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4) Myb-transformed haematopoietic cell (MTHC) lines have been developed and clones selected based on their ability to respond to tumor necrosis factor (TNF)-a. MTHC-A cells underwent apoptosis in response to TNF-a (MTHC-A). The apoptotic effect of TNF-a 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-a, 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. 〔J Clin Exp Hematop 53(2) : 115-120, 2013〕

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)-γ 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-γ-receptor knockout (IFN-γR−/−) 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-γR−/− 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.

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-2 d ) proislet allografts was analyzed in control, CD4 T cell-depleted, and CD8 T cell-depleted CBA/H (H-2 k ) 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-γ, and IL-3 mRNA) and a CD8 T cell-dependent spectrum of cytokine gene expression (IL-2, IFN-γ, 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 IL-4 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.

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.

The effects of rat stem-cell factor (SCF) and interleukin-3 (IL-3), alone or in combination, on the in vitro growth and serine proteinase expression of rat serosal/connective-tissue mast cells (CTMC) or bone marrow-derived mast cells (BMMC) were examined. Rat SCF stimulated the growth of both CTMC and BMMC. IL-3 stimulated BMMC growth to a lesser extent than did SCF, whereas CTMC numbers did not increase in IL-3. However, SCF and IL-3 had synergistic effects on the growth of both BMMC and CTMC. SCF favoured the maintenance of rat mast cell proteinase- I (RMCP-I) in CTMC, but did not induce detectable production of RMCP-I in BMMC. In contrast, when IL-3 or lymph node-conditioned medium (LNCM) was added to SCF, a subpopulation of CTMC expressed and stored the soluble proteinase RMCP-II. In BMMC, the RMCP-II content of cells maintained in SCF was significantly less than that of cells maintained in IL-3 or LNCM. RMCP-II also appeared in the supernatants of BMMC, especially when BMMC numbers were increasing rapidly in SCF with or without IL-3 or LNCM. Thus, SCF and IL-3 can regulate the growth of rat BMMC and CTMC, as well as influence their production and release of proteinases.

Murine hematopoietic cells can be transformed in vitro by recombinant retroviruses that express the myb oncogene, and hematopoietic growth factor (HGF)-dependent myeloid cell lines can be derived from these transformed primary cells. In this study, the differentiation state and responses of myb-transformed hematopoietic cells (MTHCs) have been investigated. We find that MTHCs exhibit properties of early myeloid progenitors including synergistic responses to combinations of HGFs and expression of certain surface markers. As reported previously, MTHCs respond well to granulocyte-macrophage colony-stimulating factor (GM- CSF) but can also respond to interleukin-3 (IL-3); the response to the latter factor depends on the mouse strain from which the cells are derived. Although these single factors stimulate MTHCs, combinations of these factors with colony-stimulating factor-1 (CSF-1 or M-CSF) or Steel factor (SLF or SCF) act synergistically to promote colony formation. The surface markers expressed by MTHCs include both granulocyte-macrophage lineage specific antigens Gr-1, 7/4, F4/80, and Mac-1, as well as two antigens found on early progenitors and stem cells--Thy-1 and Sca-1 (Ly6E). Expression of the latter markers is often heterogeneous and can be modulated by the growth factors to which the cells are exposed. Finally, we show that monocytic differentiation of MTHCs can be induced by exposure to tumor necrosis factor (TNF alpha). Taken together, these results suggest that MTHCs will be a useful model for studying HGF/cytokine responses in both proliferation and differentiation.

Summary. 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.

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 × 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.

Summary 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 enhances 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.

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 hemopoietic 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-α and IL-4 will differentiate into cells that have many of the properties of macrophages. When the same MTHC lines are exposed to TNF-α in combination with IFN-γ, 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-γ-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. J. Leukoc. Biol. 66: 217–223; 1999.

