High Concentrations Of IFN-gamma Research Articles

Direct recognition of LPS by human but not murine CD8+ T cells via TLR4 complex

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8(+) T cells are also capable of doing so. We report here that naive human CD8(+) T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8(+) T cells can then secrete high concentrations of IFN-gamma, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergize with IL-12 to polarize the CD8(+) T cells into cytotoxic T-cell 1 (Tc1) that produce IFN-gamma but not IL-4, with or without TCR activation. Moreover, CD8(+)CD45RO(+) memory T cells constitutively expressed TLR4 and markedly enhanced IFN-gamma production when stimulated with LPS. In contrast, activated murine CD8(+) T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8(+) T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8(+) T cells.

Characterization of effector functions of human peptide-specific CD4+ T-cell clones for an intracellular pathogen

CD4+ T cells are believed to play a dominant role in human defenses against Mycobacterium tuberculosis through production of interferon (IFN)-gamma, cytolytic T-cell (CTL) activity, and inhibition of intracellular mycobacterial growth. Most functional studies of CD4+ cells have used bulk T-cells that recognize crude mycobacterial antigens, and the functional capacity of individual human T cells is not well defined. We studied the functional capacity of human CD4+ T-cell clones that recognize a specific mycobacterial peptide. Clone B9 produced high concentrations of IFN-gamma and exhibited potent CTL activity, whereas clone D3 produced IFN-gamma but showed poor CTL activity. The CTL activity of clone B9 was inhibited by SrCl(2) and concanamycin A but not by anti-Fas antibodies. Clone B9 also reduced the mycobacterial burden in dendritic cells by more than 90%, and this antimycobacterial activity was inhibited by SrCl(2) and concanamycin A. We conclude that: (1) individual human peptide-specific CD4+ T-cell clones have differential capacity to produce Th1 cytokines and to lyse M tuberculosis-infected target cells; and (2) both granulysin and perforin contribute to the capacity of human CD4+ T-cells to lyse infected targets and to inhibit intracellular mycobacterial growth.

Tonsilar NK Cells Restrict B Cell Transformation by the Epstein-Barr Virus via IFN-γ

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56brightCD16− NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-γ (IFN-γ) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-γ concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.

Pulmonary epithelial cells are a source of interferon‐γ in response to Mycobacterium tuberculosis infection

Recent report from our laboratory showed that A549 cells representing alveolar epithelial cells produce chemokine interleukin-8 and nitric oxide (NO) when challenged with Mycobacterium tuberculosis. Interferon-gamma (IFN-gamma) played a critical role in priming these cells to generate NO in vitro. In the present study, we report that M. tuberculosis-infected A549 cells are capable of elaborating IFN-gamma as shown by enzyme-linked immunosorbent assay and intracellular staining for IFN-gamma. Secretion profile indicated that M. tuberculosis-infected A549 released significantly high concentration of IFN-gamma at 48 and 72 h post-infection. Low level of IFN-gamma release was also seen to be induced by gamma-irradiated M. tuberculosis and subcellular components of M. tuberculosis. Cell surface receptor analysis showed that the M. tuberculosis-infected A549 cells expressed enhanced levels of IFN-gamma receptors. This observation suggests that the endogenously produced IFN-gamma in response to M. tuberculosis infection plays a role in intracellular regulation of innate immunity against intracellular pathogen such as M. tuberculosis. This observation is further strengthened by the fact that infected A549 cells expressed signal transducer and activator of transcription 1 (STAT1), an important mediator for IFN-gamma signaling pathway, leading to expression of inducible NO synthase and subsequent release of NO in sufficient concentration to be mycobactericidal. Our results show that production of IFN-gamma and enhanced expression of IFN-gamma receptors by infected A549 cells is a local phenomenon occurring as de novo intracellular activity, in response to M. tuberculosis infection. To the best of our knowledge, this is the first report to show that A549 cells interact actively with M. tuberculosis to produce IFN-gamma that might play an important role in innate immunity against tuberculosis.

