Gamma interferon prevents the inhibitory effects of oxidative stress on host responses to Escherichia coli infection.

Oxidative stress occurs in animals challenged with bacterial endotoxin and can affect the expression of important host inflammatory genes. However, much less is known about the effects of oxidative stress on responses to gram-negative bacteria. The current study compared the effects of redox imbalance on hepatic responses of mice to Escherichia coli bacteria versus purified endotoxic lipopolysaccharide (LPS). Oxidative stress induced by glutathione depletion virtually eliminated hepatic tumor necrosis factor alpha responses to both E. coli and LPS. Inducible NO synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) expression was also markedly inhibited by glutathione depletion in LPS-challenged mice, but was unaffected in E. coli-infected animals. Three findings suggested that gamma interferon (IFN-gamma) production explained the differences between LPS and bacterial challenge. Glutathione depletion completely inhibited the IFN-gamma response to LPS, but only partially inhibited IFN-gamma production in infected mice. Exogenous IFN-gamma restored iNOS and ICAM-1 responses to LPS in stressed mice. Conversely, IFN-gamma-deficient, glutathione-depleted mice showed a marked decrease in iNOS and ICAM-1 expression when challenged with E. coli. These findings indicate that both the nature of the microbial challenge and the production of IFN-gamma can be important in determining the effects of redox imbalance during gram-negative bacterial infections.

Inhibition of nuclear factor-kappab activation in mouse macrophages and the RAW 264.7 cell line by a synthetic adenyl carbocyclic nucleoside.

Adenyl carbocyclic nucleosides have potent anti-inflammatory effects on a number of cell types. Notable in this regard is their ability to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) by mouse macrophages that have been activated with bacterial lipopolysaccharide (LPS). Because the transcriptional activation of the mouse TNF-alpha gene is highly dependent on kappaB enhancers, the present study determined whether the synthetic carbocyclic nucleoside 9-[(1S,3R)-cis-cyclopentan-3-ol]adenine (cPA) inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation in these cells. Stimulation of either mouse peritoneal macrophages or RAW 264. 7 macrophage-like cells with LPS led to the appearance of four distinct kappaB-binding nucleoprotein complexes detected by gel mobility shift assays. Cells treated with 100 microM cPA showed significantly reduced levels of NF-kappaB activation as evidenced by measurements of nucleoprotein kappaB-binding activity and diminished kappaB-dependent transcriptional activation. However, both the LPS-induced degradation of the cytoplasmic NF-kappaB inhibitor IkappaBalpha and the nuclear translocation of the NF-kappaB p50, p65, and c-Rel peptides were unaffected by treatment of the cells with the nucleoside. These findings suggest that certain adenyl carbocyclic nucleosides inhibit the activation of NF-kappaB/Rel complexes by a novel mechanism that results in an inhibition of their DNA-binding activities, without blocking their dissociation from IkappaBalpha or their nuclear translocation.

Redox imbalance differentially inhibits lipopolysaccharide-induced macrophage activation in the mouse liver.

Endotoxemia is accompanied by significant changes in the reductive-oxidative (redox) balance of critical target organs. Redox stress has been shown to regulate the expression of proinflammatory genes that are induced by endotoxic lipopolysaccharide (LPS) in vitro; however, much less is known about the effects of redox imbalance on LPS-induced gene expression in vivo. To assess the effects of redox stress on inflammatory responses in endotoxemia, mice were treated with either diethyl maleate (DEM), a glutathione-depleting agent, or buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, and challenged with LPS. While serum tumor necrosis alpha (TNF-alpha) responses and the appearance of TNF-alpha-positive Kupffer cells in the liver were virtually eliminated by DEM or BSO treatment, the expression of both CD14 and inducible NO synthase (iNOS) by Kupffer cells was unaffected by glutathione depletion. By contrast, LPS-induced hepatocyte and hepatic sinusoidal endothelial cell iNOS expression was significantly inhibited in DEM-treated mice. Hepatocyte iNOS induced by recombinant mouse TNF-alpha was also inhibited by DEM treatment. These results indicate that the effects of oxidative stress in this organ are cell type specific and suggest that both the production and the action of TNF-alpha are substantially influenced by the redox state of the liver during endotoxemia.