Publisher Summary This chapter discusses the role of cytokines in immunology. Cytokines are made by virtually every cell of the body. The cytokines that regulate aspects of immunity are derived primarily from blood cells, and the reticulo-endothelial system. They include cytokines produced at sites of tissue damage to attract and activate granulocytes and monocytes, cytokines produced by granulocytes and monocytes to attract lymphocytes (the chemokines), cytokines produced by lymphocytes and macrophages that act on myeloid cells and their progenitors, and cytokines that are produced by lymphocytes and monocytes that act predominantly on lymphocytes to regulate the immune response. In addition, factors, such as tumor necrosis factor α (TNF a) and lymphotoxin (TNF β), transforming growth factors (TGF), and other molecules can play a role in modulating immunity. Lymphokines are cytokines that are made by lymphocytes, most of which also act on lymphocytes. The primary source of lymphokines is antigen-triggered immune T cells. While there is enormous potential to use cytokines or their inhibitors in vivo to modulate the immune response, at the time of writing, few cytokines have yet been through extensive clinical trials. Of these, only interleukin (IL)-2, interferon-gamma (IFN-γ) and the colony-stimulating factors G- and granulocyte-macrophage colony-stimulating factor and IL-3 have effects that are related to improved immune function. For the most part, IL-2 has been used in an attempt to augment anti-tumor immunity, sometimes in conjunction with administration of activated natural killer cells or T cells.

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 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.

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.

In the present study, we show that recombinant human interleukin-1 beta (rhIL-1 beta), which has no effect on the proliferation of human progenitor cells, has synergistic effects on the expansion of human progenitor cells induced by rhIL-3 in liquid cultures. The synergistic effects of rhIL-1 beta with rhIL-3 were observed in liquid cultures using not only fresh bone marrow mononuclear cells, but also selected populations of nonadherent cells, non-T nonadherent cells, and CD34- positive cells. Anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF) antibody partially blocked the synergistic effects of rhIL-1 beta on the proliferation of colony-forming unit (CFU)-GM burst- forming unit-erythroid (BFU-E), and CFU-Mix in liquid cultures in the presence of rhIL-1 beta plus rhIL-3, suggesting that the synergistic effects of rhIL-1 beta plus rhIL-3 are explained in part by the secondary production of GM-CSF. Limiting dilution assays and liquid culture assays using CD34-positive cells indicate that rhIL-1 beta directly increases the numbers of colony-forming cells in liquid cultures. These results suggest that rhIL-1 beta has unique direct and indirect effects on the expansion of hematopoietic progenitor cells in liquid cultures.

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.

We have generated a series of myb transformed myeloid haematopoietic cell (MTHC-D) lines that can be induced to differentiate into macrophage-like or dendritic cells upon exposure to TNF in combination with IL-4 or interferon gamma. Substractive cDNA libraries made between parent and mature cells or between the two alternate differentiated cell types confirm the lineage relationships of these cells. The two mature cell types may represent different stages of dendritic cell maturation since the DC phenotype is rapidly converted to the macrophage phenotype by the addition of IL-4. Some clones of MTHC fail to differentiate in response to TNF and instead undergo apoptosis (MTHC-A). Subtractive cDNA libraries suggest that MTHC-A cells are more closely related to granulocytes than they are to macrophages. Engagement of TNF receptor type I is sufficient to induce differentiation, but both type I and type II receptors must be engaged to induce apoptosis. Several families of genes that are differentially expressed in ‘macrophage’ and dendritic cells have been isolated, including a large number of novel genes, some of which will be discussed.

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.

Human bone marrow cells cultured for 21 days in the presence of recombinant human interleukin-3 (IL-3) produced up to 28 times more colony-forming cells (CFC) than could be obtained from cultures stimulated with granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage CSF (GM-CSF). IL-3-cultured cells retained a multipotent response to IL-3 in colony assays but were restricted to formation of granulocyte colonies in G-CSF and granulocyte or macrophage colonies in GM-CSF. Culture of bone marrow cells in IL-3 also led to accumulation of large numbers of eosinophils and basophils. These data contrast with the effects of G-CSF, GM-CSF, and IL-3 in seven-day cultures. Here both GM-CSF and IL-3 amplified total CFC that had similar multipotential colony-forming capability in either factor. G-CSF, on the other hand, depleted IL-3-responsive colony-forming cells dramatically, apparently by causing these cells to mature into granulocytes. The data suggest that a large proportion of IL-3- responsive cells in human bone marrow express receptors for G-CSF and can respond to this factor, the majority becoming neutrophils. Furthermore, the CFC maintained for 21 days in IL-3 may be a functionally distinct population from that produced after seven days culture of bone marrow cells in either IL-3 or GM-CSF.

