein ligands, one would propose an alternative interpretation of apparent ligand monovalency in other receptor activation studies as well.

The effects of cAMP upon cell proliferation, cytotoxic activity, and regulation of IL-2R expression was investigated in normal human, IL-2-dependent natural killer (NK) cell clones. We report here that addition to the cultures of Bt2cAMP, a cell permeant analogue of cAMP, results in inhibition of IL-2-dependent proliferation, as assessed by [3H]Thymidine incorporation, in both NK and T cell clones. In addition, Bt2cAMP was shown to block the cytotoxic activity of NK cell clones at the level of the lytic phase. Contrasting with these inhibitory effects, cAMP induces an upregulation of the membrane expression of the IL-2R alpha chain (p55, Tac) in normal NK cell clones, which correlates with an accumulation of Tac mRNA. This is clearly at variance with T cell clones in which no such effect of cAMP alone is observed. In both cell types however, cAMP appears to synergize with IL-2 to increase IL-2R alpha mRNA expression. In addition, we demonstrate, using a cDNA probe to the IL-2R beta, that expression of this second component of the high affinity IL-2R, does not appear to be co-regulated together with IL-2R alpha in response to cAMP or/and IL-2 in cultured NK cells. Thus the effects of cAMP on human NK cell clones are complex. cAMP is inhibitory of proliferation and cytolytic function, whereas it is stimulatory of IL-2R alpha expression in these cells.

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (Fc gamma R). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fc gamma R. This domain was mapped for interaction with mouse Fc gamma R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human Fc gamma R1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237----Ala, Asn 297----Ala, and Glu 318----Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse Fc gamma R11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by Fc gamma R11 of the P388D1 cell line.

A cell surface receptor that binds to the Fc region of IgA is expressed by certain strains of group A streptococci. The physico-chemical properties and binding characteristics of this receptor, called protein Arp, were studied. Like bacterial receptors that bind IgG, protein Arp has an elongated shape and no disulfide bonds. The affinity constant of protein Arp for three different molecular forms of IgA was determined, and was found to be more than ten-fold higher for serum IgA than for two complexed forms of IgA: secretory IgA and IgA bound to alpha 1-microglobulin. Cleavage of protein Arp with CNBr resulted in a peptide corresponding to the region located outside the cell wall, except for the N-terminal 52 amino acids. This CNBr-fragment did not bind IgA, which strongly suggests that the IgA-binding region of protein Arp is located in the N-terminal part of the molecule. In addition to the binding of IgA, protein Arp also binds to IgG weakly. The pH-dependence of these two types of binding is different, with maximal binding of IgA at neutral pH (5-7) and maximal binding of IgG at acidic pH (3-5). Both for IgA and IgG, protein Arp shows strong specificity for immunoglobulins of human origin.

When exposed to hen ovalbumin (OA) complexes of IgG antibodies, guinea-pig macrophages were found to augment the phosphatidylinositol (PI) turnover. This response depended on the IgG isotype of antibodies used; OA complex of IgG2 antibody (OA gamma 2) triggered it about 3 times more effectively than did OA complex of IgG1 antibody (OA gamma 1). The inhibition experiments with monoclonal antibodies to Fc gamma 1/gamma 2R and Fc gamma 2R showed that Fc gamma 2R triggered activation of the PI turnover more intensively than did Fc gamma 1/gamma 2R. The same results were also obtained by the cross-linking of Fc gamma 2Rs or Fc gamma 1/gamma 2Rs by a combination of anti-mouse IgG F(ab')2 and anti-Fc gamma 2R F(ab')2 or anti-Fc gamma 1/gamma 2R F(ab')2. As the number of Fc gamma 2R molecules per macrophage is about one-half that of Fc gamma 1/gamma 2R molecules, the ability of Fc gamma 2R to trigger the response was found to be much greater than that of Fc gamma 1/gamma 2R. Despite this difference, neither the activity of Fc gamma 2R nor that of Fc gamma 1/gamma 2R to augment the PI turnover were affected by depletion of the intracellular Ca2+ by incubating the cells with Ionomycin and EGTA, and also by the treatment with pertussis toxin.