Mechanism by which U937 promonocytic cells inactivate human interferon-gamma.

Mononuclear phagocytes produce proteinases that are thought to play a role in regulating the activity of cytokines. Activated macrophages secrete urokinase-type plasminogen activator (uPA), which mediates the formation of the serine proteinase plasmin from the ubiquitous zymogen plasminogen. We previously observed a correlation between in vitro plasminogen activation by the promonocytic cell line U937 and the apparent ability of these cells to inactivate recombinant interferon-gamma (rIFN-gamma) by proteolysis. The present study was designed to test the hypothesis that plasmin, generated in U937 cell cultures, is both necessary and sufficient to inactivate rIFN-gamma by limited proteolysis. The following observations are consistent with this hypothesis: (1) inactivation of rIFN-gamma was prevented by inhibitors of serine proteinases or an antibody that specifically immunodepleted plasmin activity; (2) purified plasmin inactivated rIFN-gamma as efficiently as U937 culture supernatants and was similarly sensitive to serine proteinase inhibitors; and (3) plasmin removed an 11 amino acid carboxyl-terminal peptide from rIFN-gamma, which included a region known to be required for bioactivity. Overall, these data indicate that plasminogen activation by U937 promonocytic cells leads to the proteolytic inactivation of IFN-gamma by a process that only requires the production of plasmin.

Transforming growth factor beta 1 and gamma interferon provide opposing signals to lipopolysaccharide-activated mouse macrophages.

Bacterial lipopolysaccharides (LPS) are potent inducers of macrophage activation, leading to the production of a number of proinflammatory mediators. Although several cytokines that prime macrophages for enhanced LPS-triggered responses have been identified, far less is known regarding the role that cytokines play in down-regulating macrophage responses to LPS. This study was designed to determine the effects of recombinant transforming growth factor beta 1 (rTGF-beta 1) on macrophage activation by LPS. Pretreatment of either mouse peritoneal macrophages or cells of the RAW 264.7 macrophage-like cell line with rTGF-beta 1 inhibited their ability to produce both tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) in response to LPS. These inhibitory effects were reversed by increasing the concentration of LPS or by priming cells with optimal concentrations of recombinant gamma interferon (rIFN-gamma). Pretreatment of cells with rTGF-beta 1 had only a modest inhibitory effect on the expression of TNF-alpha mRNA. By contrast, the expression of mRNA for the inducible form of nitric oxide synthase (iNOS), which is responsible for NO production in activated macrophages, was significantly inhibited by rTGF-beta 1 pretreatment. Thus, rTGF-beta 1-dependent suppression of macrophage TNF-alpha biosynthesis was manifest at a posttranscriptional level, whereas the inhibition of NO production correlated with a direct effect on iNOS gene expression. Importantly, both of these suppressive effects of rTGF-beta 1 were reversed by exposing the cells to priming concentrations of rIFN-gamma. As with NO production, immunocytochemical analysis of iNOS expression in LPS-stimulated macrophages revealed that rIFN-gamma and rTGF-beta 1 had antagonistic effects, with the former increasing, and the latter reducing, the number of iNOS-expressing cells induced by LPS. These data suggest that a balance between the priming effects of IFN-gamma and the inhibitory effects of TGF-beta 1 can determine the overall level of macrophage activation induced by LPS.

Regulation of plasminogen activation by human U937 promonocytic cells.

