Search

Neuroinflammation may be a pathogenic mediator and biomarker of neurodegeneration at the boundary between mild cognitive impairment (MCI) and early-stage Alzheimer's disease (AD). Whether neuroinflammatory processes are endogenous to the central nervous system (CNS) or originate from systemic (peripheral blood) sources could impact strategies for therapeutic intervention. To address this issue, we measured cytokine and chemokine immunoreactivities in simultaneously obtained lumbar puncture cerebrospinal fluid (CSF) and serum samples from 39 patients including 18 with MCI or early AD and 21 normal controls using a 27-plex XMAP bead-based enzyme-linked immunosorbent assay (ELISA). The MCI/AD combined group had significant ( p < 0.05 or better) or statistically trend-wise (0.05 ≤ p ≤ 0.10) concordant increases in CSF and serum IL-4, IL-5, IL-9, IL-13, and TNF-α and reductions in GM-CSF, b-FGF, IL-6, IP-10, and MCP-1; CSF-only increases in IFN-y and IL-7 and reductions in VEGF and IL-12p70; serum-only increases in IL-1β, MIP-1α, and eotaxin and reductions in G-CSF, IL-2, IL-8 and IL-15; and discordant CSF-serum responses with reduced CSF and increased serum PDGF-bb, IL-17a, and RANTES. The results demonstrate simultaneously parallel mixed but modestly greater pro-inflammatory compared to anti-inflammatory or neuroprotective responses in CSF and serum. In addition, the findings show evidence that several cytokines and chemokines are selectively altered in MCI/AD CSF, likely corresponding to distinct neuroinflammatory responses unrelated to systemic pathologies. The aggregate results suggest that early management of MCI/AD neuroinflammation should include both anti-inflammatory and pro-neuroprotective strategies to help prevent disease progression.

Unraveling the mechanisms of hematopoiesis regulated by multiple cytokines remains a challenge in hematology. IL-3 is an allergic cytokine with the multilineage potential, while CSF-1 is produced in the steady state with restricted lineage coverage. Here, we uncovered an instructive role of CSF-1 in IL-3-mediated hematopoiesis. CSF-1 significantly promoted IL-3-driven CD11c+ cell expansion and dampened basophil and mast cell generation from C57BL/6 bone marrow. Further studies indicated that the CSF-1/CSF-1R axis contributed significantly to IL-3-induced CD11c+ cell generation through enhancing c-Fos-associated monopoiesis. CD11c+ cells induced by IL-3 or IL-3/CSF-1 were competent in cellular maturation and endocytosis. Both IL-3 and IL-3/CSF-1 cells lacked classical dendritic cell appearance and resembled macrophages in morphology. Both populations produced a high level of IL-10, in addition to IL-1, IL-6 and TNFα, in response to LPS, and were relatively poor T cell stimulators. Collectively, these findings reveal a role for CSF-1 in mediating the IL-3 hematopoietic pathway through monopoiesis, which regulates expansion of CD11c+ macrophages.

Dengue virus (DV) is the most widespread arbovirus, being endemic in over 100 countries, and is estimated to cause 50 million infections annually. Viral factors, such as the genetic composition of the virus strain can play a role in determining the virus virulence and subsequent clinical disease severity. Virus vector competence plays an integral role in virus transmission and is a critical factor in determining the severity and impact of DV outbreaks. Host genetic variations in immune-related genes, including the human leukocyte antigen, have also been shown to correlate with clinical disease and thus may play a role in regulating disease severity. The host's immune system, however, appears to be the primary factor in DV pathogenesis with the delicate interplay of innate and acquired immunity playing a crucial role. Although current research of DV pathogenesis has been limited by the lack of an appropriate animal model, the development of DV therapeutics has been a primary focus of research groups around the world. In the past decade advances in both the development of vaccines and anti-virals have increased in dramatically. This review summarises the current understanding of viral, vector and host factors which contribute to dengue virus pathogenesis and how this knowledge is critically important in the development of pharmaceutical interventions., (Copyright © 2012. Published by Elsevier Inc.)

Myb-transformed haematopoietic cell (MTHC) lines have been developed and clones selected based on their ability to respond to tumor necrosis factor (TNF)-α. MTHC-A cells underwent apoptosis in response to TNF-α (MTHC-A). The apoptotic effect of TNF-α in MTHC-A was mimicked by a specific inhibitor of protein kinase A (PKI 5-24) and by the tyrosine kinase inhibitor genistein, suggesting that phosphorylation of tyrosine and PKA activity were important in protecting MTHC from apoptosis. Agents that elevate intracellular levels of cAMP, e.g. cholera toxin and dibuteryl cAMP, protected MTHC-A from the apoptotic effects of TNF-α, and also reduced the apoptotic effects of PKI and genistein. MTHC-A thus provides a useful model for investigating the role of TNF-mediated apoptosis in regulation of the myeloid lineage of cells.

