Showing papers in "European Journal of Immunology in 1990"
TL;DR: Findings suggest that DHEA, presumably through receptor‐mediated mechanisms similar to other types of seroid hormones, may represent a natural and important regulator of IL 2 production in both normal and pathologic conditions.
Abstract: Dehydroepiandrosterone (DHEA) is an adrenal steroid hormone produced in abundance by humans and most other warm-blooded animals, is uniquely sulfated (DHEAS) prior to export into the plasma, and exhibits an age-related decline in production with progressive age. No major physiological functions have been ascribed to the activity of this steroid, although DHEA is known to serve as an intermediary in sex steroid synthesis. Studies on the effects of glucocorticoids (GCS) on the immune system led us to question whether DHEA had effects on the ability of activated lymphocytes to produce interleukin 2 (IL 2). We determined that (a) lymphocytes from DHEA- or DHEAS-treated mice consistently produced much greater levels of IL 2 than normals in response to stimulation, (b) direct lymphocyte exposure to DHEA at low doses (10(-10)-10(-7) M) caused an enhanced capacity to secrete IL 2 following activation, (c) IL 2 production by activated cloned T cell lines could be augmented by DHEA treatment, and (d) GCS-induced depressions in IL 2 synthesis by T cells or T cell clones could be overcome in vitro and in vivo by exposure to the effects of DHEA. These findings suggest that DHEA, presumably through receptor-mediated mechanisms similar to other types of steroid hormones, may represent a natural and important regulator of IL 2 production in both normal and pathologic conditions.
343 citations
TL;DR: This report shows that peripheral (post‐thymic) T cells of mice can become tolerant to a range of antigens, and proposes that anergic cells may themselves participate in reinforcing the tolerant state by competing at sites of antigen presentation.
Abstract: Our goal has been to develop ways to tolerize the mature immune system to any defined antigen. In this report we show that peripheral (post-thymic) T cells of mice can become tolerant to a range of antigens (human and rat immunoglobulins, and bone marrow and skin grafts that differ at multiple minor transplantation antigens). In the case of human gamma globulin (HGG), this required that the antigen be given under the cover of a short course of non-depleting anti-CD4 antibody, while for tolerance to skin and marrow grafts anti-CD8 antibody was also required. Tolerance to HGG could be reinforced by repeated injections of HGG, but was lost in the absence of any further exposure to antigen. This reversal of tolerance with time was due to new T cells being exported from the thymus, as it was not observed in tolerized, adult thymectomized mice. In contrast, tolerance to marrow and skin grafts was permanent, presumably because the established grafts acted as a continuous source of antigen to reinforce the tolerant state. Tolerance could not be broken by the infusion of unprimed spleen cells and in one example (tolerance to Mls-1a) there was clear evidence that specific peripheral T cells were anergic. We propose that anergic cells may themselves participate in reinforcing the tolerant state by competing at sites of antigen presentation.
285 citations
TL;DR: Evidence is provided that MEA is identical with the recently cloned mouse T cell growth factor P40, indicating that the two molecules interact with the same receptor.
Abstract: We have previously shown that certain bone marrow-derived mast cell (BMMC) lines proliferate in response to a mast cell growth-enhancing activity (MEA) that is distinct from interleukin (IL) 3 and IL 4. Here we provide evidence that MEA is identical with the recently cloned mouse T cell growth factor P40. The evidence is as follows: (a) recombinant P40 displayed all the biological activities ascribed to MEA: it supported the growth of MEA-sensitive BMMC lines, it induced IL 6 secretion by these cells, and it enhanced survival of primary mast cell cultures; (b) highly purified MEA stimulated the growth of P40-dependent cell lines; (c) a rabbit monospecific antiserum directed against P40 specifically inhibited the action of MEA on BMMC; (d) specific binding sites for P40 were detected on BMMC and (e) MEA competed with P40 for binding to P40-dependent T cells, indicating that the two molecules interact with the same receptor. These observations further extend the range of biological activities ascribed to P40 and warrant its proposed designation as IL 9.
276 citations
TL;DR: Neither the level of chemotactic activity nor IL8 protein levels correlated with neutrophil or leukocyte infiltration, indicating that the mechanism of migration into the inflammatory environment of the joint is complex.
Abstract: The presence of neutrophils in the synovial joint of patients with rheumatoid arthritis (RA) is thought to be due to the activity of chemotactic factors released by activated cells in the joint. We have shown in this report, for the first time, the abundance of one such factor, interleukin 8 (IL 8), in the synovial fluid of patients both with RA and other non-RA joint diseases, and the spontaneous production of IL 8 mRNA by RA synovial cells in culture. There was no correlation between the levels of chemotactic activity and IL 8 protein, suggesting that other factors with similar neutrophil chemotactic activity are also present in the synovial fluid exudate. In support of this concept neither the level of chemotactic activity nor IL 8 protein levels correlated with neutrophil or leukocyte infiltration, indicating that the mechanism of migration into the inflammatory environment of the joint is complex. Such migration is likely to be due to a number of chemotactic signals in addition to IL 8, which may either synergize with, or inhibit, the action of IL 8.
274 citations
TL;DR: There was no evidence that normal human TcR γ/δ+ intestinal IEL might use preferential variable segments of γ genes, and even the major population of IEL differed from peripheral blood T lymphocytes by their peculiar expression of surface antigens associated with activation.