Urokinase-type plasminogen activator (uPA) is initially produced by cells as a single-chain precursor (pro-uPA), which can be cleaved by the serine protease plasmin to form the two-chain molecule uPA. This latter protease efficiently converts the inactive zymogen plasminogen into plasmin. Cell surface binding of both pro-uPA and plasminogen is known to enhance the rate of plasminogen activation. It has been postulated that this may be due, in part, to an enhanced plasmin-mediated feedback activation of pro-uPA. This study directly demonstrates the enhancement by cells of this feedback activation loop by showing that uPA is generated more rapidly from pro-uPA and plasminogen in the presence of human promonocytic U937 cells than it is under fluid-phase conditions. Moreover, the enhanced activation of pro-uPA and plasminogen observed in the presence of cells was significantly less susceptible to inhibition by alpha 2-antiplasmin. Finally, the presence of cells not only potentiated the production of plasmin, as measured using a plasmin-specific peptide substrate, it also potentiated the cleavage of a natural protein substrate, 125I-labeled recombinant interferon-gamma, even in the presence of alpha 2-antiplasmin or alpha 2-macroglobulin. These results demonstrate that cell-associated plasmin mediates a positive feedback amplification of plasminogen activation and thereby potentiates the proteolysis of natural plasmin substrates, even in the presence of plasma protease inhibitors.

Effect of the carbocyclic nucleoside analogue MDL 201,112 on inhibition of interferon-gamma-induced priming of Lewis (LEW/N) rat macrophages for enhanced respiratory burst and MHC class II Ia+ antigen expression.

The effects of the carbocyclic nucleoside MDL 201,112 and the purine nucleoside adenosine on the interferon-gamma (IFN-gamma)-induced priming of macrophages (m phi s) for the respiratory burst and major histocompatibility class II (MHC class II) Ia+ antigen expression were compared. Priming of purified, peritoneal m phi s from Lewis (LEW/N) rats for 18 h with recombinant rat IFN-gamma (rRaIFN-gamma) in the presence of either adenosine (100 microM) or MDL 201,112 (10 microM) resulted in a fourfold decrease in superoxide anion (O2-) production after stimulation with opsonized zymosan. Both agents were effective even when added 2 or 4 h after rRaIFN-gamma treatment. Peritoneal m phi s from LEW/N rats stimulated with LPS/rRaIFN-gamma were observed to secrete immunoreactive and bioactive TNF-alpha over 18 h in vitro and this cytokine could be dose-dependently inhibited by MDL 201,112. MDL 201,112 did not bind to classical A1 or A2 receptors on rat brain homogenates. Physiological levels of adenosine deaminase, or treatment with the nucleoside transport inhibitor dipyridamole, reversed the effects of adenosine; however, these agents at physiological concentrations had little or no effect on the inhibition of O2- release mediated by MDL 201,112. Furthermore, incubation of LEW/N m phi s for 18 h in vitro with rRaIFN-gamma resulted in significant enhancement of MHC class II Ia+ antigen expression, and these levels could be blocked by nearly 50% by either MDL 201,112 (10 microM) or adenosine (100 microM). MDL 201,112 and adenosine were also effective in decreasing m phi opsonized zymosan-stimulated O2- levels and MHC class II Ia+ antigen expression in vivo. The effects of MDL 201,112 on the down-regulation of heat-killed M. tuberculosis-activated LEW/N m phi MHC class II Ia+ antigen expression in vitro appear to be mediated by a novel pathway, because there was no rank order of potency of ADO A1 or A2 agonist/antagonists (CCPA, NECA, XAC, or CPT) in our in vitro system. In summary, our data provide compelling evidence that immunoregulatory carbocyclic nucleoside analogues such as MDL 201,112 or adenosine appear to regulate LEW/N rat m phi activation through novel molecular mechanisms and may have important therapeutic implications for acute and chronic inflammatory diseases.

U937 cells can utilize plasminogen activator to regulate human interferon-gamma.