IL-3 is a recently described lymphokine that induces the expression of the enzyme 20-α-hydroxysteroid dehydrogenase (20αSDH) in an early T-cell precursor. We demonstrate that purified IL-3 can be used to establish continuous cultures of a discrete subpopulation of T cells with virtually 100% efficiency from normal, unstimulated splenic lymphocyte populations. Once established over a period of approximately 4–6 weeks, the cultures can be readily cloned either in soft agar or by limiting dilution. All of the established lines are Lyt1 + ,2 − , la − , Ig − , Tdt − , 20αSDH + , a phenotype characteristic of helper T cells; they are therefore distinct from continuous cultures of T cells established with IL-2. Although initiation of these cell lines was absolutely dependent on IL-3, once established all of the cell lines were independent of exogenously added lymphokines for their growth in vitro. However, all of the cell lines were found to constitutively produce IL-3 at high levels. None of the cell lines constitutively produced IL-2, but could be readily induced to produce this lymphokine by treatment with phorbol myristic acetate. The ability to produce IL-3 and IL-2 is a further indication that all the cell lines are helper T cells. The possible mechanisms by which IL-3 allows the specific differentiation and/or amplification of T cells of helper phenotype in tissue culture are discussed.

Adherent monolayer cultures of human blood monocytes, peritoneal macrophages, bone marrow macrophages, and colonic mucosa macrophages were examined for their ability to produce and secrete minactivin, a specific inactivator of urokinase-type plasminogen activator. All except colonic mucosa macrophages produced and secreted appreciable amounts of minactivin, but only blood monocytes were stimulated by muramyl dipeptide (adjuvant peptide) to increase production. The minactivin from each of these populations could be shown to preferentially inhibit urokinase-type plasminogen activator and not trypsin, plasmin, or “tissue”-type plasminogen activator (HPA66). A plasminogen-activating enzyme present in monocyte cultures appeared unaffected by the presence of minactivin and could be shown to be regulated independently by dexamethasone.

The cell lines FDC-Pl and 32D cl-23 have previously been used as unique indicators for the growth-promoting activity of interleukin-3. We show that FDC-Pl cells respond to granulocyte/macrophage colony-stimulating factor (GM-CSF, CSF-2) as well as to interleukin-3. In keeping with this finding, FDC-Pl cells express the macrophage-specific marker, F4/80. FDC-Pl cells do not, however, respond to macrophage CSF (M-CSF, CSF-1). In contrast, 32D cl-23 cells do not respond to GM-CSF and lack F4/80. Instead, 32D cl-23 cells respond to an as yet undefined factor in conditioned medium (CM) from the primate T cell line, MLA-144, and CM from mitogen-stimulated human lymphocytes (HLCM). 32D cl-23 cells are Lyt-1+. Both FDC-Pl and 32D cl-23 cells consume interleukin-3, but only FDC Pl cells consume GM-CSF. Similarly, 32D cl-23, but not FDC-Pl, cells consume 32D cl-23 growth factor from MLA-144 CM and HLCM. Interleukin-3-dependent cell lines must therefore concurrently express different functional cell surface receptors for a variety of biochemically distinct growth factors.

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.

Priming CBA mice (H-2k) with infectious ectromelia virus (EV) and C3H mice (H-2k) with noninfectious vaccinia virus (VAC) resulted in a transient virus-specific splenic cytotoxic T lymphocyte (CTL) response followed by persisting CTL memory. When unfractionated spleen cells from these mice were cultured with mitomycin-C-treated stimulator L fibroblasts (H-2k) exposed previously to the homologous noninfectious virus, potent secondary (2°) virus-specific CTL were induced. CTL induction was not affected by depleting the responders of phagocytic and/or adherent cells (presumed to be macrophages) if either virus was presented on L cells. However, only unfractionated responders yielded 2° CTL when cultured with free EV or VAC, indicating that fibroblasts could substitute for responder macrophages as antigen-presenting cells. The failure of virus to induce 2° CTL when presented on either allogeneic fibroblasts or Sepharose provided clear evidence that triggering of CTL precursors required the recognition of antigen in some closely linked association with a “self” marker. This requirement also held for stimulating the responders to undergo virus-specific proliferative responses although proliferation was not a prerequisite for 2° CTL induction. By exposing L cells to defined numbers of purified noninfectious VAC for only 10 min and then after increasing intervals fixing them with glutaraldehyde, it was shown that viral adsorption alone was necessary for the cells to become capable of both stimulating the induction of 2° CTL and inhibiting competitively their lytic activity. After adsorption, this dual capability of the cells increased at a rate that correlated inversely with the concentration of virions to which they were initially exposed. Thus, the stimulator or competitor efficacy of cells exposed to a poxvirion multiplicity of 5000 to 10,000 for 10 min was reached 90 min later by cells exposed to a multiplicity of 50 to 100. Collectively, these experiments demonstrate that only a brief exposure of host cell membranes to intact poxvirions is required to form the antigenic configuration recognized by poxvirus-specific CTL or their precursors. Since this event can occur under conditions that do not favor viral modification of native host cell membrane antigens, it supports the concept that antigen recognition occurs through T lymphocyte receptors with separate specificities for “self” and viral determinants.