Abstract: Intestinal intraepithelial lymphocytes (IEL) were studied, after isolation in humans, for their surface antigens with a large variety of monoclonal antibodies. They show peculiar characteristics when compared with peripheral blood lymphocytes and intestinal lamina propria lymphocytes. Although a majority of human intraepithelial lymphocytes (IEL) express an alpha/beta type of T cell receptor (TcR), 13% express a gamma/delta TcR, a percentage which was significantly higher than that found in blood and in lamina propria. In contrast to observations in mice, there was no evidence that normal human TcR gamma/delta+ intestinal IEL might use preferential variable segments of gamma genes. About 10% of human intestinal IEL expressed the alpha chain but not the beta chain of CD8, thus resembling a subset of CD8 alpha+beta- IEL, which was recently described in mice and found to be of thymoindependent origin. In addition, 10% of human IEL had a unique phenotype of immature T cells, as they bore only CD7, but no other T cell or natural killer cell markers. Finally, even the major population of IEL which expressed the usual markers of the T cell lineage (CD3, TcR alpha/beta, CD2, CD4 or CD8 alpha/beta) differed from peripheral blood T lymphocytes by their peculiar expression of surface antigens associated with activation. Indeed, 80% of IEL were CD45R0+, CD45A-, but co-expression of CD11a, CD29 and LFA-3 was inconstant. In addition, 90% of IEL expressed HML-1.
256 citations
TL;DR: In vivo injection of the hamster anti‐murine CD3 monoclonal antibody 145 2C11 into BALB/c mice induces a massive systemic release of several cytokines, but this cytokine release is transient since none of these cytokines are still present 12 to 24 h post‐injection.
Abstract: In vivo injection of the hamster anti-murine CD3 monoclonal antibody 145 2C11 into BALB/c mice induces a massive systemic release of several cytokines. Very high circulating levels of tumor necrosis factor are detected both by enzyme-linked immunosorbent assay and L-929 bioassay 90 min following a single injection of 10 micrograms/mouse 145 2C11. Peak circulating levels of exclusively T cell-derived products such as interferon-gamma, interleukin 2 and interleukin 3 are also detected 90 min to 8 h post-injection. Importantly, this cytokine release is transient since none of these cytokines are still present 12 to 24 h post-injection. In parallel to cytokine release, 145 2C11-treated mice (10 micrograms/mouse) exhibit somnolence, hypomotility (quantified by actimetry), hypothermia, diarrhea and piloerection. At this dosage, the physical reaction is not lethal and reverses in all mice by 48 h post-injection. Severe but again reversible anatomopathological changes are also observed: massive cellular depletion, necrosis and edema of lymphoid organs, leakage syndrome and inflammatory cell infiltrates of the lung, cell vacuolization, necrosis and vascular congestion of the liver. All these data are similar to the clinical and immunological manifestations of the OKT3-induced reaction in patients and, thus, provide an invaluable experimental tool to study its mechanisms and explore its prevention.
254 citations
TL;DR: It is shown that anti‐γ antibodies induce DNA cleavage into oligonucleosomal fragments characteristic of programed cell death (apoptosis) in both cell lines, although WEHI‐231 cells are less susceptible than CH31, indicating that these lymphomas afford a potentially interesting model to study the mechanisms of programing cell death induced by ligation of the antigen receptors on normal B cells.
Abstract: WEHI-231 and CH31 are phenotypically immature sIgM+ murine B cell lymphomas whose growth is inhibited by anti-immunoglobulin (Ig) antibodies. These lines have therefore been used as models for studying the role of surface Ig receptors in the induction of B cell tolerance. We show here that anti-mu antibodies induce DNA cleavage into oligonucleosomal fragments characteristic of programmed cell death (apoptosis) in both cell lines, although WEHI-231 cells are less susceptible than CH31. This effect was reversed by lipopolysaccharide, in agreement with the known effects of lipopolysaccharide on anti-Ig-induced growth inhibition. These results therefore indicate that these lymphomas afford a potentially interesting model to study the mechanisms of programmed cell death induced by ligation of the antigen receptors on normal B cells.
248 citations
TL;DR: The data indicate that, unlike α/β T cells, γ/δ T cells respond to mycobacterial components which are resistant to vigorous protease digestion.
Abstract: T lymphocyte subsets expressing either T cell receptor alpha/beta or gamma/delta were selected from human peripheral blood T cells and proliferative responses to molecular mass-fractionated mycobacterial lysates were determined. alpha/beta T cells primarily responded to fractions greater than 30 kDa whereas gamma/delta T cells preferentially reacted to fractions less than 3 kDa. Protease digestion abolished the stimulating activities for alpha/beta T cells, confirming that alpha/beta T cells respond to protein components. In contrast, components recognized by gamma/delta T cells proved resistant to protease digestion. In limiting dilution studies, frequencies of proliferating gamma/delta T cells remained virtually unaltered by protease treatment of stimulating lysates, while those of alpha/beta T cells became almost undetectable. Furthermore, only few gamma/delta T cells responded to the 65-kDa heat-shock protein. Our data indicate that, unlike alpha/beta T cells, gamma/delta T cells respond to mycobacterial components which are resistant to vigorous protease digestion.