Susceptibility of Borna disease virus to the antiviral action of gamma-interferon: Evidence for species-specific differences

Borna disease virus (BDV) infection in its predominant natural host - horses and sheep - leads to fatal meningoencephalomyelitis. The immune-mediated disease can also be induced experimentally in rats following intra- cerebral BDV infection. Despite a vigorous immune response, BDV persists in the central nervous system (CNS) in surviving rats. However, immunization of rats with BDV-specific T-cells prior to challenge with BDV prevents neurological disease and results in virus clearance from the CNS. To analyze whether interferon gamma (IFNgamma) might contribute to viral clearance in the rat brain, we tested the susceptibility of BDV to the antiviral action of rat IFNgamma using different rat cell lines. Even at high concentrations of IFNgamma, BDV infection of astrocyte and fibroblast cell lines as well as of rat embryo cells could not be inhibited efficiently. Similarly, infection of cultured rat hippocampal slices with BDV was not inhibited by rat IFNgamma. In contrast, de novo BDV infection of monkey kidney cells as well as human oligodendroglial cells was blocked by preincubation with human IFNgamma. Furthermore, IFNgamma reduced the BDV load in persistently BDV-infected human oligodendroglial cells but not in infected rat astrocytes. These data suggest species-specific differences in the susceptibility of BDV to the antiviral action of IFNgamma.

Interferons in enteroviral heart disease: modulation of cytokine expression and antiviral activity.

Interferon (IFN)-beta has a more than 120-fold higher antiviral activity than the closely related IFN-alpha in human myocardial fibroblasts infected with the cardiotropic enterovirus coxsackievirus B3 (CVB3). CVB3 replication induces interleukin (IL)-6 and IL-8 expression in myocardial fibroblasts, and suppresses the expression of monocyte chemoattractant protein-1 (MCP-1). We investigated whether the higher antiviral activity of IFN-beta compared to IFN-alpha was a result of a suppression of IL-8 expression by IFN-beta since previous studies had indicated that IL-8 stimulates enterovirus replication. Human myocardial fibroblasts were treated with either IFN-alpha, IFN-beta or IFN-gamma (0, 10, 100, or 1,000 IU/ml) and the concentrations of IL-6, IL-8 and MCP-1 were measured in culture supernatants by immunoassays. Both IFN-beta and IFN-gamma reduced IL-6 and IL-8 expression significantly. In addition, neutralization of IL-8 in culture supernatants of myocardial fibroblasts using a monoclonal antibody demonstrated a significant reduction of CVB3 titers. Antiproliferative effects of all three IFNs were very low (<30% with 1,000 IU/ml), indicating that the suppression IL-6 and IL-8 was not related to cytotoxicity. MCP-1 expression was increased only by high concentrations of IFN-gamma (1,000 IU/ml). By contrast, IFN-alpha had no significant effect on IL-6, IL-8 and MCP-1 expression. In conclusion, suppression of IL-8 expression is an "immuno-modulating" feature of IFN-beta in human myocardial fibroblasts, which is similar to the activity of IFN-gamma. This feature of IFN-beta contributes to its high antiviral activity against CVB3 and may be useful in the treatment of enteroviral heart disease.

Spectrum of manifestations of Mycobacterium tuberculosis infection in primates infected with SIV.

To characterize the manifestations of coinfection with M. tuberculosis and SIV infection, we studied 12 SIV-infected rhesus monkeys, six of which were infected intrabronchially with a low dose of Mycobacterium tuberculosis H37Rv. In the six coinfected animals, M. tuberculosis antigen-stimulated lung and blood cells produced high concentrations of IFN-gamma but not IL-4 8-16 weeks after infection. Of the three coinfected animals with high levels of plasma viremia, two developed disseminated tuberculosis and the other died of bacterial peritonitis. Of three coinfected animals with moderate levels of plasma viremia, two had no clinical or radiographic evidence of tuberculosis or progressive SIV infection for 6 months after infection. At neuropsy, pulmonary granulomata were observed and acid-fast organisms or M. tuberculosis were present. These clinical, immunologic and pathologic findings are consistent with those in humans with latent tuberculosis infection (LTBI), and suggest that a model of LTBI in SIV-infected primates can be developed. Such a model will permit delineation of the immunologic and microbial factors that characterize LTBI in HIV-infected persons.