Murine interferon-inducible T cell alpha chemokine (I-TAC) is a potent non-ELR Cys-X-Cys (CXC) chemokine that predominantly attracts activated T lymphocytes and binds to the receptor CXCR3. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) we analysed murine I-TAC expression in two different progenitor dendritic cell (DC) lines, MTHC-D2 and JAWS II which were exposed to various cytokines, and Con A-activated splenocytes from a panel of knockout mice. Analysis of the progenitor DC lines and Con A cultures demonstrated that murine I-TAC is primarily regulated by interferon (IFN)-gamma via interferon regulatory factor (IRF)-1. It has been proposed that I-TAC may have a role in autoimmune diseases such as multiple sclerosis (MS). Because I-TAC appears to be secreted from antigen-presenting cells (APCs) and attracts activated T cells, we examined the level of murine I-TAC mRNA in the central nervous system (CNS) of wild-type and IFN-gamma-receptor knockout (IFN-gammaR-/-) mice with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE). Peak I-TAC expression was detected in wild-type mice on day 14 when the mice begin to recover, whereas very low levels of I-TAC were detected in the CNS of IFN-gammaR-/- mice which develop severe EAE and die. The expression characteristics of murine I-TAC suggest an important mediator of immune cell communication that could augment vaccines and autoimmune therapies.

The effects of colony-stimulating factor 1 (CSF-1), the primary regulator of mononuclear phagocyte production, are thought to be mediated by the CSF-1 receptor (CSF-1R), encoded by the c-fms proto-oncogene. To investigate the in vivo specificity of CSF-1 for the CSF-1R, the mouse Csf1r gene was inactivated. The phenotype of Csf1(-)/Csf1r(-) mice closely resembled the phenotype of CSF-1-nullizygous (Csf1(op)/Csf1(op)) mice, including the osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes. Compared with their wild-type littermates, splenic erythroid burst-forming unit and high-proliferative potential colony-forming cell levels in both Csf1(op)/Csf1(op) and Csf1(-)/Csf1r(-) mice were significantly elevated, consistent with a negative regulatory role of CSF-1 in erythropoiesis and the maintenance of primitive hematopoietic progenitor cells. The circulating CSF-1 concentration in Csf1r(-)/Csf1r(-) mice was elevated 20-fold, in agreement with the previously reported clearance of circulating CSF-1 by CSF-1R-mediated endocytosis and intracellular destruction. Despite their overall similarity, several phenotypic characteristics of the Csf1r(-)/Csf1r(-) mice were more severe than those of the Csf1(op)/Csf1(op) mice. The results indicate that all of the effects of CSF-1 are mediated via the CSF-1R, but that subtle effects of the CSF-1R could result from its CSF-1-independent activation.

Ross River virus (RRV) is an Australian alphavirus that is often responsible for chronic epidemic polyarthritis and myalgia in humans. Past studies have shown severe disruption of striated muscle fibers to be prominent in RRV pathology in mice; in the present study, macrophages were directly implicated as the primary mediators of muscle damage. General immunosuppressive therapies had only minor effects on mortality and morbidity in RRV-infected mice, with no inhibition of muscle damage. Treatment of mice with macrophage-toxic agents (e.g., silica) prior to RRV infection completely abrogated disease symptoms without significantly affecting titers of virus in organs. Further studies found that clinical signs of infection and muscle damage correlated with a massive influx of macrophages into hind leg muscle, whereas no such infiltrate or damage was observed for silica-treated mice. These observations are significant for the human disease context, as monocytic cells have been detected in the synovial effusions of persons with epidemic polyarthritis.

Immune induction is effected through the interaction of antigen-presenting cells with specific receptors on the surface of thymus-derived lymphocytes. Cells most able to ingest, process, and present antigen appear to be related to the mononuclear phagocyte/neutrophil series. For example dendritic cells (DC) can be found in colonies of GM-CSF-responsive bone marrow cells, and under experimental conditions are routinely expanded as a population in vitro from GM-CSF-responsive progenitor cells. To address the question of DC lineage and to determine what genes are involved in lineage commitment, we have generated a series of GM-CSF-responsive cell lines that can be induced to differentiate in a homogeneous manner in vitro. The cloned cell lines are derived from 12-day fetal liver and are transformed with a truncated form of c-myb, which lacks the normal autoregulatory sequences. As far as we know, these myb-transformed hemopoi-etic cells (MTHC) differ from normal only in the unregulated expression of myb, a gene whose expression is obligatory for proliferation of hemopoietic cells. MTHC in the presence of TNF-alpha and IL-4 will differentiate into cells that have many of the properties of macrophages. When the same MTHC lines are exposed to TNF-alpha in combination with IFN-gamma, the cells instead become DC. The differentiated DC are potent presenters of antigen in mixed lymphocyte reactions and of soluble antigen to specific T cell lines. Thus, cells with the properties of both macrophages and DC can be derived from a single type of GM-CSF-responsive progenitor cell. We have used this MTHC system to analyze differences in gene expression as the cells mature along the DC and macrophage pathways. A distinctive pattern of differentially expressed cDNAs is evident where macrophage-specific cDNAs are homologous to genes encoding cytoskeletal and cell-surface proteins, whereas the DC-specific cDNAs are homologous to signaling, chemokine, and IFN-gamma-inducible genes. We discuss the utility of MTHC in analyzing the relationships between DC and macrophages, and suggest that DC and macrophages represent extreme phenotypes in a spectrum of antigen handling cells that are somewhat interchangeable, depending on their immediate environment.