243 citations
TL;DR: It is shown that anti‐mIg treatment causes DNA fragmentation (apoptosis) in immature B lymphocytes such as WEHI‐231 cells, and co‐treatment with the protein kinase C activator phorbol 12‐myristate 13‐acetate prevents apoptosis induced by anti‐ mIg.
Abstract: Anti-membrane immunoglobulin (anti-mIg) antibodies exert inhibitory effects in immature B lymphocytes such as WEHI-231 cells. We show here that anti-mIg treatment causes DNA fragmentation (apoptosis) in these cells. We also report that co-treatment with the protein kinase C activator phorbol 12-myristate 13-acetate prevents apoptosis induced by anti-mIg. These results are in agreement with our initial proposal that sensitivity to the toxic effects of anti-mIg reflects, at least partially, altered signal transduction in immature B lymphocytes. Variations in signal transduction pathways during B lymphocyte ontogeny may, therefore, play a critical role in determining whether B cells should be activated or inhibited via their mIg.
241 citations
TL;DR: It is demonstrated that DM via a receptormediated mechanism inhibits IL 6 production at the transcriptional level, and this may contribute to the anti‐inflammatory and immunosuppressive effect of glucocorticoids.
Abstract: We have examined the effect of dexamethasone (DM) and cortisol on the production of interleukin (IL)6 from the murine macrophage cell line RAW264.9, human monocytes, human endothelial cells and the human fibroblast cell line FS4. In RAW264.9 cells DM in the concentration range 10(-9) M to 10(-6) M inhibited the lipopolysaccharide (LPS)-induced production of IL6 by 10% to 90%. Cortisol had a similar effect, but was about 25 times less potent than DM. Also, when 10(-6) M of DM was added to the cultures after addition of LPS, it completely inhibited the residual 24-h production of IL6. Corresponding to the effect on IL6 production, DM (10(-6) M) reduced the mRNA levels for IL6 in the RAW264.9 cells. The glucocorticoid analogue RU 486 competes with DM and cortisol for the glucocorticoid receptor and reversed the inhibitory effect of DM, demonstrating that DM exerts its effect via the glucocorticoid receptor. DM also had an inhibitory effect on LPS-stimulated IL6 production in freshly isolated human monocytes, and on IL 1-stimulated IL6 production in human endothelial cells and FS4 fibroblasts. These results demonstrate that DM via a receptor-mediated mechanism inhibits IL6 production at the transcriptional level, and this may contribute to the anti-inflammatory and immunosuppressive effect of glucocorticoids.
228 citations
TL;DR: Cells of the expanded abnormal T cell subset were shown to express genes encoding interferon (IFN)‐γ, tumor necrosis factor (TNF)‐β,TNF‐α and interleukin (IL) 6, cytokines which are associated with inflammatory immune responses, and may play an important role in exacerbation of the pathological symptoms of the systemic autoimmune disease.
Abstract: Expression of cytokine genes in freshly isolated T cell subsets in the autoimmune lpr mouse has been studied to determine what factors may be produced by these cells in vivo. RNA prepared from T cell subsets from diseased lpr mice and from the normal congenic strain, MRL/n, was tested for the presence of cytokine-specific message using the polymerase chain reaction. Cells of the expanded abnormal T cell subset were shown to express genes encoding interferon (IFN)-gamma, tumor necrosis factor (TNF)-beta, TNF-alpha and interleukin (IL)6, cytokines which are associated with inflammatory immune responses. These cells may thus play an important role in exacerbation of the pathological symptoms of the systemic autoimmune disease. These cells expressed no detectable IL1, IL2, IL3, IL4 or IL5. Phenotypically normal CD4+ and CD8+ T cells from both lpr and MRL/n also contained transcripts for IFN-gamma, TNF-alpha, TNF-beta and IL6. IL2 mRNA was found almost exclusively in the CD4+ subset, indicating that the CD8+ T cells in the lpr mouse are not highly activated through their class I major histocompatibility complex molecules to produce IL2, as could occur if a virus infection was inducing autoimmunity in these mice. Similar levels of IL2 mRNA were present in the CD4+ T cells of lpr and MRL/n mice, demonstrating that these cells are not defective in IL2 production in vivo.
TL;DR: A new surface molecule has been discovered on mouse intestinal intraepithelial lymphocytes (IEL) using a rat anti‐mouse IEL monoclonal antibody, M290, which was expressed at high levels on nearly all IEL and on a majority of T cells in the gut lamina propria.
Abstract: A new surface molecule has been discovered on mouse intestinal intraepithelial lymphocytes (IEL) using a rat anti-mouse IEL monoclonal antibody, M290. It was expressed at high levels on nearly all IEL and on a majority of T cells in the gut lamina propria. M290 stained, with lower intensity, a small minority of T cells in other lymphoid tissues. Expression was biased towards the CD8+ subset. Stimulation of peripheral T cells with mitogens did not induce expression of the new antigen but addition of transforming growth factor beta to stimulated T cells had a marked inductive effect. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of IEL surface components precipitated with M290 showed principal bands at 135, 120, 28 and 24 kDa (reduced) and 135, 100, 24 and 21 kDa (nonreduced). Precipitation with antibodies to integrin subunits showed that the new molecular complex was not a member of the beta 1, beta 2, or beta 3 integrin families although all of these were represented on IEL. A 13-amino acid N-terminal sequence obtained from the 120-kDa beta subunit of the antigen prepared from an M290+ T hybridoma (MTC-1) did not show homology with integrins. Pulse-chase studies using MTC-1 cells showed that the 135-kDa alpha subunit was derived from a 147-kDa precursor. The function of this new molecular complex is not yet known.