Glycodelin A (GdA) is one of the progesterone inducible endometrial factors that protect the fetal semiallograft from maternal immune rejection. The immumoregulatory effects of GdA are varied, with diverse effects on the fate and function of most immune cell types. Its effects on T cells are particularly relevant as it is capable of regulating T cell activation, differentiation, as well as apoptosis. We have previously reported that GdA triggers mitochondrial stress and apoptosis in activated T cells by a mechanism that is distinct and independent of its effects on T cell activation. In this study we describe the characterization of a cell surface receptor for GdA on T cells. Our results reveal a novel calcium-independent galactose-binding lectin activity of GdA, which is responsible for its apoptogenic function. This discovery adds GdA to a select group of soluble immunoregulatory lectins that operate within the feto-placental compartment, the only other members being the galectin family proteins. We also report for the first time that both CD4(+) and CD8(+) T cell subsets are equally susceptible to inhibition with GdA, mediated by its novel lectin activity. We demonstrate that GdA selectively recognizes complex-type N-linked glycans on T cell surface glycoproteins. and propose that the galectin-1 glycoprotein receptor CD7 maybe a novel target for GdA on T cells. This study, for the first time, links the lectin activity of GdA to its biological function.

Human monoclonal IgG1 and IgG3 antibodies specific for the Rh antigen D (anti-D) were tested for their ability to promote the binding of D-positive red cells to peripheral blood monocytes and Fc receptor (FcR)-bearing cell lines (U937, K562 and Daudi). Monocyte-mediated antibody-dependent cell-mediated cytotoxicity and metabolic (chemiluminescent) responses were also determined. By comparing the activity of different cell lines in rosette assays, and by using murine myeloma IgG2a and IgG1 to block FcRI and FcRII respectively, these functional interactions of sensitized red cells (E-IgG1 and E-IgG3) with monocytes or cell lines were shown to be mediated predominantly and perhaps solely by FcRI. E-IgG3 bound to human monocytes and cell lines to a greater extent than E-IgG1. Rosette formation by E-IgG3 was relatively less susceptible to inhibition by fluid-phase murine IgG2a than was rosette formation by E-IgG1. These findings may be due to the long hinge region of IgG3 which enables it to bridge the gap between two negatively charged cells more efficiently than IgG1. Consistent with this hypothesis was the greatly increased rosette formation achieved by treating monocytes or U937 cells with neuraminidase or bromelain, procedures shown to reduce the zeta potential of these cells. The lytic and metabolic activities of untreated human monocytes were also greater towards E-IgG3 than E-IgG1, red cell binding being a prerequisite for these responses. However, after pretreatment of monocytes with neuraminidase, these responses were greater with E-IgG1 than with E-IgG3. Further, the addition of polybrene to non-specifically enhance cell to cell binding also resulted in greater lysis and chemiluminescence with E-IgG1 than with E-IgG3. These results indicate that, although E-IgG3 are more effective than E-IgG1 in promoting red cell binding to monocytes, E-IgG1 are more efficient at activating the lytic and metabolic processes providing the steric disadvantages of the shorter hinge region of cell-bound IgG1 are circumvented.

Human interleukin 4 (IL-4) upregulates Fc epsilon R2/CD23 expression on the surface of B lymphocytes. Here it is shown that IL-4 induces expression of CD23 mRNA in normal human B lymphocytes whereas recombinant IL-1, IL-2, IL-5, IFN gamma, IFN alpha 2b and semi-purified low molecular weight B cell growth factor were unable to do so. CD23 mRNA expression could be observed in B cells after 6 hr incubation with IL-4 and was maximal for 24-72 hr. Costimulation of the B cells with anti-IgM antibody enhanced the IL-4 induced CD23 mRNA expression. In contrast, IFN gamma and IFN alpha 2b inhibited IL-4 induced CD23 mRNA expression in normal B lymphocytes. Thus the regulatory effects of IL-4 and interferons on the CD23 membrane expression are linked to an increase and a decrease of CD23 transcripts respectively.

Various methods for studying the IgG interaction with its placental receptor have been examined to establish kinetic parameters. IgG binding has an average rate constant k 1 , of (2.57 ± 0.94) × 10 7 / M per min. However, high displacement doses (1000 μ/g) of unlabelled IgG resulted in a curvilinear dissociation curve with two binding sites: one with a fast dissociation rate constant, k 2 of 0.100/min and a slow one with k 2 of 0.010/min. IgG binding was associated with a high average affinity of (3.70 ± 1.65) × 10 8 / M and a total number of receptor binding sites of (2.07 ± 0.93) × 10 14 sites/mg of membrane protein, in close agreement with previous results. The stoichiometry of IgG to Fcγ-receptor was established as a 4:1 binding ratio from log dose-response curves as opposed to a 1:1 binding ratio from previous reports.