Background: Previous in vivo depletion studies of CD4 and CD8 T cells indicated that different rejection mechanisms operate for proislet allografts and xenografts. The cellular and molecular mechanisms of acute proislet allograft and xenograft rejection have therefore been characterized and directly compared., Methods: The intragraft cytokine mRNA profile in rejecting BALB/c (H-2d) proislet allografts was analyzed in control, CD4 T cell-depleted, and CD8 T cell-depleted CBA/H (H-2k) recipient mice using semi-quantitative reverse transcriptase-assisted polymerase chain reaction (RT-PCR). The cytokine profiles for proislet allografts and pig proislet xenografts at 3-10 days posttransplant were directly compared and correlated with graft histopathology., Results: Allograft rejection was protracted (2-3 weeks), characterized by infiltrating CD8 T cells and CD4 T cells (no eosinophils) and was associated with a Th1-type CD4 T cell response (IL-2, IFN-gamma, and IL-3 mRNA) and a CD8 T cell-dependent spectrum of cytokine gene expression (IL-2, IFN-gamma, IL-3, and IL-10 mRNA). Xenograft rejection was rapid (6-8 days), involved predominantly CD4 T cells and eosinophils, and in contrast to allografts, exhibited intragraft mRNA expression for the Th2 cytokines IL-4 and IL-5., Conclusions: Proislet allograft and xenograft rejection differ in the tempo of destruction, phenotype of the cellular response and intragraft profile of cytokine mRNA. The recruitment of eosinophils only to the site of xenorejection correlates with IL4 and IL-5 mRNA expression. These findings suggest that different anti-rejection strategies may need to be developed to optimally target the allograft and the xenograft response.

The rejection of pig proislet (islet precursor) xenografts in CBA/H mice is a CD4+ T cell-dependent process. The molecular mechanisms of xenograft rejection, xenograft survival during anti-CD4 mAb therapy and xenograft tolerance post-withdrawal of anti-CD4 mAb administration, were examined by using a semiquantitative PCR method. Temporal analysis of intragraft cytokine mRNA demonstrated a Th0-like pattern of expression (IL-2, IFN-gamma, IL-3, IL-4, IL-5, and IL-10) on day 4 of the acute xenograft rejection process. From day 5, however, only Th2-associated transcripts (IL-3, IL-4, IL-5, and IL-10) were enhanced in xenografts compared with isograft controls. Immunohistochemistry showed that the principal participants in the rejection infiltrate were CD4+ T cells and eosinophils, with smaller numbers of CD8+ T cells. In vivo depletion of CD4+ T cells prevented xenograft rejection but had minimal effect on the peak levels of IL-2, IFN-gamma, and IL-10 mRNA; in contrast, the enhanced expression of IL-3, IL-4, and IL-5 transcripts seen in rejecting xenografts was abrogated. This established a positive correlation between acute xenograft rejection, presence of CD4+ T cells, and enhanced intragraft expression of mRNA for the Th2-type cytokines IL-3, IL-4, and IL-5. In tolerant hosts, long-term proislet xenograft survival and function (> 190 days) was accompanied by intragraft expression of IL-2 and IL-10 mRNA; IFN-gamma, IL-3, IL-4, and IL-5 mRNA were either undetected or not enhanced. The induced rejection of long-term functioning xenografts (> 170 days) in nontolerant hosts resulted in selective enhancement of IL-4 transcript expression. This study suggests that Th2-like CD4+ T cells are differentially activated in response to xenoantigen and that xenograft tolerance is associated with lack of expression of the Th2 cytokine, IL-4.

We have shown that pertussis toxin (PTx) modulates the effect of tumor necrosis factor-alpha (TNF-alpha) in inducing monocytic differentiation of WEHI-3B (JCS) myeloid leukemic cells in vitro. PTx (0.1-2 ng/ml) alone was not cytotoxic and did not induce morphological changes in JCS cells. In the presence of a suboptimal concentration of TNF-alpha (25 U/ml), however, PTx (1 ng/ml) acted synergistically in inhibiting proliferation and in inducing monocytic differentiation of the JCS cells. Expression of the macrophage differentiation marker (Mac-1) on JCS cells was increased by the combination of PTx and TNF-alpha, and phagocytic activity of the cells was also enhanced. Moreover, JCS cells treated with PTx and TNF-alpha had reduced tumorigenic capacity in vivo. The data suggest that a PTx-sensitive G protein may be involved in regulating the TNF-alpha-induced monocytic differentiation of the myeloid leukemic JCS cells and that combination of PTx and TNF-alpha may be useful in the treatment of some forms of myelomonocytic leukemia.

We have previously shown that non-cytotoxic concentrations (600-1200 U/ml) of recombinant mouse tumour necrosis factor-alpha (TNF-alpha) can induce differentiation of a subclone (JCS) of the WEHI-3B myelomonocytic leukaemia cell line into mature cells with the characteristics of macrophages. In the present study, the effects of recombinant mouse interleukin-4 (IL-4), either alone or in combination with mouse TNF-alpha, on the growth and differentiation of JCS cells were examined. IL-4 alone (20-5000 U/ml) inhibited the growth of JCS cells in a dose-dependent manner but did not induce cell differentiation. However, combinations of IL-4 and TNF-alpha acted in synergy to inhibit cell proliferation and induce monocytic differentiation of JCS cells, as shown by increased expression of the macrophage differentiation antigens (F4/80, Mac-1), stimulation of phagocytic activity, induction of non-specific esterase and NBT-reducing activities, increased plastic adherence and morphological criteria. Similar synergistic interactions were also shown by human TNF-alpha and mouse IL-4, indicating that TNF-alpha might exert its effects through the low-affinity (p55) TNF receptors. Moreover, the clonogenicity of JCS cells in vitro and their tumorigenicity in vivo were significantly reduced by combined TNF-alpha and IL-4 treatment. Our results indicate that TNF-alpha can act as a differential signal for JCS cells and that its effects are modulated by IL-4. Therefore, the combination of TNF-alpha and IL-4 may be useful in the treatment of some forms of myelomonocytic leukaemia.