TL;DR: Results indicate that the IL‐A29 and CC15 antibodies define a unique population of CD4−CD8−, γ/δ T cells.
Abstract: In this study, two monoclonal antibodies, IL-A29 and CC15, are described that identify a novel bovine cell surface marker of 215/300 kDa. The antibodies reacted with a discrete population of resting lymphocytes in peripheral blood which, in young animals, constituted about 25% of the mononuclear cells. Thymus, lymph nodes and spleen contained less than 5% positive cells. These cells were negative for surface Ig, a monocyte/granulocyte marker, and the T lymphocyte antigens CD2, CD6, CD4 and CD8. Immunohistological analyses revealed the presence of IL-A29/CC15-positive lymphocytes in the thymic medulla, in the outer cortex of lymph nodes, in the marginal zones of the spleen, in the dermal and epidermal layers of the skin and in the lamina propria of the gut. The IL-A29/CC15+ cells in unfractionated blood mononuclear cells responded in autologous and allogeneic mixed lymphocyte cultures, and when purified they responded to concanavalin A in the presence of recombinant interleukin 2. These observations suggested this population of cells belonged to the T cell lineage. In order to unambiguously define their lineage, cDNA clones encoding bovine T cell receptor (TcR) and CD3 proteins were isolated. Northern blot analyses of IL-A29/CC15+ cell populations and of established cell lines of various lineages demonstrated that they expressed TcR delta and CD3 gamma, delta and epsilon mRNA: TcR alpha was not expressed, whereas only a truncated form of TcR beta mRNA was present. These results indicate that the IL-A29 and CC15 antibodies define a unique population of CD4-CD8-, gamma/delta T cells.
TL;DR: The results indicate a new role of the FcϵRII/CD23 molecules in the uptake of antigen by APC which might be of importance in the maintenance of an ongoing immune response against allergens.
Abstract: In this study we investigated the role of the low-affinity receptor for IgE (Fc epsilon RII, CD23) on Epstein-Barr virus (EBV)-transformed human B cells in the uptake and presentation to T cells of antigen after complexing with IgE Cloned EBV-transformed B cells were incubated for 5 h with (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP)-haptenized tetanus toxoid (NIP-TT) or NIP-TT complexed with a chimeric human IgE/mouse anti-NIP monoclonal antibody (IgE x NIP-TT) and then contacted for 2 min with autologous cloned TT-specific T cells Intracellular Ca2+ mobilization in T cells was determined as an early indicator of T cell activation The antigen-presenting capacity of B cells was significantly increased by complexing the antigen with IgE This effect could be selectively reversed in a dose-dependent manner by blocking the Fc epsilon RII with an anti-CD23 monoclonal antibody The IgE-mediated increased capacity for presenting antigen became particularly evident when B cells were incubated with NIP-TT or IgE x NIP-TT for only 1 h at 4 degrees C, washed and then cultivated for 6 h at 37 degrees C allowing uptake and processing of the antigen These results indicate a new role of the Fc epsilon RII/CD23 molecules in the uptake of antigen by APC which might be of importance in the maintenance of an ongoing immune response against allergens
TL;DR: Despite its lack of direct triggering, the capacity of human recombinant C5a (hrC5a) was able to act synergistically with LPS, leading to higher IL 1 and TNF release by human monocytes and mouse peritoneal macrophages.
Abstract: Lipopolysaccharide (LPS) is a potent inducer of interleukin 1 (IL 1) synthesis and release, and of tumor necrosis factor (TNF) secretion. Many signals can enhance the LPS-induced production of these cytokines. We have previously observed that addition of low amounts of normal human serum to the culture medium enhances IL 1 production. Among serum factors, anaphylatoxins C3a and C5a and/or their desArg derivatives have been shown to enhance LPS-induced IL 1 and TNF production. However, the capacity of natural anaphylatoxins to induce by themselves the production of cytokines remains a controversial issue. We have investigated the capacity of human recombinant C5a (hrC5a) to induce IL 1 and TNF production. Despite its lack of direct triggering, hrC5a was able to act synergistically with LPS, leading to higher IL 1 and TNF release by human monocytes and mouse peritoneal macrophages. As assessed by the comitogenic assay, hrC5a increased IL 1 release, whereas cell-associated IL 1 activity was not significantly modified. Measurement by enzyme-linked immunosorbent assay of human IL 1 beta led to similar conclusions, whereas measurement of IL 1 alpha by radioimmunoassay indicated, in addition, an increase in intracellular IL 1 alpha.
TL;DR: Disease progression in susceptible mice seems to be a consequence of a deficiency of IFN‐γ and a predominance of interleukin 4 rather than the result of an excess amount of TNF‐α, which is curcial for TNF-mediated killing of L. major parasites.