Urokinase-type plasminogen activator (uPA) converts the proenzyme plasminogen to plasmin and thereby contributes to processes like cell migration, tissue remodeling, and cytokine processing. We report here that uPA produced by the human U937 promonocytic cell line also initiated the inactivation of recombinant interferon-gamma (rIFN-gamma) by plasmin-mediated proteolysis. When cultured serum-free with plasminogen, U937 promonocytic cells generated measurable levels of plasmin activity and destroyed the antiviral activity of exogenously added rIFN-gamma. This effect was not seen in the absence of plasminogen, was prevented by inhibitors of uPA and plasmin, and was accompanied by changes in the electrophoretic mobility of rIFN-gamma on polyacrylamide gels, consistent with limited proteolysis of the lymphokine. Culturing U937 cells or blood monocytes for 48 h led to an elevated expression of their surface uPA and an increase in their capacity to produce plasmin and inactivate rIFN-gamma. The ability of rIFN-gamma to induce Fc receptors on U937 cells could also be prevented by providing the cells with a source of exogenous plasminogen, indicating that U937 cells could control their own activation in vitro through the action of uPA. The results of these studies support the conclusion that mononuclear phagocytes have the capacity to use uPA to regulate cytokine activity in vitro.

Adenosine and a related carbocyclic nucleoside analogue selectively inhibit tumor necrosis factor-alpha production and protect mice against endotoxin challenge.

Adenosine (ADO) and its structurally related analogues are known to regulate the activities of immune and inflammatory cells, including a number of key functions of mononuclear phagocytes. In this study ADO and the synthetic ADO analogue MDL201112 inhibited TNF-alpha, but not IL-1, production by activated mouse peritoneal macrophages and the macrophage-like cell lines J774 and RAW-264. Northern blot analysis indicated that MDL201112 selectively inhibited the expression of steady-state TNF-alpha RNA in LPS+IFN-gamma-activated J774 and RAW-264 cells. This effect could not be attributed to changes in TNF-alpha RNA stability. In contrast, ADO had no effect on RNA levels for TNF-alpha and IL-1, suggesting that ADO acts at a post-transcriptional biosynthetic step. To determine whether either compound inhibited TNF-alpha-mediated inflammatory responses, mice were treated with ADO or MDL201112 and challenged with a lethal dose of endotoxic LPS and D-galactosamine, an hepatotoxin that sensitizes mice to lethal LPS challenge. A single i.p. injection of MDL201112 (100 mg/kg) protected over 90% of the mice, whether injected 1 h before or at the time of LPS challenge. MDL201112 also inhibited the appearance of TNF-alpha in the serum of LPS-challenged animals. The compound did not block D-galactosamine sensitization nor did it prevent lethality caused by the injection of rTNF-alpha. ADO failed to protect animals against endotoxin lethality, most likely due to the rapid metabolism of the nucleoside in vivo. These results establish ADO and MDL201112 as potent inhibitors of TNF-alpha biosynthesis and suggest that MDL201112 or similar analogues warrant further study as potential agents for the treatment of endotoxin shock and other diseases in which TNF-alpha plays an important pathogenic role.

Human herpesvirus 6 inhibits the proliferative responses of human peripheral blood mononuclear cells.

Human herpesvirus 6 (HHV-6) was recently isolated from the blood of immunosuppressed individuals and children with exanthem subitum. In this study, the effect of HHV-6 on the proliferative response of human peripheral blood lymphocytes was tested. T cell lines or human peripheral blood lymphocytes were infected with HHV-6 variant A, strain GS, or variant B, strain Z29. Infected and uninfected cell lysates were UV inactivated and sonicated; when lysates were added to peripheral blood mononuclear cells from HHV-6-seropositive adults, lymphoproliferative responses to antigens (tuberculin purified protein derivative or mumps) and mitogens (phytohemagglutinin or interleukin-2) were inhibited in a dose-dependent manner. This inhibition was reversed by a rabbit anti-HHV-6(GS) antiserum, and inhibition was not due to cell lysis induced by viral infection, since cell death was not observed. This in vitro suppression of cellular immune responses by HHV-6 proteins may be relevant to the in vivo pathogenesis of HHV-6 and its relationship to human diseases.