We have isolated a subclone (JCS) of the WEHI 3B myelomonocytic leukemia, which acquires the characteristics of mature macrophage lineage cells in the presence of PMA or noncytotoxic concentrations of TNF-alpha (600-1200 U/ml). JCS cells were compared with D+ and D- subclones of WEHI 3B. Unlike D+ cells, JCS cells did not produce differentiated granulocyte-macrophage colonies in the presence of postendotoxin serum or recombinant G-CSF. Stimulation with PMA or TNF-alpha reduced proliferation of JCS cells. TNF-alpha decreased the level of cell surface J11D antigen with concurrent increased expression of Mac-1 and FcR antigens and phagocytic activity. These TNF-alpha-mediated effects were enhanced by addition of IFN-gamma to the cultures. Furthermore, differentiation-inducing activity of PMA could be prevented using neutralizing anti-TNF-alpha antibodies. The results indicate that exogenous TNF-alpha can act as a differentiative agent for JCS cells and that endogenous TNF-alpha is the active substance when PMA is used to stimulate macrophage differentiation of JCS cells.

We have examined the role of Gi alpha in haemopoietic cells using the myelomonocytic progenitor cell lines FDC-P1 and WEHI-3B (JCS). During growth factor-dependent proliferation of FDC-P1 cells Gi alpha was found predominantly in the nucleus and associated with the plasma membrane. Following removal of growth factor, Gi alpha accumulated in the cytoplasm and at the plasma membrane. Treatment of FDC-P1 cells with pertussis toxin (PT) completely inhibited translocation of Gi alpha to the nucleus and reduced the sensitivity of FDC-P1 cells to the proliferative effects of growth factors, indicating that translocation of Gi alpha plays a regulatory role in, but may not be essential for, cell division. Gi alpha initially associated with DNA during S/G2 of the FDC-P1 cell cycle but separated from condensing chromosomes during mitosis. In contrast to FDC-P1 cells, WEHI-3B (JCS) cells proliferate in the absence of added growth factors but can be induced to differentiate by TNF-alpha. In proliferating JCS cells Gi alpha was again associated with the nucleus but when proliferation was inhibited by TNF-alpha, Gi alpha accumulated in the cytoplasm with none detected in the nucleus. Thus the cytokine regulated accumulation of Gi alpha at different intracellular sites correlated with the ability of the cell to progress through the proliferative cycle. When the tyrosine kinase inhibitor genistein was added to FDC-P1 cells prior to stimulation with IL-3 or GM-CSF, proliferation was almost completely inhibited but translocation of Gi alpha was not affected, suggesting that tyrosine phosphorylation was not involved in G protein translocation but was essential for cytokine induced cell division. Cholera toxin (CT) also inhibited proliferation of FDC-P1 cells but had no effect on translocation of Gi alpha to the nucleus. The near complete inhibition of cell division by genistein and CT without a corresponding effect on Gi alpha movement indicates that Gi alpha can be regulated independently of tyrosine kinase and adenylyl cyclase activities, respectively.

Haemopoietic cells from A/J mice do not form colonies (proliferate) in response to interleukin-3 (multi-CSF, IL-3). We have examined different populations of cells from A/J mice and shown that, despite their failure to proliferate in response to IL-3, cells from bone marrow, spleen and the peritoneum all bound 125I-labelled IL-3. A wide variety of cell types bound IL-3 as determined by autoradiography, including promyelocytes, myelocytes, metamyelocytes, polymorphs, promonocytes, monocytes, eosinophils and lymphocytes, but not nucleated erythroid cells, and the proportion of each cell type binding label was similar when cells from A/J mice were compared with those of C57B1/6 and Balb/c mice. Bone marrow cells from A/J mice internalized interleukin-3 with normal kinetics and mRNA extracted from these cells contains the same species of IL-3 receptor and IL-3 receptor-like mRNAs as are found in the other strains. Collectively the data suggest that the failure of haemopoietic cells from A/J mice to proliferate in response to IL-3 is related to a selective defect in signalling to proliferation specific genes. This defect is apparently not related to internalization or processing of the IL-3/IL-3-receptor complex, but may be due to failure to activate appropriate accessory molecules in the cell.

We investigated whether increased concentrations of circulating cytokines may be responsible for exercise-induced priming of blood neutrophils (J. A. Smith et al. Int. J. Sports Med. 11: 179-187, 1990). The plasma concentrations of tumor necrosis factor-alpha, interleukin- (IL) 1 beta, IL-6, granulocyte-macrophage colony-stimulating factor, and neopterin in trained and untrained human subjects were measured by immunoassay before and after 1 h of cycling at 60% of maximal oxygen uptake. C-reactive protein and creatine kinase (CK) were also measured before and 24 h after exercise as markers of the "acute-phase response" and muscle damage (C. Taylor et al. J. Appl. Physiol. 62: 464-469, 1987), respectively. The small changes in the plasma concentrations of cytokines or neopterin observed after exercise in both trained and untrained subjects were not significantly different to those found in a control group of nonexercised subjects. However, untrained subjects did exhibit an acute-phase response (P = 0.04) 24 h after exercise without additional release of CK into plasma. Baseline training differences were confined to a twofold elevation in CK activity (P = 0.04). The results show that circulating cytokines are unlikely to be responsible for the priming of neutrophil microbicidal activity observed after moderate endurance exercise (J. A. Smith et al. Int. J. Sports Med. 11: 179-187, 1990).