Extracellular heat shock proteins have been reported to participate in both innate and adaptive immune responses. We have found that recombinant mouse inducible heat shock protein 72 (Hsp72) bound to lymphoid neoplastic P388D1 cells. In the present study, we examined which region of mouse Hsp72 interacted with this cell line by using truncated variants that are sequentially lacking sections of the C-terminal region. The full-length mouse Hsp72 specifically bound to P388D1 cells, but not to mastocytoma P815 cells. Deletion of the C-terminal tail portion of mouse Hsp72 markedly decreased the binding to P388D1 cells and the sequential truncation of the C-terminal helical region led to a loss of binding activity. Specific binding was not observed for either the variant with a minimal substrate-binding structure or the ATP-binding domain alone. On the other hand, two truncated variants lacking the ATP-binding domain significantly bound to P388D1 cells. However, the variant lacking the substrate-binding domain did not show any binding to this cell line. These results suggest that the activity to bind P388D1 cells is attributable to the C-terminal region of mouse Hsp72 in combination with the substrate-binding domain. Interestingly, the binding of mouse Hsp72 to P388D1 cells was competed by the variant with the C-terminal flexible tail sequence, but not by the variant without that sequence. These competitive experiments imply that there may be at least two membrane receptors on P388D1 cells and also that both receptors may recognize the various structures in the C-terminal region of the Hsp70 family for regulation of innate immunity.

Toll-like receptor (TLR) family members are pattern-recognition receptors and very important molecules in innate immunity. Although TLRs are originally type I transmembrane receptors, soluble forms of TLRs are detected in human plasma and milk. This study showed that soluble TLR2 (sTLR2) is detected in human parotid saliva. Western blotting with anti-TLR2 antibodies (Abs) showed that three polypeptides are detected as sTLR2 with molecular weights of 55, 40 and 27kDa, respectively. Parotid saliva neutralized the binding of anti-TLR2 polyclonal Ab to cell-surface TLR2 on THP-1, a human monocytic cell line. Immunohistochemical analysis revealed that TLR2 is expressed in serous and interlobular ductal cells of human salivary gland. Human salivary gland cell lines, AZA3 and HSY, constitutively expressed TLR2. Parotid saliva augmented IL-8 production of THP-1 cells stimulated with a synthetic TLR2 ligand, Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)). Depletion of sCD14 from parotid saliva by immunoprecipitation eliminated the augmentation of IL-8 production, indicating that the augmentable effects depended on sCD14 in parotid saliva. On the other hand, preincubation of Pam(3)CSK(4) with parotid saliva abrogated the augmentation of IL-8 production, indicating that sTLR2 in saliva bound to Pam(3)CSK(4) and neutralized its function. These results suggest that parotid saliva modulates the TLR2-mediated immune responses with binary mechanisms via sTLR2 and sCD14 in the oral cavity.

The T cell receptor (TCR)-CD3 complex represents on of the most intricate membrane receptor structures since it is built from six distinct chains. This complexity led to a number of different proposals for the arrangement of the receptor subunits, its stoichiometry and the mechanisms responsible for receptor triggering. Early work had demonstrated that basic and acidic transmembrane (TM) residues were involved in the assembly but the molecular arrangement could not be deduced due to the complexity of the receptor. Using a novel method for the isolation of intact radiolabeled protein complexes, we demonstrated that the complex assembled in the ER contains only a single TCRalphabeta heterodimer and one copy of each of the CD3deltaepsilon, CD3gammaepsilon and zeta-zeta signaling dimers. Surprisingly, assembly of each of the three signaling dimers with TCR was dependent on one of the three basic TCR TM residues as well as both acidic residues located in the TM domains of the interacting signaling dimer. Each assembly step thus results in the formation of a three-helix interface in the membrane that involves one basic and two acidic TM residues, and this arrangement effectively shields these ionizable residues at protein-protein interfaces from the lipid. Since proteins whose TM domains have exposed ionizable residues are not stably integrated into the lipid bilayer, assembly based on shielding of ionizable residues permits full equilibration of the receptor into the lipid bilayer and prevents degradation. Assembly, export of intact receptor complexes and degradation of unassembled components thus rely on the same organizing principle.