Maternal and neonatal IL-4 and IFN-gamma production at delivery and 3 months after birth

Problem: To study the production, interrelationships and determinants of maternal and neonatal cytokines (IL-4 and IFN-gamma) in whole blood cultures during labor, after vaginal delivery and at 3 months after delivery. Major results: The concentrations of maternal IFN-gamma and IL-4 in peripheral blood were down-regulated at delivery compared with values 3 months postpartum. The concentrations of neonatal IFN-gamma and IL-4 were down-regulated at birth and were still at a low level 3 months later. The concentrations of IFN-gamma in maternal and umbilical cord blood samples correlated at delivery ( r=0.43; P<0.03). Maternal IL-4 concentrations correlated immediately after delivery and 3 months later ( r=0.46; P<0.02) as did those of IFN-gamma ( r=0.57; P<0.002). Neonates who were delivered at less than 40 weeks of gestation had higher IFN-gamma concentrations 3 months after birth than those who were delivered after a longer duration of gestation (341 vs. 157 pg/ml; P<0.01). Conclusions: Broad immune activation, reflected in increased production of IL-4 and IFN-gamma, can be detected in women during the postpartum period. Labor-related factors had little effect on ranking of mothers in terms of their IL-4 or IFN-gamma levels, since maternal production of these cytokines correlated immediately after delivery and 3 months later. In neonates, the production of IFN-gamma 3 months after birth was dependent on gestational age at delivery.

Higher concentrations of interferon-gamma enhances uptake and transport of dietary antigens by human intestinal cells: a study using cultured Caco-2 cells.

Besides functioning as a mucosal barrier and transporting nutrients, intestinal epithelial cells (IECs) also serve as antigen-presenting cells (APCs). Modification of protein antigens by proteolysis is one of the principal steps in antigen presentation to Th cells. We used a Caco-2 intestinal epithelial cell line to investigate the transepithelial transport of the dietary antigen, ovalbumin (OVA). We also examined the effects of the proinflammatory cytokine interferon-gamma (IFN-gamma) on the antigen transport process in Caco-2 cell layers. Caco-2 cell layers transferred both intact and degraded OVA from the mucosal to the serosal side. IFN-gamma stimulated OVA transport and most of the transported OVA in such cells were degraded. We also examined OVA uptake by Caco-2 cells using immunohistochemical means. Caco-2 cells incorporated OVA in a time-dependent manner and IFN-gamma significantly enhanced antigen internalization. Flow cytometry also demonstrated that IFN-gamma elevated the internalization of FITC-OVA. We also determined the effects of low and high concentrations of IFN-gamma on mucosal permeability and internalization of FITC-OVA. Although both 10 and 50 ng/mL IFN-gamma stimulated mucosal permeability to the same extent, more FITC-OVA was internalized by Caco-2 cells incubated with 50 than with 10 ng/mL IFN-gamma. These results suggest that the effects of IFN-gamma on mucosal permeability and the internalization of antigens by intestinal epithelial cells are brought about by different mechanisms. Therefore, higher concentrations of IFN-gamma stimulate the uptake, processing, and transport of dietary antigens by IECs.

Th1-like dominance in high-risk first-degree relatives of type I diabetic patients.

The humoral part of the immune system, including autoantibodies, is known to predict manifest Type I (insulin-dependent) diabetes mellitus in first-degree relatives but the cell-mediated immune process preceding the manifest disease still is not known. The aim of this investigation was to estimate the immunological balance of Th-like lymphocytes (Th1/Th2) in high-risk first-degree relatives of Type I diabetic children. Peripheral blood mononuclear cells (PBMC) from 21 healthy high-risk first-degree relatives (ICA > or = 20) were examined and compared with the response seen in PBMC from children with newly diagnosed Type I diabetes and healthy control subjects of similar age, sex and HLA-type. Expression of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA were determined by RT-PCR and as protein by ELISPOT after stimulation with specific epitopes of GAD65 (a.a. 247-279, 509-528, 524-543), bovine serum albumin, the ABBOS peptide (a.a. 152-169) and insulin. High-risk relatives had a high ratio of IFN-gamma:IL-4 compared with both diabetic children (p = 0.0005) and healthy control subjects (p = 0.004). Production of IFN-gamma seen in high-risk relatives was negatively correlated to production of GADA (r = -0.44, p = 0.05). The high concentration of IFN-gamma from high-risk relatives, decreased after stimulation with peptides of GAD65, the ABBOS peptide, BSA and insulin. Increased secretion of IL-4 was observed after stimulation with two peptides of GAD65 (a.a. 509-528 and 524-543), the ABBOS peptide and insulin. Overwhelming production of IFN-gamma seen in peripheral blood mononuclear cells from high-risk first-degree relatives of children with Type I diabetes suggests a Th1-like immune deviation in the prediabetic phase.