A set of primers (MF43, MF44 and MF45) were designed and used in the polymerase chain reaction to distinguish between the expression of mouse IL-3 receptor and mouse IL-3 receptor-like mRNAs. Primers MF43 and MF45 were specific for IL-3 receptor mRNA while the primers MF44 and MF45 were specific for IL-3 receptor-like mRNA. Primers MF44 and MF45 could not amplify IL-3 receptor cDNA even at an annealing temperature of 46 degrees C which is 20 degrees C below the melting temperature of the primers, or at high template concentrations (up to 100 ng cDNA). The optimal range of Mg2+ concentrations for the two pairs of primers MF43, MF45 and MF44, MF45, were essentially the same and this permits comparisons of the expression level of these two mRNAs under identical PCR conditions. Both the IL-3 receptor and IL-3 receptor-like mRNAs could be detected in normal bone marrow cells and IL-3-dependent cell lines (FDC-P1 and 32D cl-23), as well as in the IL-3 independent cell lines P388D1 and WEHI-3B, the latter being a constitutive producer of IL-3. In contrast, neither species of mRNA was detected in the T lymphoma cell line (EL-4). The ratio of IL-3 receptor-like mRNA to IL-3 receptor mRNA was usually greater than 1, except in 32D cl-23 cells where it was 0.66.

Four myeloid cell lines (M1, WEHI-3B D+, FDC-P1, and 32D) were screened for the presence of J11d antigen. One of these cell lines, the myeloid leukemia M1, was found to express a high level of J11d antigen on the cell surface. Recombinant mouse leukemic inhibitory factor (rm-LIF), recombinant human LIF (rh-LIF), and steroids (hydrocortisone and dexamethasone) could induce M1 cells to undergo monocytic differentiation. The level of J11d antigen was greatly reduced after treatment of the cells with LIF or steroids. Western blotting revealed that the apparent molecular weight of the J11d antigen on M1 cells was 45-48 kDa. Furthermore, the level of J11d mRNA was also reduced during LIF-induced differentiation of M1 cells.

Proliferation, maturation, chemotaxis, and activation of neutrophils and monocytes are mediated largely by cytokines, including colony-stimulating factors and lymphokines. Cytokines produced in the intestinal mucosa contribute to the increased migration of neutrophils and monocytes into the lesion of inflammatory bowel disease and to the activation of these inflammatory cells. Lamina propria mononuclear cells isolated from colon tissue from 14 patients with inflammatory bowel disease (IBD) and from histologically normal controls were studied. Cells from IBD-affected tissue produced significantly more colony-stimulating factor activity (1402 +/- 252 U) per 2 x 10(6) cells than those from normal mucosa (362 +/- 85 U), mainly because of the increased production of granulocyte colony-stimulating factor and interleukin 1. This was accompanied by increases in the amount of specific messenger RNA for these two cytokines in lamina propria mononuclear cells from mucosa of patients with Crohn's disease (CD) compared with normal controls. By contrast, there was a substantial reduction in interleukin 3 production in CD and in ulcerative colitis lamina propria mononuclear cells, and this was reflected in significantly reduced expression of interleukin 3 messenger RNA in CD cells. Of the agents used in the therapy of IBD, hydrocortisone and 5-aminosalicylic acid, but not cyclosporin A, markedly suppressed in vitro production of cytokines by lamina propria mononuclear cells, suggesting that their therapeutic efficacy in vivo may be due in part to down-regulation of cytokine production in the inflamed mucosa.

The role of non-specific cytotoxicity in the pathogenesis of inflammatory bowel disease (IBD) was investigated by assaying the natural killer (NK) and lymphokine-activated killer (LAK) cell activity of lamina propria mononuclear cells (LPMC) from 22 specimens of intestinal mucosa affected by IBD. Only minimal levels of NK activity were detected against K562 cells, as well as colon carcinoma cells, adenoma cells and fibroblasts freshly isolated from the intestinal mucosa. Culture of LPMC from IBD in the presence of interleukin-2 (IL-2) generated LAK cells that mediated high levels of activity against K562 cells and against neoplastic epithelial cells and fibroblasts derived from the intestinal mucosa. A group of 20 histologically normal specimens of intestinal mucosa showed similar levels of LAK activity against the K562 and intestinal cell targets. The minimal mucosal NK activity in IBD suggests that the cytotoxic properties of NK cells are not important in the pathogenesis of IBD. The presence of LAK precursor cells in the inflamed mucosa of IBD and their ability to lyse biologically relevant targets in vitro suggests that LAK cells have the potential to contribute to intestinal mucosal injury in IBD.

Gliotoxin, an epipolythiodioxopiperazine, is a fungal metabolite that causes genomic DNA degradation preferentially in certain blood cell types including T lymphocytes and macrophages. Gliotoxin has previously been used to treat murine allogeneic bone marrow prior to transplantation into irradiated recipients, and in this situation the drug prevents development of graft-versus-host disease, and permits the establishment of allogeneic bone marrow chimeras. We have examined the nature of the cells that survive gliotoxin treatment and report here that gliotoxin selectively spares a unique class of haemopoietic stem cell that forms large (HPP) colonies in the presence of mixtures of M-CSF and IL-3. We confirm that the cells which survive gliotoxin treatment are capable of reconstituting the haemopoietic system in allogeneic lethally irradiated mice.