Abstract: We have previously shown that during an infection with Leishmania major, susceptible BALB/c mice, as opposed to mice of a resistant strain (C57BL/6), are primed by lipopolysaccharide for the production of high levels of tumor necrosis factor-alpha (TNF-alpha) which is known to be a potent macrophage (M phi) stimulator in other parasitic diseases. In the present study we investigated whether TNF-alpha activates M phi for killing of L. major parasites. In the absence of interferon-gamma (IFN-gamma) or lipopolysaccharide, TNF-alpha (0.025-25,000 U/ml) failed to activate peritoneal exudate M phi from BALB/c mice for killing of L. major amastigotes. In the presence of suboptimal doses of IFN-gamma (5 or 10 U/ml), however, TNF-alpha mediated a rapid elimination of intracellular parasites, which was highly significant compared to IFN-gamma alone. The combination of TNF with interleukin 4, in contrast, was inactive in this respect and allowed survival of intracellular parasites. From these data we conclude that the presence of IFN-gamma is crucial for TNF-alpha-mediated killing of L. major parasites by M phi. Disease progression in susceptible mice therefore seems to be a consequence of a deficiency of IFN-gamma and a predominance of interleukin 4 rather than the result of an excess amount of TNF-alpha.
TL;DR: It is shown that co‐culture of an HLA‐DR1/4‐restricted, influenza hemagglutinin‐specific T cell clone with a specific peptide presented by interferon‐γ‐induced DR4‐expressing keratinocytes causes tolerance induction.
Abstract: Antigen recognition by interleukin 2 (IL 2)-producing T lymphocytes can lead to two distinct outcomes, depending on the nature of the antigen-presenting cell. Recognition of antigen presented by specialized antigen-presenting cells leads to T cell activation; in contrast, antigen presentation by cells which lack "accessory function" can lead to a state of specific nonresponsiveness, which is characterized by a failure to produce IL 2. We have shown in this study that co-culture of an HLA-DR1/4-restricted, influenza hemagglutinin-specific T cell clone with a specific peptide presented by interferon-gamma-induced DR4-expressing keratinocytes causes tolerance induction. This effect was DR restricted, in that it required pre-incubation of the T cell clone with keratinocytes expressing an appropriate DR type (DR4Dw14). The induction of T cell tolerance was also antigen specific; no inhibition resulted from pre-incubation of the clone with an irrelevant peptide. Furthermore cell to cell contact appeared to be necessary, and the addition of supernatant from interferon-gamma-induced keratinocytes did not cause any inhibition. This phenomenon may have relevance to the immunogenicity of transplanted cultured keratinocytes and to the effects of major histocompatibility complex class II induction on non-bone marrow-derived cells. Presentation of tissue-specific autoantigens by cells such as keratinocytes may provide a mechanism of avoiding, rather than stimulating, autoimmune reactions in the context of a local inflammatory response.
TL;DR: Recombinant interleukin 4 down‐regulates the expression of CD 14 on normal human monocytes, as assessed by flow cytometry, binding assays with radiolabeled anti‐CD 14 monoclonal antibody (mAb), and immunoprecipitation of 125I‐labeled monocytes with anti-CD14 mAb.
Abstract: Recombinant interleukin 4 (IL4) down-regulates the expression of CD14 on normal human monocytes, as assessed by flow cytometry, binding assays with radiolabeled anti-CD14 monoclonal antibody (mAb), and immunoprecipitation of 125I-labeled monocytes with anti-CD14 mAb. In parallel, CD23 expression on monocytes was strongly increased by IL4 stimulation, as assessed by both flow cytometry and immunoprecipitation. Down-regulation of surface CD14 was first detectable after 24-36 h of incubation with rIL4, and was almost complete after 4 days of culture. None of the other recombinant lymphokines tested (IL1, IL2, IL3, IL5, IL6, interferon-gamma, tumor necrosis factor alpha and beta, granulocyte-macrophage colony-stimulating factor) decreased CD14 expression. Metabolic labeling studies with [35S]methionine showed that both the membrane-associated and the soluble form of CD14 are decreased by IL4 stimulation. Northern blot analysis showed that incubation of monocytes with IL4 induced a marked decrease in CD14 mRNA. Nuclear run-off assays revealed that the IL4-dependent down-regulation of CD14 resulted from decreased transcription. Thus, IL4 exerts specific and opposite effects on the expression of monocytic antigens.
TL;DR: IL 2, IL 4 and IFN‐γ accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.
Abstract: The production of interleukin 2 (IL 2), IL 4 and interferon-gamma (IFN-gamma) by in vitro activated unselected human blood mononuclear cells was studied at a single-cell level. Individual lymphokine-synthesizing cells were identified by intracellular immunofluorescent staining using cytokine-specific monoclonal or polyclonal antibodies. Cultures from adult blood donors revealed a biphasic kinetic production pattern for IL 2 and IFN-gamma with peaks occurring 4-6 and 24-30 h after initiation of the cultures. Approximately 20%-40% of the lymphocytes produced IL 2 and IFN-gamma. In contrast, only 1%-3% of the lymphocytes synthesized IL 4 with maximal frequency after 6 h of culture. CD4+ as well as CD8+ T cells contributed to the synthesis of all three lymphokines studied. CD4+CD45R- T cells were the major producers of IL 2 and IL 4, while CD8+CD45R- T cells were the most common phenotype of IFN-gamma-synthesizing cells. By performing two-color immunofluorescence studies we observed that among IL 4-producing cells every second one made simultaneously IL 2 and every fourth one made IFN-gamma. Mononuclear cells from umbilical cord blood could be stimulated to make IL 2 to the same extent as cells from adult blood donors. No IL 4 production and a strikingly reduced frequency of IFN-gamma producers were noted in cell cultures from neonates. IL 2, IL 4 and IFN-gamma accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.