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.

In mice with meningitis induced by ectromelia virus, the influx of inflammatory cells into cerebrospinal fluid (CSF) was shown to be triggered by an immunologically specific, T cell-dependent mechanism. These cells were counted, and their cytotoxicity assayed in vitro. Cell numbers reached a peak 6 days after infection and showed specific cytotoxicity for ectromelia-infected target cells, an effect that reached a peak at day 6 and was retained for at least a further 4 days. Treatment with anti-thymocyte serum 2 to 3 days before harvest of CSF cells depressed the numbers found and their cytotoxicity, suggesting that they reached the exudate from the blood.

Interleukin-3 (multi-CSF) is a multilineage haematopoietic growth regulator that initiates the proliferation and differentiation of multipotential stem cells. Complementary DNA clones encoding interleukin-3 (IL-3) have recently been isolated and the structure of the IL-3 gene determined. IL-3 is produced by T lymphocytes or T lymphomas only after stimulation with antigens, mitogens or chemical activators such as phorbol esters. The myelomonocytic leukaemia line WEHI-3B also produces IL-3 but its production is constitutive and the WEHI-3B cells do not appear to produce significant levels of any of the other lymphokines normally secreted by T lymphocytes after stimulation. It has been proposed that the genetic change leading to the constitutive expression of IL-3 may have been a key event in the development of this leukaemia. We report here that the constitutive synthesis of IL-3 by the WEHI-3B cell line is due to the insertion of an endogenous retrovirus-like element close to the 5' end of the gene. The insertion, an intracisternal A particle (IAP) genome, is positioned with its 5' long terminal repeat (LTR) close to the promoter region of the IL-3 gene, resulting in constitutive synthesis of IL-3.

The inflammatory response in mouse brain and the elimination of virus from the brain following intracerebral inoculation of a group A arbovirus has been shown to be T-cell dependent. Virus persists in the brain of T-cell depleted mice, and inflammation is depressed. Virus persists in the brain of T-cell depleted mice, and inflammation is depressed. Inflammation is restored by transfer of immune cells but not by immune serum, and the transferred cells are effective in reducing virus titres in the absence of circulating antibody. Transferred antibody is not efficient in protecting infected mice. The cells that restore inflammation can come from mice immunized with a group A arbovirus of the same subtype but not from mice immunized with more distantly related viruses. Macrophages may be important effector cells working in collaboration with T cells.

Interleukin-3 (IL-3) (multipotential colony-stimulating factor [multi- CSF]) is an important regulator of hemopoiesis. We recently isolated a cDNA clone of this gene and describe in this manuscript the biologic properties of the expressed gene product. An SV40 expression vector carrying cDNA encoding murine interleukin-3 was constructed so that expression of the IL-3 gene was placed under the control of the SV40 early promoter. When the expression vector was transfected to COS-1 monkey cells, IL-3 activity was secreted into the medium, reaching maximal levels 72 hours after transfection. The IL-3 produced by the COS-1 cells was partially purified using diethylaminoethyl Sephacel and phenyl-Sepharose, and its chromatographic properties were the same as IL-3 produced by the WEHI-3 cell line. The biologic activities of the “expressed” IL-3 include the “induction”of 20-alpha-hydroxysteroid dehydrogenase (20-alpha-SDH) in splenic lymphocytes from nu/nu mice, proliferative activity for 32D cl-23 and FDC-P1 cell lines, and colony- stimulating activity for granulocyte-macrophage, eosinophil, megakaryocyte, natural killer-like, erythroid, and multipotential colony-forming cells from murine fetal liver and adult bone marrow.