Murine interferon-gamma enhances resistance to infection with strains of Pseudomonas aeruginosa in mice.

This study focused on the role of recombinant murine interferon-gamma (rMuIFN-gamma) in modulating resistance to infections produced by Pseudomonas aeruginosa in mice. CBA/C mice were treated with rMuIFN-gamma, exposed to thermal injury, and challenged with P. aeruginosa organisms. Survival of infected, burned mice was determined over a period of 14 days and compared with control animals not receiving rMuIFN-gamma. Two clinical isolates of P. aeruginosa that differed originally in the ability to produce metalloproteases, were used to infect the mice. When the mice were pretreated with rMuIFN-gamma before the infection, no statistically significant differences in survival were observed with either of the two strains of bacteria compared to controls. However, when mice were pretreated and injected therapeutically with rMuIFN-gamma (continued receipt of rMuIFN-gamma after infection), there was a significant protective effect in animals infected with one of the strains of P. aeruginosa. We conclude that rMuIFN-gamma may be useful in treating infections with certain strains of P. aeruginosa in mice.

Regulation by transforming growth factor-beta 1 of expression and function of the receptor for IFN-gamma on mouse macrophages.

Transforming growth factor-beta (TGF-beta) is known phenomenologically as a negative regulator of several functions of mouse bone marrow macrophages. The studies reported here extend this list by showing that TGF-beta can suppress cytolytic activity of mouse bone marrow culture-derived macrophages that already have become activated by IFN-gamma and LPS for tumor cell killing, as well as confirm that this cytokine can interfere with the induction of activation. Suppression was caused by a shift in the dose response curve for IFN-gamma rather than absolute inhibition; the 50% effective dose for IFN-gamma was increased approximately fourfold by treatment with TGF-beta. TGF-beta also decreased the absolute number of IFN-gamma R on the surfaces of pretreated macrophages by approximately 30 to 35%, without altering the affinity with which IFN-gamma bound. The increased concentration of IFN-gamma needed to produce the higher level of receptor occupancy explained the observed shift in the IFN-gamma dose response curve. These results suggest that TGF-beta mediates its negative regulatory effects on macrophage activation by interfering with coupling of the IFN-gamma R to the pathways that induce and maintain macrophage activation for tumor cell killing. Such effects are consistent with the view that TGF-beta is a negative regulator of macrophage activation for tumor cell killing. Because of this fact, neoplastic cells that secrete this cytokine may have a distinct survival advantage.

Proteolytic inactivation of cytokines by Pseudomonas aeruginosa.

Pseudomonas aeruginosa alkaline protease and elastase are thought to contribute to bacterial invasiveness, tissue damage, and immune suppression in animals and patients infected with the bacterium. This study examined the ability of the two proteases to inactivate a number of cytokines that mediate immune and inflammatory responses. Human recombinant gamma interferon (rIFN-gamma) and human recombinant tumor necrosis factor alpha were inactivated by both proteases. Murine rIFN-gamma was relatively resistant to alkaline protease but was inactivated by elastase, and human recombinant interleukin-1 alpha and recombinant interleukin-1 beta were resistant to the effects of both proteases. Western immunoblots suggested that cytokine inactivation by these proteases, where it occurred, required only limited proteolysis of the polypeptides. The ability of different P. aeruginosa strains to inactivate IFN-gamma appeared to require the production of both proteases for optimum activity. These results indicate that in vitro cytokine inactivation by Pseudomonas proteases is selective, requires only limited proteolysis, and in certain instances reflects the cooperative effects of both proteases.

Inactivation of human gamma interferon by Pseudomonas aeruginosa proteases: elastase augments the effects of alkaline protease despite the presence of alpha 2-macroglobulin.