TL;DR: The murine Mx‐1 protein is one of the best biochemically and functionally characterized interferon (IFN)‐induced proteins that is necessary for providing resistance to murine cells against viral influenza infection and is a strictly by type I IFN‐regulated protein in human peripheral blood lymphocytes.
Abstract: The murine Mx-1 protein is one of the best biochemically and functionally characterized interferon (IFN)-induced proteins that is necessary, and sufficient, for providing resistance to murine cells against viral influenza infection. Recently an intracellular human protein homologous to the murine Mx-1 protein has been identified by means of a specific monoclonal antibody. The restricted induction of this intracellular protein in human mononuclear cells (MNC) by various cytokines was investigated. MNC from 26 of 28 healthy people and 35 of 36 cancer patients before IFN-alpha therapy had no detectable Mx-homologous protein. Incubation of human MNC with IFN-alpha and IFN-beta for 24 h at different concentrations led to a dose-dependent induction of the Mx-homologous protein. All IFN-alpha or IFN-beta preparations tested were equally effective in eliciting this intracellular protein. IFN-gamma induced only 1% of the Mx amount elicited by type-1 IFN compared on a weight basis. Neither interleukin (IL) 1 nor IL3, IL4, IL5, IL6, tumor necrosis factor-alpha/beta, granulocyte colony-stimulating factor (CSF) or granulocyte macrophage-CSF at any of the concentrations tested were capable of eliciting any detectable amount of the Mx homolog, while IL2 was a poor Mx-homologous protein inducer. In the presence of high-titered IFN-alpha antisera both IL2 and IFN-gamma were unable to stimulate this protein, proving that IFN-gamma and IL2 indirectly induce the Mx homolog via IFN-alpha. Therefore, the human Mx-homologous protein is a strictly by type I IFN-regulated protein in human peripheral blood lymphocytes.
TL;DR: The findings suggest that modulation of cell‐mediated immune reactions may be achieved by relatively low doses of heparin which inhibit expression of T lymphocyte heparanase.
Abstract: Previously we reported that activated T lymphocytes express a heparanase enzyme that degrades the heparan sulfate moiety of the proteoglycan of the extracellular matrix (ECM). Expression of the heparanase enzyme was found to be associated with the ability of activated T lymphocytes to penetrate blood vessel walls and accumulate in target organs. We recently found that relatively low doses of heparin administered to mice or rats inhibited T cell-mediated immune reactions. In the present study we investigated the effects in vitro and in vivo of the heparanase inhibitor, heparin, on the expression of T lymphocyte heparanase and on the ability of T lymphocytes to mediate a delayed-type hypersensitivity (DTH) reaction. We found that heparanase was induced by immunizing mice with antigen in vivo or by activating T lymphocytes with concanavalin A in vitro. Relatively low doses of heparin administered once daily in vivo (5 micrograms) or present in vitro (0.1 microgram/ml) inhibited the expression of heparanase induced by immunization or by concanavalin A incubation. Higher or lower doses of heparin did not have these effects. The same doses of heparin that inhibited expression of heparanase also inhibited the ability of the lymph node cells to migrate to a site of antigen and adoptively produce a DTH reaction. These findings suggest that modulation of cell-mediated immune reactions may be achieved by relatively low doses of heparin which inhibit expression of T lymphocyte heparanase.
TL;DR: Since blast cells in acute lymphoblastic leukaemia do not express functional immunoglobulin, it is inferred that the tumor cell‐associated VH family repertoire is determined through antigen‐independent mechanisms.
Abstract: During B cell development, immunoglobulin heavy chain (IgH) variable region (VH) genes are rearranged and expressed in a programed manner and accumulating evidence suggests recurrent utilization of developmentally restricted VH genes in malignant B lymphoid populations. We have used polymerase chain reaction gene amplification in conjunction with a panel of VH family-specific amplimers to directly compare the repertoire of VH region rearrangement in mature, CD5+ B cell chronic lymphocytic leukemia with that in immature, CD5 B lineage acute lymphoblastic leukemia. The results revealed a diverse pattern of VH family utilization common to both disease groups in which VH regions most proximal to the IgH joining locus were preferentially rearranged relative to their family sizes with recurrent utilization of several known developmentally restricted VH genes in close to germ-line configuration. These results indicate that biased VH family usage is independent of tumor cell phenotype in B lineage leukemias. This bias may reflect similar stages or compartments in normal B lymphopoiesis from which diverse types of B cell malignancy may arise. Moreover, since blast cells in acute lymphoblastic leukaemia do not express functional immunoglobulin, we infer that the tumor cell-associated VH family repertoire is determined through antigen-independent mechanisms.