Mast cells participate in inflammation and allergies by releasing biologically active mediators stored in numerous cytoplasmic granules. Degranulation is tightly controlled and requires activation of cell surface receptors, such as the high affinity IgE receptor (FceRI). Here, we discuss some of the key components of the molecular machinery that regulates the final steps of fusion between the granular and plasma membrane based on results obtained with the rat mast cell line RBL-2H3. We emphasize the role of soluble N -ethylmaleimide attachment protein receptors (SNAREs) proteins such as syntaxin 4 that can promote membrane fusion through formation of a stable complex with SNAP-23. We also highlight the role of a Ser/Thr kinase found to be associated with Rab3D, a negative regulator of degranulation. Associated kinase activity, which diminishes after stimulation as a consequence of intracellular calcium increases, specifically phosphorylates syntaxin 4 thereby affecting its capacity to bind to its t-SNARE partner SNAP-23. Our results suggest a new way of how Rab3 GTPases may intersect with the function of SNAREs thought to be universal mediators of membrane fusion.

The generation of signals following engagement of cell surface receptors is an ordered process that requires tight regulation as spurious signals could result in unwanted, and possibly deleterious, cellular responses. Like other cell surface receptors, stimulation of a mast cell via the high affinity IgE receptor (FcepsilonRI) causes multiple biochemical events that ultimately result in cell activation and effector responses. Recently, our knowledge of how these events are ordered has increased. We now have identified some of the molecules involved, how they are organized into macromolecular complexes by FcepsilonRI stimulation, and the role of some of the constituents of these macromolecular signaling complexes in mast cell effector responses. In brief, we review the knowledge on macromolecular signaling complexes used by FcepsilonRI in mast cell activation and provide our view on the regulation of signal generation and its effect on mast cell activation.

Complement activation by bound IgG in serum at physiological concentrations is reflected in the deposition of C3b/iC3b in the absence of antigenic expression of the IgG or of any bound C1q on the target. The aim of this study was to investigate the functional requirements for this phenomenon and to establish its relationship to a release or concealment of the antigens. Microtiter wells coated with IgG by direct adsorption or by binding of IgG antibodies to pre-adsorbed homologous antigen were incubated with serum or serum reagents at 37 °C. The complement reaction was analyzed by ELISA to quantitate bound or released reaction products, and the release of IgG from the coated microtiter wells was gauged radiometrically. In the presence of serum, rapid binding of C1q and C3b occurred and was soon followed by a rapid loss of C1q expression; C3b binding remained high. Loss of IgG paralleled that of C1q.The functional requirement for the reaction was restricted to the activation and deposition of C3b/iC3b but was dependent of the combined function of the classical and alternative complement pathways. The loss of the IgG antigen was solely the result of antigen concealment, whereas the loss of C1q was only partly so. In biological terms, the concealment of bound IgG and C1q may reflect mechanisms by which complement down-regulates leukocyte responses stimulated by ligand-cell membrane receptor interactions.

Quantitative relationships based on multivalent ligand receptor binding equilibria are developed which can describe the enhanced binding observed when polyvalent ligands are used as inhibitors of cell/cell interactions. This theory is able to explain the many orders of magnitude difference reported between the apparent dissociation constants determined for the interaction of Entamoeba histolytica membrane receptors with mono and multivalent N-Acetylgalactosaminide inhibitors. Given two experimentally accessible constants the theory provides a framework for the quantitative evaluation of custom designed polyvalent inhibitors of lectin and antibody mediated interactions.

Regulation of CD44-mediated binding to hyaluronan is critical in normal and diseased immune cell function. In earlier work by others (Shepley and Racaniello, J. Virol., 68, 1301 1309), anti-CD44 mAb blocked poliovirus binding to CD155 (the poliovirus receptor) in HeLa cells, suggesting that CD155 and CD44 may be physically associated. Here, we present evidence that CD155 and CD44 are physically associated in human monocytes. In co-modulation experiments in U937 monocytic cells, CD155 and CD44 reciprocally co-modulated. In primary human monocytes, CD 155 syn-capped with CD44. In immunofluorescence flow cytometric experiments, anti CD44 mAb inhibited up to 94% of binding by anti-CD155 mAb which blocks poliovirus binding to CD155. This inhibition was specific for CD155. Culturing monocytes increased the extent of inhibition. In addition, mAb against PRR2, a novel molecule that is related to CD 155, was inhibited by anti-CD44 in a dose-dependent manner, but not by anti-CD14. These data support the interpretation that CD155 (and related proteins) are physically associated with CD44 on monocyte cell surfaces. Although the current study does not address functional significance, we speculate that this interaction may have a role in regulating monocyte CD44 ligand binding which may be critical in pathological processes such as tumor metastasis and arthritis.