Pseudomonas aeruginosa alkaline protease (AP) has recently been shown to produce limited proteolysis of human gamma interferon (IFN-gamma) and thereby destroy the antiviral and macrophage-activating activities of the lymphokine. In the present study we describe some of the characteristics of Pseudomonas elastase (E) with regard to inactivation of human IFN-gamma. The inhibitory effect of E on IFN-gamma bioactivity differed from that of AP in that the direct effects of E were reduced in the presence of human serum. That this property of human serum was in large part attributable to the protease inhibitor alpha 2-macroglobulin (alpha 2-M) was suggested by the following observations: (i) methylamine treatment of serum reduced its effect on E, (ii) E interacted directly with alpha 2-M to induce a characteristic conformational change in the protease inhibitor, and (iii) preformed E-alpha 2-M complexes lacked IFN-gamma-degrading activity. Despite these findings, anti-E antiserum partially neutralized the effect that a Pseudomonas filtrate showed on IFN-gamma, suggesting that E contributes to the activity of bacterial filtrates. Treatment of IFN-gamma with E in the presence of a suboptimal concentration of AP resulted in an E dose-dependent inactivation of the lymphokine. Preformed E-alpha 2-M complexes, although ineffective by themselves at cleaving IFN-gamma, degraded the lymphokine, providing AP was also present in the reaction mixture. These data demonstrate that the destruction of small, biologically significant peptides by Pseudomonas proteases can involve protease-protease synergy that acts even in the presence of the serum protease inhibitor alpha 2-M.

Pseudomonas aeruginosa alkaline protease degrades human gamma interferon and inhibits its bioactivity.

This study was performed to determine the effect of Pseudomonas aeruginosa on gamma interferon (IFN-gamma) production by antigen-stimulated human T-cell clones. Crude bacterial filtrates prepared from certain strains of P. aeruginosa inhibited IFN-gamma production by T cells and reduced the antiviral activity of preformed IFN-gamma. Bacterial filtrates prepared from mutant strains that did not produce the exoenzyme alkaline protease (AP) did not inhibit IFN-gamma activity. The inhibitory activity of bacterial filtrates was heat and trypsin sensitive and was neutralized by an antiserum to AP. Crystalline AP mimicked the effects of the bacterial filtrates, and an inactive filtrate from a protease-deficient mutant strain was reconstituted by the addition of AP. AP-treated recombinant IFN-gamma showed altered migration on Western blots (immunoblots) of polyacrylamide gels, and this modification correlated with a dose-dependent loss of antiviral activity. The ability of recombinant IFN-gamma to elevate the expression of Fc receptors on cells of the U-937 histiocytic cell line was also diminished by AP treatment. These results indicate that the Pseudomonas protease AP can inhibit the antiviral and immunomodulatory activities of IFN-gamma.

Antigenic specificities of Pseudomonas aeruginosa alkaline protease and elastase defined by human T cell clones.

Virulent strains of Pseudomonas aeruginosa derive their pathogenicity, in part, from their secretion of two proteolytic enzymes, alkaline protease (AP) and elastase (E). Human T lymphocytes specific for AP and E can be detected in the blood of immune donors and have afforded the opportunity to characterize the antigenicity of these proteins. To accomplish this goal, we have recently selected 68 human T cell clones from five different Pseudomonas-immune donors and determined their fine specificities. Fifty-five (81%) were found to be protease specific, demonstrating the immunogenicity of the exoenzymes in humans. These clones defined five AP and three E specificities and suggested the existence of at least five allomorphic determinants expressed on the proteases of various Pseudomonas strains. Limiting dilution analysis confirmed a number of antigenic relationships suggested by the long-term T cell clones and revealed that T cells specific for allomorphic protease determinants were at least as frequent in the blood of immune donors as were T cells committed to conserved determinants. Thus, both primary and long-term human T cell clones showed specificity patterns that distinguished proteases from different Pseudomonas strains. These observations describe a heretofore unknown antigenic system of Pseudomonas aeruginosa that should assist in defining the nature and specificity of Pseudomonas immunity in humans.