TL;DR: Comparisons of the effects of the immunosuppressants cyclosporin A, FK506 and rapamycin using murine B cells activated with a variety of mitogens suggest that although CsA, F k506 andRapamycin are all inhibitors of B cell activation, the inhibitory activity of rapamyccin can be clearly distinguished from that of Cs a and Fk506.
Abstract: The effects of the immunosuppressants cyclosporin A (CsA), FK506 and rapamycin have been compared using murine B cells activated with a variety of mitogens. FK506 is a macrolide antibiotic that has been recently shown to inhibit T cell activation by a mechanism that appears similar to that of CsA. Rapamycin is a macrolide structurally related to FK506 whose mechanism of T cell suppression appears to be distinct from that of FK506 and CsA. While CsA and FK506 were found to preferentially inhibit B cell activation caused by stimuli which induce a rise in intracellular calcium, rapamycin partially inhibited activation by all stimuli tested, including those which are not associated with a calcium flux. All three compounds were found to inhibit cell cycle progression within the G1 phase; however, the rapamycin-sensitive event within G1 was completed earlier than the G1 events inhibited by CsA and FK506. In addition, inhibition of anti-IgM-activated B cells with CsA and FK506, but not with rapamycin, resulted in cell death. These data suggest that although CsA, FK506 and rapamycin are all inhibitors of B cell activation, the inhibitory activity of rapamycin can be clearly distinguished from that of CsA and FK506. Although the suppressive effects of CsA and FK506 on B cell proliferation were nearly identical in this study, their biological activities were distinguishable since FK506, but not CsA, could antagonize rapamycin-mediated suppression.
TL;DR: Findings suggest that CD27+ B cells are predisposed to form cell‐cell interactions, and within 3 h of cell culture CD27+, but not CD27−, B lymphocytes were found to form LFA‐1‐mediated homotypic B cell clusters.
Abstract: CD27 is present on the surface of a major subset of peripheral blood T lymphocytes. In this report we show that CD27 is also expressed on a subpopulation of the normal human B cell lineage which is absent from cord blood but present in tonsils and in the peripheral blood of adult individuals. CD27+ B lymphocytes are characterized by the following criteria: (a) in terms of physical properties, the CD27+ B cells form a population with an increased cell size combined with a decreased cell density; (b) the CD27 expression of tonsillar B lymphocytes is positively correlated with mIgA but negatively correlated with membrane IgM/membrane IgD positivity; (c) CD27 on B cells can be induced selectively by the combination of Staphylococcus aureus plus interleukin 2, but not by either treatment alone, and (d) CD27+ B lymphocytes express high levels of the adhesion structures LFA-1 (CD11a), ICAM-1 (CD54), LFA-3 (CD58) and of the lymphocyte homing receptor CD44. These latter findings suggest that CD27+ B cells are predisposed to form cell-cell interactions. Accordingly, within 3 h of cell culture CD27+, but not CD27−, B lymphocytes were found to form LFA-1-mediated homotypic B cell clusters.
TL;DR: It is demonstrated that operational transplantation tolerance can be achieved with simple, non‐toxic antibody therapy, and the introduction of comparable tolerance‐inducing regimens in clinical organ transplantation could obviate the need for long‐term immunosuppression and its unfortunate side effects.
Abstract: Mice given short courses of anti-CD4 and anti-CD8 monoclonal antibodies became tolerant of allogeneic skin grafted at the same time. Tolerance could be obtained without T cell depletion across multiple minor antigen mismatches, both in naive and primed animals, demonstrating that peripheral T cells could be tolerized, even if they had been previously activated. Where donor and recipient were incompatible across the whole major histocompatibility complex, specific tolerance could be achieved by using a combination of depleting followed by non-depleting antibodies, where each alone was unsuccessful. Although mice clearly tolerated their original skin grafts, we observed in some strain combinations that a second fresh, but genotypically identical graft, was slowly rejected. Such mice also possessed T cells which could proliferate to donor-type stimulator cells in vitro. Whatever the mechanisms, we have demonstrated that operational transplantation tolerance can be achieved with simple, non-toxic antibody therapy. The introduction of comparable tolerance-inducing regimens in clinical organ transplantation could obviate the need for long-term immunosuppression and its unfortunate side effects.
TL;DR: Findings are indicative of local production of IL 6 in the CNS during EAE, and represent the first demonstration of IL6 production in non‐infectious CNS inflammatory disease.
Abstract: Interleukin 6 (IL6) is one of the major inflammation-associated cytokines. Elevated serum or tissue levels of IL 6 have been reported to occur in several human diseases, including infections of the central nervous system (CNS), but not in non-infectious CNS inflammation, e.g. multiple sclerosis. While studying experimental autoimmune encephalomyelitis (EAE) as an animal model for autoimmune inflammation of the CNS, we found increased IL 6 levels in the CNS of mice suffering from a lethal form of the disease. IL 6 levels in the spleens and sera were not significantly increased. These findings are indicative of local production of IL6 in the CNS during EAE, and represent the first demonstration of IL6 production in non-infectious CNS inflammatory disease.
TL;DR: A differential loss of T cell functions in the course of HIV infection which is predominantly caused by a lack of IL2 production after stimulation via the CD3/T cell receptor complex is demonstrated.