Receptor tyrosine kinases (RTK), like the PDGF-receptor, translate information from the extracellular environment into cytoplasmic signals that regulate a spectrumof cellular functions. RTK molecules consist of ligand binding extracellular domains, cytoplasmic kinase domains and tyrosine phosphorylation sites [Ullrich and Schlessinger, 1990 (Cell61, 203–212); Heldin, 1992 (EMBO J.11, 4251–4259)]. Upon ligand-induced RTK oligomerization, the kinase domains will become activated and induce auto(trans)phosphorylation of a number of cytoplasmic tyrosine residues. These phosphorylated tyrosine residues are incorporated in distinct sequence motifs and act as specific docking sites for SH2 domain-containing proteins [Songyang et al., 1993 (Cell72, 767–778)]. In contrast to single- or oligo-chain RTK, immunological receptors such as antigen receptors, FcR and cytokine receptors are multi-chain complexes in which distinct receptor functions appear to be compartimentalized in distinct polypeptides. Here, we summarize current knowledge on the structural and functional characteristics of the B-cell antigen receptor complex (BCR) and address the specific ability of accessory molecules to recruit intracellular signaling intermediates towards the activated receptor complex.

In addition to the antigen receptor, resting T cells express a number of receptors that can be stimulated to generate proliferative signals. These "accessory" receptors require co-expression of the T cell receptor (TCR), suggesting that they channel their signals via secondary activation of the signal transduction function of the CD3-TCR complex. Little is known about how different receptors control each other's function when one or more stimuli are presented at the same time. In order to study the regulation of accessory receptors by the CD3-TCR and vice versa, we have investigated the activation of the CD2 cell adhesion molecule receptor and the pertussis toxin receptor, a 43 kDa plasma membrane protein. Both receptors can activate signal transduction pathways in T cells similar to that of the CD3-TCR, including increases in Ca2+ and phosphatidylinositol turnover. They are also similar in that they utilize the antigen receptor to transmit their signals to the cell since CD3-TCR(-) mutants cannot be activated via either CD2 or the toxin receptor. We have previously shown that submaximal stimulation of the CD3-TCR blocks second messenger generation and proliferation in response to pertussis toxin. This heterologous desensitization was unidirectional since activation of the toxin receptor had no effect on CD3-TCR function. Here we extend these studies to show that activation of both CD2 and the toxin receptor led to rapid tyrosine phosphorylation of three similar proteins. Submaximal stimulation of the CD3-TCR completely inhibited toxin receptor-stimulated tyrosine protein kinase activity but did not desensitize CD2 function as determined by activation of tyrosine protein phosphorylation. Furthermore, CD2 stimulation did not lead to desensitization of the pertussis toxin receptor. These data support a system of complex regulatory relationships between different signaling receptors and suggest a model for signal integration and inter-receptor cross-talk in T cell activation.

Recombinant human gamma interferon (IFN gamma) was used to study IFN gamma receptors (IFN gamma-R) on human CD4+ and CD8+ peripheral blood T lymphocytes. When cell-bound 125I-IFN gamma was cross-linked by disuccinimidyl suberate, the receptor-ligand complex migrated under nonreducing conditions as major bands of 155 kD and 90 kD and a minor band of 65-70 kD. Under reducing conditions, only the 90 and 65 kD complexes were found; the 155 kD band was not seen. In contrast, complexes from WISH and Raji human lines exhibited a single band of a 125 kD under both reducing and nonreducing conditions. The molecular pattern of the receptor ligand complex on WISH cells was not altered when these cells were mixed with T lymphocytes during the extraction procedure. The results suggest that the IFN gamma receptor on T lymphocytes differs from those previously described.