Whilst B cells in human blood can be shown to express interleukin-2 receptor (IL-2R) p55 and p75 chains, using a high-sensitivity immunofluorescence procedure, fresh tonsil B cells did not show detectable levels of expression. Culture of tonsil B cells led to low levels of expression of the p55 chain of the IL-2R, an effect which was dependent on protein synthesis. The level of expression of IL-2R chains could be modulated by culturing in the presence of a number of factors which activate B cells. p55 levels were more readily modulated than p75 levels. IL-4 and combinations of IL-4 with anti-IgM, IL-2 or tumour necrosis factor-beta (TNF-beta) modulated p55 levels, but IL-5 did not. Changes in IL-2R expression were small when compared with other B-cell activation markers such as CD23. When unfractionated tonsil cells were activated with a polyclonal stimulus, the major change was the expression of p55 by T-cell blasts--p75 expression remained low in T and B cells, and p55 expression by B cells remained low.

The number of colonies produced by bone marrow cells in response to interleukin 3 (IL-3) in soft agar cultures varies according to the strain of the donor mice. A/J, AKR, A.TH and A.TL bone marrow cells are particularly hyporesponsive, producing only occasional colonies in the presence of IL-3. Bone marrow cells from all strains of mice, including A/J, produce distinctively large colonies in response to the combination of IL-3 and macrophage colony stimulating factor (M-CSF). In cultures of A/J bone marrow cells, the synergy between IL-3 and M-CSF is further reflected in an increase in both the number and the variety of colonies produced. The increase in colony numbers may be due to the priming of a population of A/J colony-forming-cells (CFCs) by IL-3, enabling them to respond to M-CSF. In support of this notion, IL-3 enhanced the level of c-fms (M-CSF receptor) messenger RNA in cultures of A/J bone marrow cells. It is also possible that a subpopulation of CFCs requires both IL-3 and M-CSF as co-mitogens. The A/J strain provides a novel system for studying the mechanisms involved in the interaction between IL-3 and M-CSF in haemopoiesis.

The cloning and expression of the genes for rat, mouse, and human IL-3 have resulted in major advances in our knowledge of this lymphokine and the biological processes in which it is involved. Such advances include the primary structures of the IL-3 proteins, the nucleotide sequences of the IL-3 genes, and the elucidation of the retroviral insertion that resulted in constitutive synthesis of IL-3 by the leukemic cell line WEHI-3B. These advances have facilitated studies of the control of IL-3 gene expression in normal and malignant cells, which is an ongoing area of interest. The use of the cloned IL-3 gene in a retroviral expression vector has allowed the generation of experimental autocrine leukemias from IL-3-dependent cell lines. The use of recombinant rat, mouse, and human IL-3 has verified that IL-3 is a multilineage hemopoietic regulator in each of these species. Although it is clear that in each of the above species IL-3 promotes the growth of a wide variety of myeloid progenitors, the regulation of stem-cell division and maturation by IL-3 and its synergism with other regulators requires clarification and remains an active area of research. The discovery that hemopoietic stem cells from certain murine strains do not proliferate in response to IL-3 alone has provided a model system in which to analyze the synergistic activities of IL-3. It will be interesting to see whether analogous differences in the responsiveness of stem cells to IL-3 also exist in humans. Our work on the ontogeny of blood cell development in embryonic and fetal mice indicates that in every strain, early hemopoietic progenitor cells respond to IL-3 plus M-CSF, or to IL-3 plus IL-4, rather than to IL-3, M-CSF, or IL-4 alone. The difficulties encountered in acquiring sufficient cells to work with from embryonic sources could possibly be overcome by developing a fetal sheep model in which access to the embryo and fetus can be achieved at several sequential points in development. It may then be possible to determine the molecular events that regulate primitive stem-cell responsiveness to IL-3 as well as other hemopoietic cytokines. Recombinant IL-3 proteins should continue to prove valuable in further biological studies of IL-3, in the generation of monoclonal antibodies against IL-3, and for further studies of the IL-3 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

The activity of natural effector (NE) cells was studied in lamina propria lymphocytes (LPL) obtained from 61 histologically normal specimens of human intestine, which included 45 resected for colon carcinoma and 16 resected for nonmalignant conditions. The mean spontaneous natural killer (NK) cell activity in LPL (1.7 X 10(2) cytotoxic units (C.U.)/10(5) cells) was very low in contrast to that found in peripheral blood mononuclear cells (PBMC) (38.5 X 10(2) C.U./10(5) cells). Significant NK activity was detected in only 16 (47%) of the tissues resected for carcinoma, and in five (38%) of those removed for nonmalignant conditions. Exposure to human leucocyte interferon resulted in only minimal increases in cytotoxicity for K562 target cells. Consistent with these findings, large granular lymphocytes represented less than 0.5% of freshly isolated LPL. Cultures of LPL from both carcinoma and nonmalignant conditions in MLA144-conditioned medium (CM), a source of interleukin 2 (IL 2), generated marked increases in cytotoxicity to levels comparable with or exceeding those found in PBMC. (Mean cytotoxicities were 90.4 X 10(2) and 49 X 10(2) C.U./10(5) cells, respectively.) Cytotoxicity induced by culture in MLA144-CM could be blocked by pretreatment of LPL with the monoclonal antibody anti-Tac directed against the IL 2 receptor. In addition, LPL cultured in recombinant human IL 2 were induced to levels of cytotoxicity that were similar to those induced by MLA144-CM. These data indicate that IL 2 is the factor in MLA144-CM responsible for generating lymphokine-activated killer (LAK) cells in LPL. The IL 2-activated LPL killer cells were OKT11+, OKT3-, Leu-7-, Leu-11b-, as determined by antibody and complement-mediated lysis, and the precursor cells in the lamina propria necessary for generation of killer cells by IL 2 were also OKT11+, OKT3-, Leu-7-, Leu-11b-. These studies indicate that LAK cells may be an important potential source of nonspecific cytotoxicity in the intestinal mucosa.