Abstract: To investigate the effects of persistant human immunodeficiency virus (HIV) infection on T cell reactivity, functional properties of peripheral blood T cells from HIV-seropositive homosexual men in various stages of infection were studied. T cell activation via CD3 resulting in proliferation and differentiation was measured in a model system independent of accessory cells, using immobilized anti-CD3 monoclonal antibodies (mAb). T cells from HIV-infected asymptomatic men had a decreased proliferative response compared to HIV-negative controls. T cells from AIDS-related complex (ARC) and AIDS patients, compared to T cells from asymptomatic HIV-infected men, had a significantly lower proliferative response to anti-CD3 mAb. This diminished response to anti-CD3 mAb was shown to be due to decreased interleukin (IL) 2 production and could be enhanced by co-stimulation with anti-CD28 mAb or by adding IL 2. Anti-CD3-induced generation of cytotoxic T lymphocytes was fully intact in early infection but was severely decreased in T cells from ARC and AIDS patients. Cytotoxic activity could be restored to near normal levels after co-stimulation with either anti-CD28 mAb or IL 2. Our data demonstrate a differential loss of T cell functions in the course of HIV infection which is predominantly caused by a lack of IL 2 production after stimulation via the CD3/T cell receptor complex. In early HIV infection this seems to be predominantly caused by a specific loss of memory T cells. However, in later stages of infection when both naive and memory T cell subsets are depleted, resulting in a normal naive/memory T cell ratio, T cell functions further deteriorate probably due to intrinsic activation defects. These findings may be of pathogenic relevance since diminished T cell reactivity may facilitate spreading and replication of virulent HIV variants heralding development of ARC and AIDS.
TL;DR: The results suggest that the γ/δ T cells precede the α/β T cells in appearance during listerial infection and may be involved at the first line of the host‐defense against Listeria.
Abstract: To search for a potential role of T cell antigen receptor (TcR) gamma/delta-bearing cells in host-defense against Listeria monocytogenes, we analyzed the sequential appearance of gamma/delta and alpha/beta T cell in the peritoneal exudate cells (PEC) during an i.p. infection with sublethal dose (2 X 10(3) of viable Listeria organisms in mice. The PEC on day 1 after the infection consisted of 48% macrophages and 50% lymphocytes, most of which were surface IgM+ (B) cells. The number of PEC increased to the maximal level by day 3. The PEC at this stage contained an appreciable number of CD3+ T cells in addition to a large number of macrophages. Of the CD3+ cells, the proportion of CD4- CD8- cells, most of which expressed no TcR alpha/beta, increased to the maximal level on day 3 after the infection. In correlation with an increased number of CD3+ CD4- CD8- TcR alpha/beta- cells, high level of TcR gamma/delta chain gene messages was detected in the nonadherent population of the PEC on this stage. On the other hand, the PEC on day 8 contained an increased number of CD4+ CD8- and CD4- CD8+ cells which expressed TcR alpha/beta chain on their surface. These results suggest that the gamma/delta T cells precede the alpha/beta T cells in appearance during listerial infection. The gamma/delta T cells may be involved at the first line of the host-defense against Listeria.
TL;DR: The ability of the T cell epitope‐containing peptides 12 and 21 to interact with many different HLA alleles means they may potentially be very useful as “universal carrier molecules” in synthetic vaccines.
Abstract: Tetanus toxoid-specific T cell clones were isolated from a human donor. To determine the T cell epitopes recognized by the clones, 30 peptides representing amphipathic alpha helical regions of the tetanus toxin were screened for ability to induce proliferation of the clones. Two T epitopes were identified. These occurred within peptides 12 and 21, and had the amino acid sequences NSVDDALINSTKIYSYFPSV and PGINGKAIHLVNNESSE, respectively. An unusual feature was that both peptides could be presented to their respective T cell clones by antigen-presenting cells of many HLA specificities. Further investigation of peptide 12 showed that the epitope was only seven amino acids in length and had a very hydrophobic sequence, namely YSYFPSV. The ability of the T cell epitope-containing peptides 12 and 21 to interact with many different HLA alleles means they may potentially be very useful as "universal carrier molecules" in synthetic vaccines.
TL;DR: With the use of the immunospot assay antigen‐specific T cells could be detected even in the absence of detectable IFN‐γ in the culture supernatants, however, the ELISA assay should be more convenient for screening large clinical material.
Abstract: Activation of T cells results in intracellular expression and secretion of cytokines such as interferon (IFN)-gamma. Here we have used three different assays for determination of IFN-gamma in tetanus toxoid- or mitogen-activated human T cell cultures. Two of these assays [intracytoplasmic immunofluorescence and enzyme-linked immuno spot assay (ELISPOT)] determined the expression and secretion of IFN-gamma at the single-cell level while the third assay enzyme-linked immunosorbent assay (ELISA) measured IFN-gamma secreted into the culture supernatant. Comparison of all three tests revealed a good correlation between the ELISPOT assay and the ELISA, whereas expression of intracellular IFN-gamma showed a qualitative but not a quantitative correlation with the latter. Both the immunospot assay and the immunofluorescence may be used to detect approximate numbers of specific T cells even when present at low frequencies. With the use of the immunospot assay antigen-specific T cells could be detected even in the absence of detectable IFN-gamma in the culture supernatants. However, the ELISA assay should be more convenient for screening large clinical material.