Kinetics of mammary and intestinal IgA-producing cells in the lactating rat.

IgA-producing B lymphocytes in the lactating mouse mammary gland are thought to be derived from lymphoid tissues associated with the small intestine. By contrast, adoptive transfer studies in the rat have failed to confirm a simple entero-mammary migration of IgA plasma cell precursors during lactation. To explain this discrepancy we postulated that recent cIgA+ cell immigrants undergo extensive local proliferation in the rat mammary gland. The present study tested this hypothesis by determining mitotic indices of cIgA+ cells in the gland during lactation. The results did not support this contention and suggest that rat mammary cIgA+ B cells are not derived from the same pool of precursors providing IgA-producing B cells for the small intestine.

Lymphocyte supernatant-induced human monocyte tumoricidal activity: dependence on the presence of gamma-interferon.

Recently, gamma-interferon (IFN-gamma) has been shown to be have the capacity to activate macrophages in several murine and human systems. The studies reported here were undertaken to determine the identity of the lymphokine responsible for activation of human monocytes to a tumoricidal state. Macrophage-activating factor (MAF) activity was assessed using a 24-h 51Cr release assay with human monocytes as effector cells and K-562 targets. Stimulated lymphocyte supernatants were produced by stimulation of peripheral blood mononuclear cells with concanavalin A in serum-free media. Interferon was detected in an antiviral assay. Four lines of evidence lead to the conclusion that MAF and IFN-gamma are identical in this system: (a) fractionation of stimulated lymphocyte supernatants by adsorption chromatography, followed by anion or cation exchange chromatography (Mono-S, Mono-Q columns), resulted in nearly identical elution profiles of MAF and IFN activities. All of the individual fractions containing MAF activity were found to contain IFN in amounts corresponding to MAF activity. (b) Monoclonal antibody specific for IFN-gamma neutralized the ability of stimulated lymphocyte supernatants to induce human monocyte tumoricidal activity. This antibody also neutralized the MAF activity of purified IFN-gamma but not alpha-interferon. (c) The biological MAF activity of activated lymphocyte supernatants and IFN-gamma were similar. Dilution versus MAF activity for IFN-gamma and stimulated lymphocyte supernatants exhibited identical slopes. Lymphocyte supernatants and IFN-gamma demonstrated similar MAF activity on three effector cells: monocytes, in vitro-differentiated macrophages, and dexamethasone-differentiated macrophages. (d) Analysis of supernatants produced by five antigen-stimulated human T-cell clones demonstrated coordinate production of MAF and IFN. These results provide compelling evidence for support of the concept that IFN-gamma is the major human lymphokine capable of inducing monocyte-macrophage tumoricidal activity.

Identification of the principal T lymphocyte-stimulating antigens of Pseudomonas aeruginosa.

To aid in understanding the role of cellular immunity in limiting Pseudomonas aeruginosa infections, we have identified some of the principal antigens of the organism that are recognized by human T cells. Clones of T cells were selected in such a manner that they would provide information not only about the identity of Pseudomonas antigens, but also the T cell repertoires of immune donors. Most clones were found to be specific for Pseudomonas alkaline protease (AP). Such clones could be physically isolated by selecting with crude Pseudomonas antigens or purified AP. In either case, their fine specificities were the same when tested against a panel of Pseudomonas antigens. The conclusion that AP is the principal immunogen for many donors was confirmed by measuring the absolute frequencies of proliferating T cells committed to AP and all other Pseudomonas antigens. Frequencies of AP-specific clones (1.5-2.7 X 10(-5] were comparable to those from the same donors that were specific for all secreted Pseudomonas antigens (1.3-6.0 X 10(-5]. These results provide a model system for studying human T cell-mediated immunity to bacteria by identifying discrete antigens and measuring the repertoire diversities of cells responding to them.