The responses of popliteal lymph nodes of sheep to Ross River virus (RRV) or Kunjin virus (KV) have been studied by monitoring the cell populations, virus titre, antibody titre and plaque-forming cell (PFC) content of the efferent lymph. In a typical primary response to subcutaneous inoculation in the lower hind limb of either RRV or KV, the flow rate of lymph increased slightly. Increased cell concentrations in the lymph following inoculation caused the hourly cell outputs to rise 5.3- to 10.1-fold above pre-challenge levels. High cell outputs were sustained at greater than 3-fold above pre-challenge figures for up to 10 days. The increased number of large, blastic lymphocytes ranged from 4.0- to 14.9-fold; the increase in these cells usually followed the rise in total number of lymphocytes by 24 to 48 h and remained at greater than 3-fold higher levels for up to 12 days. Ross River virus was recovered in the efferent lymph for at least 36 h after inoculation. Sudden disappearance of the virus from the lymph was correlated with the onset of significant haemagglutination-inhibition antibody titres by 3 to 4 days after infection. In the efferent lymph, PFC were detected 5 to 7 days after primary or secondary inoculation of KV or RRV when lymphocytes were incubated with specific haemagglutinin-coated goose erythrocytes and guinea-pig complement. Antibody levels in the lymph were persistent while the occurrence of PFC was transient.

Spleen cells from mice immunized with vaccinia or influenza viruses were mixed with spleen cells from mice immunized by dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) and transferred into irradiated syngeneic recipient mice which were subsequently restimulated with trinitrophenylated (TNP) or unmodified viruses. One week later, spleens were removed and assayed for indirect anti-DNP plaque-forming cells (PFC). When spleen cells from both influenza and vaccinia primed mice were restimulated with the haptenated form of the virus used for the initial immunization there was an enhanced PFC response compared to that seen with spleen cells from unprimed mice.

Publisher Summary This chapter discusses the natural cytotoxic activity of mouse spleen cell cultures maintained with interleukin-3 (IL-3). Many of the properties of natural killer (NK) and natural cytotoxic (NC) effector functions are the same such as pre-existence in high levels in the host without priming, lack of H-2 restriction, ability to lyse more than one tumor cell type, no classical immunological memory, and no well-defined markers of T cells, B cells, or macrophages. NK and NC cells are closely related in terms of function, but enough differences exist to pose the question of whether they are from separate cell lineages or merely two variants of an effector cell type that mediate most or all spontaneous cytotoxicity. The difficulty in solving this question stems from the inability to physically separate these two cell types because of a lack of known specific markers. The observation of the selectivity of IL-3 to maintain proliferation of cells with NC activity but not NK activity provides an important feature that may be helpful to separate the two activities. The different lymphokine requirements for NK and NC cells, although suggestive of separate lineage, can be taken as two maturation pathways or stages of differentiation of a common stem cell.

Infection of confluent primary mouse muscle cell cultures with Ross river virus (RRV), an Australian alphavirus, resulted in a noncytolytic persistent virus infection. Analysis of the species of viral RNA synthesized in infected cells suggested that defective interfering particles of RRV do not play a role in the establishment or maintenance of viral persistence.

Recent studies from our laboratory concerning the molecular biology of the hemopoietic growth factor, interleukin-3 (IL-3) (multi-CSF) are described. The murine myelomonocytic leukemia line WEHI-3B has been shown to possess a rearranged IL-3 gene resulting from insertion of an endogenous retroviral element near the IL-3 gene promoter. This rearrangement which results in the constitutive expression of IL-3 is postulated to have been an important step in the development of this leukemia. A more direct investigation of the role of autostimulatory loops in leukemogenesis has been carried out by constructing a retroviral expression vector carrying the IL-3 gene and introducing it into the factor-dependent myelomonocytic cell line FDC-P1. The resulting autocrine-stimulated cell line has been shown to be leukemogenic, confirming the oncogenic potential of abnormal expression of IL-3 by hemopoietic cells. Studies on the structure and evolution of the IL-3 gene in other mammals are also presented.

Publisher Summary Theoretically, macrophage activation in one form or another is essential for recovery from any infectious disease in which the agent can survive or multiply within normal macrophages. This chapter discusses lymphocyte–macrophage interactions that lead to macrophage activation. It presents the concept that antigen presentation on cell membranes, or antigen presented in some manner controlled by genes within major histocompatibility systems, is mandatory for efficient induction of effector T cells leading to a cell mediated immunity response. The concept is supported by a limited amount of data from experimental systems involving infection of mice with lymphocytic choriomengitis or ectromelia viruses or Listeria monocytogenes. If studies of other infectious or oncogenic agents in a variety of host species further support this concept, it could provide a basis for fruitful investigation of the cellular and molecular requirements for T cell induction, the nature of T cell receptors for antigen, and the library of T cell receptor specificities in normal animals. Moreover, this could lead to a more rational approach to practical problems of immunoprophylaxis and immunotherapy.