Autonomous production of a required growth factor is one mechanism by which a cell may become tumorigenic. Several leukaemias have been described which secrete growth factors which may be involved in autocrine stimulation of cell proliferation. One of these leukaemias is WEHI-3B, a myelomonocytic leukaemia that constitutively produces interleukin-3 (IL-3). Cloning of the IL-3 gene has enabled us to investigate possible genetic changes in this gene in WEHI-3B cells which may have resulted in autonomous production of growth factor. We have shown that one of the IL-3 genes in WEHI-3B has been rearranged, as a result of the insertion of a 5.1 kilobase intracisternal A-type particle genome head to head with the 5' end of the IL-3 gene, 215 bases upstream of the IL-3 TATA box. The rearranged gene, when cloned into a lambda EMBL3A vector, could readily be expressed in COS-1 monkey cells, whereas the normal gene, in the same vector, was silent. Thus the insertion of the endogenous retroviral element has resulted in abnormal expression of the IL-3 gene and is postulated to have been a key genetic change in the development of this leukaemia. In an attempt to experimentally construct IL-3 producing leukaemias, IL-3 responsive FDC-P1 and 32D cl-23 cells were transfected with a retroviral expression vector containing the IL-3 gene. This resulted in autonomous production of IL-3 and continuous proliferation of the transfected cells. As a result of transfection, the FDC-P1 and 32D cl-23 cells became leukemogenic demonstrating the oncogenic potential of abnormal expression of IL-3. The autocrine nature of the experimental leukaemias was demonstrated by blocking their proliferation with an IL-3 neutralising antiserum. Similarly treated cultures of normal bone marrow cells also produced IL-3 and could be maintained for several months after transfection but were not leukemogenic. The factor-dependent cell lines are unable to differentiate in the presence of known CSF's and presumably have undergone other genetic changes which allow them to become leukemogenic when autocrine-stimulated. In contrast, the transfected bone marrow cells could differentiate and form colonies containing mature granulocytes and macrophages. The non-tumorigenic behaviour of the transfected bone marrow cells is consistent with the concept that several other genetic changes which effectively block differentiation are required for development of tumorigenicity in these cells.

The nature of the interaction between vaccinia virus (VAC) and fibroblastic cells that renders the latter capable of being recognized by virus-specific, H-2 identical murine T lymphocytes has been studied. L cells exposed for 10 min to VAC rendered noninfectious by exposure to ultraviolet light became susceptible targets for cytotoxic T lymphocytes (CTL) without the synthesis of new viral proteins. Susceptibility was retained even if cellular protein synthesis was irreversibly inhibited with pactamycin before virus exposure. Immobilization of cell-surface membranes by glutaraldehyde fixation before (but not after) exposure to virus severely impaired the formation of the "virus + self" complex that in vitro stimulated secondary CTL responses by H-2 identical virus-primed memory cells even though virus attachment to fixed cells were unaffected. This stimulatory complex, once formed, was maintained in membrane fragments prepared from cells previously exposed to VAC. These findings indicate that VAC-specific CTL or their immediate precursors can recognize only those viral envelope antigens that become membrane integrated and that this event requires neither host cell-specific nor virus-specific protein synthesis.

The growth factor-dependent, non-leukemogenic cell line FDC-P1, was converted to an interleukin-3 (IL-3) producing leukemogenic cell line using a retroviral expression vector carrying the IL-3 gene. The new cell line, FDC-P1-IL3 proliferated independently of exogenous IL-3 and its proliferation was inhibited by anti IL-3 antisera. This inhibition could be overcome by addition of GM-CSF to the cultures. The data indicate that insertion of the retroviral expression vector into the genome of FDC-P1 cells has established an autocrine loop involving constitutive secretion of IL-3 and that such a loop can play an important role in leukemogenesis.

An autocrine leukemia (FDC-P1-IL3) has been developed using a retroviral vector containing the interleukin-3 (IL-3) gene to transfect the IL-3-dependent cell line FDC-P1. When leukemia cells were reisolated from experimental animals, it was found that levels of interleukin-2 (IL-2) receptor (IL-2R) expression were greater on cells isolated from the lymph node than on cells isolated from the spleen. Cloned sublines of FDC-P1-IL3 were selected by flow microfluorometry for high or low levels of IL-2R expression. Those clones that expressed high levels of IL-2R grew preferentially in the lymph node. Although IL-2 is not mitogenic for FDC-P1 cells and does not increase the rate of growth of FDC-P1-IL3 cells in vitro, the cloning efficiency of FDC-P1-IL3 is increased fourfold in the presence of IL-2. These observations suggest that the IL-2R on FDC-P1-IL3 cells plays an important role in modulating the growth of this leukemia in sites that contain high levels of IL-2.