Two murine T cell lines (C30.1 and Line 1) were used to study the expression of the p55 interleukin-2 receptor gene. C30.1 is an IL-2-dependent T cell line that can be stimulated for a short period of time by IL-4. Line 1 cells are propagated in IL-4 but they also proliferate in response to IL-2. In both cell lines stimulation by IL-2 leads to a strong induction of p55 IL-2 receptor mRNA while stimulation by IL-4 leads only to a very moderate increase in expression of this mRNA. The induction of p55 IL-2 receptor mRNA by IL-4 is comparable to that of β-actin mRNA. These data confirm that IL-2 upregulates p55 IL-2 receptor gene expression while IL-4, which also activates T cells, does not lead to specific induction of this gene. We have also determined the transcription initiation sites utilized by the p55 IL-2 receptor gene in C30.1 and Line 1 cells. Seven sites were identified, one of which predominates. Resting cells, or cells stimulated with IL-2 or IL-4, display the same pattern of transcription site utilization.

Evidence from several experimental approaches allows us to conclude that the primary amino acid sequence of the lower hinge region (residues 234-237) of human IgG molecules determines recognition by human Fc gamma RI, Fc gamma RII and Fc gamma RIII. Glycosylation of the CH2 domain is also essential, although the carbohydrate is not accessible for direct interaction with ligands. The role of the carbohydrate moiety may be to maintain a protein conformation that allows accessibility to amino acid side chains essential for ligand recognition and binding. It appears logical that the evolutionarily-related Fc gamma R molecules should interact with overlapping non-identical sites on the IgG molecule.

B lymphocytes cultured with LPS show increased expression of Fc gamma R II and increased binding of Ag-IgG complexes (both greater than 200%). In contrast, B lymphocytes cultured with either IL-4 or anti-mu show a marked loss (85-90%) of binding of Ag-IgG complexes that is specific, time and temperature dependent, and reversible. Decreased binding of complexes was not due to decreased expression of the receptor and therefore appears to be due to some form of alteration of the receptor. Based on the observation that the loss of binding of complexes requires protein synthesis, we favor the view that the loss is due to association of Fc gamma R II with another membrane molecule whose expression is induced or increased by IL-4 or anti-mu. Anti-mu induced loss of Fc gamma R II ligand binding capacity does not require cross-linking of surface IgM because the effect can be generated with F(ab') anti-mu. Anti-mu induced loss of Fc gamma R II binding of complexes was substantially prevented by IFN-gamma, whereas IFN-gamma did not reduce the anti-mu caused increase in expression of MHC class II molecules. This result shows that increased expression of the latter molecules can be dissociated from loss of Fc gamma R II ligand binding capacity. A myeloid cell line was identified that constitutively expresses Fc gamma R II binds relatively few complexes. This cell line may be useful in identifying alterations of Fc gamma R II which lead to the loss of binding of complexes. These results indicate that various B lymphocyte activators have different effects on B lymphocyte expression and function, and can thereby affect Fc gamma R II generated regulatory signals.

Treatment of monocytes or K562 cells with proteolytic enzymes like pronase or trypsin, increases both the affinity of the type II Fc receptor for IgG and the signaling via this receptor. In the present study we evaluated whether other proteases could similarly enhance Fc gamma RII affinity. We furthermore assessed whether all cell types expressing Fc gamma RII display this effect. Therefore, proteins from the coagulation system and PMN-derived enzymes were tested for effects on Fc gamma RII-mediated ligand binding. Enzymes of the coagulation system were tested both in fibrinogen-depleted plasma, as well as in purified form. No effects were found on Fc gamma RII-mediated rosette formation for both situations. In contrast, supernatant of stimulated granulocytes as well as leucocyte elastase were observed to be active in augmenting EA-hIgG rosette formation of thrombocytes and myeloid cell lines K562 and U937. The B cell lines Raji and Daudi, did not show enhanced rosette formation after enzyme treatment. The active component from granulocyte supernatant was partially characterized as a serine esterase with an apparent Mw of 30 kD. We tested whether the isotype specificity of Fc gamma RII on K562 cells changes upon enzyme treatment. It was found that all three tested murine subclasses gamma 1, gamma 2a, gamma 2b, bound equally well to this receptor, and interaction with all isotypes was enhanced to the same extent.