In order to facilitate the identification of human haemopoietic growth factors (HGFs), in complex conditioned media from limited cell sources, we have developed a method for the rapid assay of HGFs using bone marrow stem cells (BMSCs) obtained by Percoll density gradient enrichment of non-adherent cells from long-term human bone marrow culture. These BMSCs provide the basis for a 72 h assay where proliferation is triggered by the presence of GM-CSF, G-CSF and IL-3. The assay is up to 1000 times more sensitive than the conventional colony assay in detecting the presence of HGFs. We also describe methods for the rapid separation and identification of HGFs using fast protein liquid chromatography (FPLC) with phenyl-Superose and Mono Q columns. This enables individual HGFs present in complex conditioned media to be rapidly identified from the elution profile of HGF activity determined in the BMSC assay.

Interleukin-3 (IL-3) is a recently described lymphokine which induces expression of the enzyme 20 alpha hydroxysteroid dehydrogenase (20αSDH) in spleen cells of nu/nu mice (1). In normal mice 20αSDH is associated with cortisone-resistant Thy 1+ cells (1–3) whereas cortisone sensitive Thy 1+ cells in the thymus are terminal deoxynucleotidyl transferase (TdT) positive and 20αSDH negative (4–6). IL-3 induces proliferation of Thy lLyt 1- 2 spleen cells, but has no effect on thymocytes (1). The factor is found in conditioned media (CM) from: a) mixed leucocyte reactions; b) antigen stimulated immune lymphocytes; c) PHA or Con A stimulated lymphocytes; and d) the WEHI-3 cell line. In the first three cases IL-3 is produced by a Thy 1+ Lyt 1+ 2- cell. Conditioned media from in vitro stimulated lymphocytes have been used to support the growth of cytotoxic Thy 1+ Lyt 1- 2+ T cell lines due to the presence of IL-2 (7, 8). Recently, Nabel et al. (9) have demonstrated that CM from Con A stimulated lymphocytes will support the growth of several distinct phenotypes of “T” lymphocytes. One interpretation of this finding is that IL-2 supports the growth of many phenotypically different subsets of T cells.

WEHI-3 cells, originally designated as a myelomonocyte cell line produce high levels of Interleukin-3 (IL-3) constitutively. EL4 cells can be induced to produce IL-3 in presence of conconavalin A (Con A) and phorbol myristic acetate (PMA). There appears to be a correlation between production of IL-3 and expression of Thy 1.2 alloantigen among WEHI-3 sublines.

Evidence is briefly reviewed which supports the following proposals: (1) There are two main subsets of thymus-derived lymphocytes (T cells), effector T cells (T E ), and helper T cells (T H ). T H cells are required for optimal T E cell and B cell responses. (There may be a third T cell subset (T D ) concerned with delayed hypersensitivity). (2) The antigen-receptor dictionaries of these subsets are different from each other and from the B cell dictionary. In the mouse, the T E cell dictionary is restricted mainly to variations of the patterns of antigens dictated by genes in the K or D regions of the H-2 complex and the T H cell dictionary is restricted mainly to variations of the patterns of antigens dictated by genes (Ir genes) in the I region (Ia antigens) and perhaps other loci such as M-locus. (3) As a consequence, T cell responses are triggered only by allogeneic cell surface antigens (or certain similar xenogeneic antigens) or antigenic patterns produced by the action of self H-2 genes in conjunction with foreign antigens. (4) The display or production of antigenic patterns of the latter type is the province of stimulator cells, probably monocytes and macrophages. (5) Ir genes may operate by producing (in association with foreign antigens) antigenic patterns in stimulator cells which dictate the amount of T H cell and B cell responses. (6) The antigenic patterns responsible for T cell triggering may be produced by glycosylation of foreign polypeptides by batteries of glycosyl transferases normally concerned with production of histocompatibility antigens in stimulator cells. (7) T cell receptors for antigen may be either wholly or partly composed of glycosyl transferases, which evolved from those involved in the production of histocompatibility antigens and associated mechanisms of cellular interaction and communication present in very primitive organisms.