The sodium iodide symporter gene and its regulation by cytokines found in autoimmunity.

Iodide concentration by the thyroid gland, an essential step for thyroid hormone synthesis, is mediated by the Na+/I- symporter (NIS). To identify factors that may regulate this process, we have studied NIS gene expression in the Fisher rat thyroid cell line (FRTL-5) by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. Increasing concentrations of bovine TSH (0.1, 1, 10, 50 and 100 mU/l), with or without tumour necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) or interleukin-1 alpha (IL-1 alpha) were added to FRTL-5 cells previously deprived of TSH for a minimum of 5 days. RNA was extracted and samples were studied for NIS expression. TSH enhanced NIS mRNA expression in a dose-dependent manner, with induction evident at 0.1 mU/l, reaching a peak at 50 mU/l, an effect detected after 6 h of stimulation, but not in the first 2 h. Both TNF alpha and, to a lesser extent, IL-1 alpha inhibited basal and TSH-induced NIS expression. High concentrations of IFN gamma also downregulated TSH-stimulated NIS mRNA expression. Using the same technique, we also investigated NIS mRNA tissue distribution in two male and one female Wistar rats. High levels of NIS expression were detected in the thyroid, stomach, and mammary gland, lower levels were found in the intestine, adipose tissue and liver, borderline levels were expressed in the salivary gland, and no expression was detected in the kidneys. In summary, we have shown that TSH upregulates rat NIS gene expression in vitro, and this induction can be modulated by cytokines. Analysis of the distribution of rat NIS mRNA ex vivo demonstrated variable levels of NIS transcription in different tissue samples.

Identification of Neospora antigens recognized by CD4+ T cells and immune sera from experimentally infected cattle.

Neospora caninum is recognized as a major cause of infectious abortion in cattle. Very little is known about immunity to Neospora. Cell mediated responses have previously been shown to be important in the development of protective immunity to the closely related parasite Toxoplasma gondii, and may therefore be an important component in the immune response to Neospora. In this paper we report that a group of low molecular weight NCI strain tachyzoite antigens (< or = 30 kDa) separated by SDS PAGE and bound to nitrocellulose membrane stimulated proliferation in vitro of CD4+ T cells from calves experimentally infected with N. caninum. Proliferation was accompanied by production of high concentrations of IFN-gamma. Several of these antigens were also recognized by antibody produced in these animals. As the most effective vaccines require the stimulation of both humoral and cell mediated immune responses, these antigens may be important in the development of a vaccine against neosporosis.

Contribution of CD4+ and CD8+ T lymphocyte subsets to the cytokine secretion patterns induced in mice during sensitization to contact and respiratory chemical allergens.

Chemical allergens of different types, those that cause in humans allergic contact dermatitis or occupational asthma induce in mice divergent immune responses characteristic, respectively, of T-helper 1 (Th1)- and Th2-type cell activation. Such responses are associated with the development of different cytokine secretion patterns by draining lymph node cells (LNC), such that contact allergens stimulate vigorous interferon-gamma (IFN-gamma) production, but little secretion of the Th2 cytokines interleukin-4 and interleukin-10 (IL-4 and IL-10), whereas the converse pattern is provoked by respiratory allergens. Using selective depletion with antibody and complement we have here examined the relative contribution of CD4+ and CD8+ T lymphocytes to the cytokine secretion patterns of draining LNC isolated from mice sensitized to chemical allergens. Mice received repeated topical applications of respiratory allergens, trimellitic anhydride (TMA) or diphenylmethane diisocyanate (MDI), or of contact allergens 2,4-dinitrochlorobenzene (DNCB) or formaldehyde. Thirteen days following the initiation of exposure the production by draining LNC of IL-10, IFN-gamma and mitogen (concanavalin A)-inducible IL-4 was measured by enzyme-linked immunosorbent assay (ELISA) after various periods of culture. It was found that the high levels of IL-4 and IL-10 secretion stimulated by TMA or MDI, and the lower levels of these cytokines induced by DNCB or formaldehyde, were in all cases dependent upon the presence of CD4- cells. In contrast, the comparatively high concentrations of IFN-gamma observed following exposure to contact allergens were found to be derived from CD4+ cells, and in the case of DNCB from CD8+ cells also. The low levels of IFN-gamma induced by treatment with TMA or MDI were associated largely or wholly with CD8+ cells. These data indicate that the type 2 cytokine responses induced to different extents by both contact and respiratory chemical allergens are almost exclusively a function of CD4+ cells, but that IFN-gamma is produced by either CD4+ cells in the case of contact allergens or largely by CD8+ cells in the case of chemical respiratory allergens.