Glycosylated chimeric mouse-human anti-NIP IgG3 antibody produced by growth of the J558L mouse B cell plasmacytoma is characterised with respect to the single carbohydrate chain at Asn-297 in the CH2 domain indicating that the mouse cell glycosyl transferases dictate the pattern of glycosylation rather than the human CH region of the heavy chain. Additionally, three unusual alpha-galactose-containing oligosaccharides are reported. Only the Fc region has detectable carbohydrate. Aglycosylated anti-NIP IgG3 antibody has been produced by cell growth in the presence of the antibiotic tunicamycin. Functionally, whilst the glycosylated intact IgG3 interacts with human Fc gamma R111 expressed on human killer (K) cells to trigger antibody-dependent cellular cytotoxicity the aglycosylated intact IgG3 fails to trigger cell lysis, localising the site on IgG for triggering human Fc gamma R111 mediated functions to the CH2 domain. The monomeric aglycosylated trypsin Fc fragment inhibits human Fc gamma R1 recognition by U937 cells 115-fold less well (K50 = 2 microM) than does glycosylated Fc (K50 = 17 nM), confirming that aglycosylation disrupts the site for human Fc gamma R1 within the CH2 domain and indicating that the trypsin Fc fragments reflect the functional properties of the intact IgG glycoforms. Structurally, 1H NMR shows that the absence of carbohydrate at Asn-297 results in a small and localised protein structural change in the vicinity of the reporter group His-268 within the CH2 domain. The site on IgG for triggering human Fc gamma R111 mediated functions is then localised to the vicinity of His-268. The profound impact of aglycosylation on human Fc gamma R1 recognition implies structural disruption of the proposed site for human Fc gamma R1 in the lower hinge region of IgG (residues 234-239), proximal to His-268.

Enriched human interferon-gamma (HuIFN-gamma) receptor preparations were obtained by affinity chromatography of non-ionic detergent solubilized COLO 205 cell membranes on immobilized recombinant HuIFN-gamma. The active fractions, identified by a competition ELISA, were used as the immunogen in a BALB/c mouse. Fusion of its splenocytes with myeloma cells yielded several hybrids secreting antibodies that inhibit the antiviral activity of HuIFN-gamma; the two most active ones were selected for further characterization. This blocking activity was restricted to both the human species and the gamma type of IFN. Affinity purification of cell membrane extracts on the immobilized monoclonal antibodies resulted in the visualization of a major protein band with an Mr of 90,000, which is in good agreement with the results obtained by other authors [Aguet M. and Merlin G. (1987) J. exp. Med. 165, 988-999; Novick D., Orchansky P., Revel M. and Rubinstein M. (1987) J. biol. Chem. 262, 8483-8487; Sheehan K. C. F., Calderon J. and Schreiber R. D. (1988) J. Immun. 140, 4231-4237].

The interaction of type I (staphylococcal protein A) and type III (streptococcal FcRc) bacterial Fc receptors with goat immunoglobulins has been studied. Staphylococcal protein A bound poorly to the majority of goat immunoglobulins at all pHs tested. There was some evidence that protein A bound IgG2 better than IgG1, particularly at pH 8 and above. One of 10 sera tested demonstrated a high level of reactivity with protein A and this was shown to correlate with the presence of a natural antibody to protein A. The streptococcal Fc receptors, FcRc, bound efficiently to all goat IgG and goat sera tested. Both goat IgG subclasses reacted efficiently with the FcRc between pH 6 and 8. Inhibition of binding of 125I-FcRc to immobilized goat IgG enabled levels of IgG in goat serum to be estimated. These results suggest the streptococcal FcRc will be of value as an immunochemical reagent in studies involving isolation and quantitation of goat immunoglobulins.

Characterization of the human placental membrane receptor for human 125 I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10 7 mole −1 , and 2.0 ± 0.16 × 10 15 IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 m M ), and periodate (4 m M ) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 m M or 20 m M ) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of 125 I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.

Actin can form specific, direct associations with immunoglobulin resulting in soluble complexes or cross-linked matrices. This interaction can be detected by four in vitro assays using purified components: (1) actin enhances the cytophilic activity of guinea pig IgG2; (2) in solutions of low ionic strength, actin and IgG2 co-precipitate: (3) soluble complexes exist in 0.1 M KCl as revealed by the displacement of actin from its expected sedimentation pattern in a gradient of sucrose when in the presence of IgG 1, IgG2, or IgM; (4) immunoglobulin (IgG1, IgG2, BGG)‡: increases the viscosity of F-actin solutions, presumably by crosslinking F-actin filaments. These data suggest that direct interaction of a cytoskeletal protein with a cell surface receptor is possible.