Infection of cells with either ectromelia or lymphocytic choriomeningitis (LCM) virus in the presence of 2-deoxy-D-glucose (2-DOG) inhibited by up to 70% the extent to which the infected cells become susceptible to virus-specific cell-mediated lysis. The concentration of 2-DOG used had little effect on the extent of total protein synthesis (incorporation of [35S] methionine) but inhibited (up to 25%) glycoprotein synthesis, as measured by incorporation of [3H] fucose. This suggested that glycoprotein synthesis was a necessary event for infected cells to become susceptible to T-cell mediated lysis. The profiles (polyacrylamide gel electrophoresis) of newly synthesized, cellular glycoproteins from unifected and ectromelia-infected cells in the presence and absence of 2-DOG were compared and found to be very complex, with only minor changes. However, when convalescent serum from infected mice was used to isolate newly synthesized components from the cell surface shortly after infection, it showed four main species ranging in size from 25,000 to 70,000 daltons. 2-DOG inhibited production of these by 70%, thus corresponding to the biological data. The nature of the new glycoproteins seen in infected cells and whether they are in fact the structures recognized by effector T cells remain to be determined.

Cyclosporine (CsA) inhibits release of interleukin 2 (IL-2) and hemopoietic growth activities such as interleukin 3 (IL-3) from major histocompatibility complex (MHC)-antigen-activated T cells. Production of both lymphokines appears to be coordinately regulated; the antigen dose response, T cell dose response, and time course of lymphokine appearance are similar. The triggering of lymphokine production by these cells is solely dependent on T cell-target cell interaction, as the T cell dose response curve indicates that no cooperation occurs between T cells, and any metabolic contribution by the target cell was eliminated by ultraviolet irradiation. This interaction triggers the transcription of lymphokine-encoding mRNA. The process of lymphokine release can be divided into 4 steps: Antigen binds to the T cell; a signal is transferred to the cell nucleus; transcription of lymphokine-encoding mRNA occurs; and intact lymphokine is synthesized and secreted. CsA inhibits antigen triggered lymphokine production. However, it does not inhibit lymphokine release from the constitutively producing tumor cell lines WEHI-3 (which releases IL-3) and MLA 144 (which produces IL-2). Thus CsA has no effects on the lymphokine secretion process or any direct action upon lymphokine-coding mRNA. CsA does not affect antigen recognition during cell-mediated cytotoxicity. Therefore, CsA acts after antigen binding and before transcription of lymphokine-encoding mRNA. That is CsA blocks the transmission of the antigen signal. This information is used to show that this CsA-sensitive signal is required continuously to maintain the T cell in a lymphokine-secreting state.

trans-4-Hydroxy-2-nonenal (HNE) is an aldehyde end-product of lipid peroxidation in biological systems which is capable of producing a range of powerful biological effects. We wish to describe a sensitive and selective strategy for the determination of HNE in biological samples. The method is based on the formation of the O-pentafluorobenzyl (O-PFB) oxime derivatives of HNE and its deuterated internal standard which, after sample clean-up by solid-phase extraction and purification by high-performance liquid chromatography (HPLC), were derivatised further to trimethylsilyl ethers. Subsequent capillary column gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-NICIMS) using selected-ion monitoring allowed quantitation in the low ng/ml range. The use of an internal standard and the O-PFB oxime derivatives circumvented the problems encountered previously by other workers because of the volatility and instability of HNE. The syn-isomer of HNE O-PFB oxime followed the anti-isomer on the HPLC and GC columns used, giving a distinctive pair of peaks of characteristic relative proportion. Moreover, the NICI mass spectra of the geometrical isomers were significantly different, providing further evidence to validate the identity of any endogenous HNE recovered. The method was used to identify and quantify HNE in platelets, monocytes, plasma and oxidised low-density lipoprotein.

Rat mast cells with the properties of mucosal mast cells (MMC) proliferate in cultures of haemopoietic tissue in the presence of conditioned medium (CM) from antigen- or mitogen-activated T lymphocytes. The present study shows that recombinant rat interleukin-3 (rIL-3) both stimulated the development of MMC from bone marrow (BM) precursors and maintained the proliferation of rat MMC lines in an identical manner to that of CM. The content per cell of the MMC granule-specific proteinase RMCPII was similar in both IL-3- and CM-stimulated cultures. Passage of CM through DEAE-cellulose separated two active peaks that stimulated autologous MMC proliferation. The biochemical properties of peak 1 were similar to those of murine IL-3 and stimulated multi-potential stem cell development in soft agar cultures of BM cells from rats treated with 5-fluorouracil (which enriches for haemopoietic stem cells). RIL-3 was also active in this assay whereas peak 2 was not, demonstrating that peak 1 contained IL-3 activity. The presence of MMC in the majority of multi-potential colonies in the soft agar cultures confirmed the early stem cell origin of the MMC lineage. The cultured BM-derived mast cells in the rat are analogues of the MMC subset that is most readily observed proliferating in the gastrointestinal tract in response to helminth parasite infection. The demonstration that IL-3 is responsible for the development and proliferation of MMC should lead to a better understanding of the functional roles of these cells.

The haemopoietic stem cell line 32D cl-23 is alcian blue positive, Lyt 1+, and requires a growth factor, interleukin-3 (IL-3). This cell has now been shown to respond to interleukin-2 (IL-2). Antibodies directed against the murine interleukin-2 receptor blocked 3H-thymidine incorporation by 32D cl-23 cells grown in interleukin-2, but had no effect on 32D cl-23 in interleukin-3. Thus the receptor for interleukin-2 on 32D cl-23 cells may be similar to the interleukin-2 receptor on murine T cells. Similar results were obtained using a different basophil cell line 32D cl-5, but not using another basophil cell line, Rotundi, or factor dependent musocal type mast cells (P cells) derived from spleen.