Interferon-γ Constitutively Expressed in the Stromal Microenvironment of Human Marrow Cultures Mediates Potent Hematopoietic Inhibition

Clinical and laboratory studies have suggested involvement of interferon-gamma (IFN-gamma) in the pathophysiology of aplastic anemia. T cells from aplastic anemia (AA) patients secrete IFN-gamma in vitro, activated cytotoxic lymphocytes infiltrate aplastic bone marrow (BM), and IFN-gamma mRNA, not detected in normal BM, is present in BM from most AA patients. Many patients respond to immunosuppressive therapy with antithymocyte globulin and cyclosporine. Using long-term BM cultures (LTBMC) as a tissue culture model of hematopoiesis, we show that IFN-gamma is a potent inhibitor in the long-term culture-initiating cell (LTC-IC) assay, the best in vitro surrogate test for human hematopoietic stem cells, as well as of the output of committed progenitor cells (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]). In LTBMC, continuous addition of relatively high IFN-gamma concentrations (1,000 U/mL weekly or 200 U/mL every 2 days) was required for inhibition of secondary colony formation, a measure of LTC-IC number and clonogenicity. To mimick local production of IFN-gamma, human stromal cells were engineered by retroviral-mediated gene transfer to express a transduced IFN-gamma gene. IFN-gamma secreted by stromal cells was far more potent than exogenous IFN-gamma in its effects in the LTC-IC assay. For purified CD34+ cells culture in the presence of IFN-gamma stroma dramatically reduced secondary colony numbers as well as production of CFU-GM and BFU-E. Supernatants from these cultures contained only about 20 U/mL of IFN-gamma; this quantity of cytokine, when added to LTBMC, had little effect on hematopoiesis. The mechanism of hematopoietic suppression was related to the inhibition of cell cycle progression and induction of apoptosis of CD34+ cells. There was no apparent effect of local low-level IFN-gamma production on stromal cell function, as reflected in cell morphology, cell surface phenotype, or expression of hematopoietic growth factor genes. LTBMC with genetically altered stromal cells offers an in vitro model of immune suppression of hematopoiesis in AA and may be helpful in testing certain therapeutic modalities. We infer from our data that local production of low levels of inhibitory cytokine is sufficient to markedly inhibit hematopoiesis and to destroy stem cells and more mature progenitor cells.

Low-dose-melphalan-induced up-regulation of type-1 cytokine expression in the s.c. tumor nodule of MOPC-315 tumor bearers and the role of interferon gamma in the therapeutic outcome.

We have previously shown the importance of endogenous tumor necrosis factor (TNF) production for the curative effectiveness of low-dose melphalan (L-phenylalanine mustard) for mice bearing a large MOPC-315 tumor. In the current study we demonstrate that low-dose melphalan is actually associated with enhanced expression of mRNA for TNF alpha in the s.c. tumor nodule. Moreover, the expression of mRNA for interferon gamma (IFN gamma) and interleukin-12 (IL-12; p40) is also elevated at the tumor site. However, while elevation in the expression of mRNA for TNF alpha and IFN gamma is evident within 24 h after the chemotherapy, elevation in the expression of mRNA for IL-12(p40) is first evident 72 h after the chemotherapy. Moreover, neutralizing anti-IFN gamma mAb, like neutralizing anti-TNF mAb but not neutralizing anti-IL-12 mAb, reduced the curative effectiveness of low-dose melphalan for MOPC-315 tumor bearers. Studies into the mechanism through which IFN gamma mediates its antitumor effect in low-dose-melphalan-treated MOPC-315 tumor-bearing mice revealed that MOPC-315 tumor cells, which are not sensitive to the direct antitumor effects of TNF, display some sensitivity to the antiproliferative activity of high concentrations of IFN gamma. However, unlike TNF alpha, IFN gamma is unable to promote the generation of anti-MOPC-315 cytotoxic T lymphocyte activity and, in fact, exerts an inhibitory activity on CTL generation. Taken together, our studies illustrate that low-dose melphalan therapy of MOPC-315 tumor bearers is associated with the rapid elevation in the expression of mRNA for IFN gamma and TNF, two cytokines which are important for the curative effectiveness of low-dose melphalan, and which mediate their antitumor effect, in part, through distinct mechanisms.

Inhibition of Type I Collagen mRNA Expression Independent of Tryptophan Depletion in Interferon-γ-Treated Human Dermal Fibroblasts

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that modulates type I collagen synthesis. In addition, IFN-gamma also exerts potent effects on cellular tryptophan levels by inducing the expression of indoleamine 2,3-dioxygenase (IDO) and tryptophanyl-tRNA synthetase. Because recent evidence indicates that IDO-mediated oxidative tryptophan catabolism is important in cellular responses to IFN-gamma, we investigated the role of IDO in the IFN-gamma-induced modulation of type I collagen gene expression. IFN-gamma ( > or = 50 U/ml) stimulated IDO expression in human dermal fibroblasts in vitro, resulting in a > 90% depletion of tryptophan in the culture media following incubation for 48 h. Higher concentrations of IFN-gamma ( > or = 500 U/ml) caused a marked decrease in type I collagen mRNA levels. Time-course studies indicated that maximal induction of IDO mRNA expression in IFN-gamma-treated fibroblast cultures (24 h) preceded the maximal decrease in collagen mRNA (96 h). Type I collagen mRNA levels were also markedly and selectively decreased in fibroblasts maintained in tryptophan-depleted cultures. Addition of exogenous tryptophan (up to 2500 microM) to IFN-gamma-treated fibroblasts restored "normal" concentrations of tryptophan in the culture media, but did not abrogate the IFN-gamma-induced decrease in collagen mRNA. Addition of the tryptophan metabolite kynurenine, in concentrations similar to those generated in fibroblast cultures following IFN-gamma treatment for 48 h, had no significant effect on type I collagen mRNA levels. These results indicate that although IFN-gamma causes activation of IDO and enhanced tryptophan catabolism in fibroblast cultures, neither the ensuing tryptophan starvation nor the accumulation of kynurenine in the culture media can fully account for the inhibitory effects of IFN-gamma on type I collagen mRNA expression.

Influence of mouse strain and vaccine viability on T-cell responses induced by Mycobacterium bovis bacillus Calmette-Guérin

C57BL/6 and BALB/c mice were vaccinated with either live or heat-killed Mycobacterium bovis bacillus Calmette-Guérin (BCG) organisms, and splenic T cells were used to screen the stimulatory potential of fractionated somatic and secreted mycobacterial proteins by production of gamma interferon (IFN-gamma). Maximum responses were obtained with fractionated secreted proteins of Mycobacterium tuberculosis. There was no single dominant antigen, but five regions of mycobacterial proteins induced high concentrations of IFN-gamma. However, only two of the five regions stimulated T cells from both mouse strains: two were exclusively recognized by T cells from BALB/c mice, and one was exclusively recognized by T cells from C57BL/6 mice. T cells from mice vaccinated with heat-killed M. bovis BCG organisms failed to respond to fractionated secreted proteins but recognized several somatic antigen fractions. As late as 1 year after primary vaccination, memory T cells responded to similar protein regions, and IFN-gamma production was intensified by secondary infection. Our data confirm a central role for secreted proteins in immunity to mycobacteria. Moreover, we demonstrate that a major set of mycobacterium-reactive T cells is stimulated only by vaccination with live but not with heat-killed M. bovis BCG organisms. Because a major impact of genetic host factors on antigen recognition was observed, we favor the use of live carrier organisms which secrete mycobacterial proteins over subunit vaccines as an improved antituberculosis vaccine.

The Influence of Tumour Necrosis Factor‐γ, Interleukin‐1β and Interferon‐γ on the Expression and Function of the Complement Regulatory Protein CD59 on the Human Colonic Adenocarcinoma Cell Line HT29

CD59 is a 18-25 kDa glycoprotein which, by inhibiting the formation of the membrane attack complex, protects homologous cells from complement mediated damage. We have described recently the expression and complement regulatory function of CD59 on colonic adenocarcinoma cells both in vivo and in vitro. In this study we have examined the influence of cytokines on the expression and complement regulatory function of CD59 on the colonic adenocarcinoma cell line HT29. CD59 expression on the HT29 cells was up-regulated after stimulation by mononuclear cells activated by mixed lymphocyte reaction and by culture supernatants from activated mononuclear cells. Similarly, a dose-dependent increase in CD59 expression was observed after stimulation with both tumour necrosis factor-alpha and interleukin-1 beta. A dose-dependent increase in the level of CD59 expression was also seen using low concentrations of interferon-gamma (IFN-gamma), while CD59 expression on cells cultured with high IFN-gamma concentrations was comparable to non-stimulated cells. Cytokine treated cells were more resistant to lysis by homologous complement than non-stimulated cells, and the increase in CD59 expression was shown to be partially responsible for this. The present data strengthen the role of CD59 as a possible participant in tumour escape.

Preferential expression of CD30 by human CD4 + T cells producing Th2‐type cytokines

A large panel of human CD4+ T helper (Th) cell clones with established Th1, Th2, or Th0 profiles of cytokine secretion were examined for the expression of CD30, a member of the tumor necrosis factor receptor superfamily. Th1 clones expressed poor or no CD30 mRNA, and showed low or undetectable expression of both membrane and soluble CD30 (sCD30) protein, whereas Th2 clones showed both CD30 mRNA and membrane CD30 and released substantial amounts of sCD30. Th0 clones exhibited an intermediate pattern of CD30 expression and release. When T cells from the same donor were stimulated with three different antigens (purified protein derivative, PPD; Toxocara canis excretory/secretory antigen, TES; Lolium perenne group I, Lol p I), production of high concentrations of IFN-gamma, but not expression of CD30 or production of IL-4 and IL-5, were observed at any time after stimulation with PPD. In contrast, both CD30 expression and production of IL-4 and IL-5, but not of IFN-gamma, were concomitantly detectable in TES- and Lol p I-reactive T cells, suggesting a temporal relationship between CD30 expression and beginning of Th2-type cytokine production. Finally, CD4+CD30+ T cells specific for Lol p I and inducible to production of Th2-type cytokines were sorted out from the circulation of grass-sensitive patients in concomitance with the onset of allergic symptoms during the seasonal exposure to grass pollen. Thus, CD30 expression appears to be associated with the differentiation/activation pathway of human T cells producing Th2-type cytokines.

Inhibition of Legionella pneumophila growth by gamma interferon in permissive A/J mouse macrophages: role of reactive oxygen species, nitric oxide, tryptophan, and iron(III)

A/J mouse macrophages infected with Legionella pneumophila and treated with gamma interferon (IFN-gamma) in vitro developed potent antimicrobial activity. This antilegionella activity was independent of the macrophage capacity to generate reactive oxygen intermediates, since the oxygen radical scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on the antilegionella activity of IFN-gamma-activated macrophages. Likewise, whereas the ability of IFN-gamma-activated macrophages to synthesize reactive nitrogen intermediates was markedly inhibited by the L-arginine (Arg) analogs, NG-monomethyl-L-arginine and L-aminoguanidine, as well as by incubation in L-Arg-free medium, their ability to inhibit the intracellular growth of L. pneumophila remained intact. The intracellular growth of L. pneumophila in A/J macrophages was inhibited by the iron(III) chelator desferrioxamine and reversed by Fe-transferrin as well as by ferric salts. Additionally, IFN-gamma-activated macrophages incorporated 28% less 59Fe(III) compared with nonactivated cells. Nonetheless, only partial blocking of growth restriction was observed when IFN-gamma-stimulated macrophages were saturated with iron(III). Indole-propionic acid, which appears to inhibit the biosynthesis of L-tryptophan (L-Trp), was an L-Trp-reversible growth inhibitor of L. pneumophila in macrophages, implying that the intracellular replication of this pathogen is also L-Trp dependent. However, an excess of exogenous L-Trp did not reverse the growth inhibition due to IFN-gamma, though a small synergistic effect was observed when the culture medium was supplemented with both iron(III) and L-Trp. We conclude that IFN-gamma-activated macrophages inhibit the intracellular proliferation of L. pneumophila by reactive oxygen intermediate- and reactive nitrogen intermediate-independent mechanisms and just partially by nutritionally dependent mechanisms. We also suggest that additional mechanisms, still unclear, may be involved, since complete reversion was never obtained and since at higher concentrations of IFN-gamma, iron(III) did not induce any significant reversion in the L. pneumophila growth inhibition.