Cytokine interactions in experimental cutaneous leishmaniasis. Interleukin 4 synergizes with interferon-gamma to activate murine macrophages for killing of Leishmania major amastigotes

We investigated the effect of recombinant murine interleukin 4 (IL 4) in the absence or presence of recombinant murine interferon-gamma (IFN-gamma) on adherent bone-marrow macrophages (M phi), peritoneal exudate and resident peritoneal M phi from susceptible BALB/c M phi, which were pulse-infected with Leishmania major amastigotes (AM), IL 4 (5-100 U/ml) failed to activate any of these M phi populations for killing of intracellular AM. However, in the presence of low concentrations of IFN-gamma (10-20 U/ml), which alone caused only a slight or intermediate reduction of the number of intracellular parasites. IL 4 led to a dramatic increase of the parasite elimination by all M phi populations. In the case of resident peritoneal M phi, the synergism of IFN-gamma and IL 4 required the incubation of the M phi with both cytokines or with IFN-gamma alone for at least 10 h prior to infection; adding both cytokines after infection of the M phi did not cause a significant reduction of the intracellular parasite burden. The synergistic effect of IL 4 and IFN-gamma was completely abrogated in the presence of anti-IL 4 antibodies. Furthermore, there was no significant difference between M phi derived from either susceptible BALB/c or from resistant C57BL/6 mice. Evidence is presented that the synergistic action of IL 4 and IFN-gamma occurs via an L-arginine-dependent killing pathway. From these data we conclude that IL 4 provides a strong stimulus for the killing of intracellular L. major AM provided low concentrations of IFN-gamma are present. Also, IFN-gamma is apparently an important priming signal for the activation of resident M phi to eliminate intracellular AM.     

Tumor necrosis factor-alpha in combination with interferon-gamma, but not with interleukin 4 activates murine macrophages for elimination of Leishmania major amastigotes

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.     

Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha

Previously, we reported that exposure of bone marrow-derived macrophages (M phi) to a phagocytic stimulus in the simultaneous presence of interferon-gamma (IFN-gamma) induced these cells to generate nitrite (NO2-). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettii) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2- secretion. As shown here, the capacity of phagocytosis to elicit NO2- production by IFN-gamma-treated M phi was inhibited by antibody to murine recombinant tumor necrosis factor-alpha (rTNF-alpha), suggesting that phagocytosis enabled IFN-gamma to activate M phi via the induction of TNF-alpha as an autocrine second signal. M phi NO2- production in response to rIFN-gamma and either exogenous TNF-alpha or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism. However, addition of either Leishmania promastigotes or latex beads to M phi cultures simultaneously exposed to both IFN-gamma and exogenous murine or human rTNF-alpha further potentiated activation as measured by NO2- release. Furthermore, anti-TNF antibody failed to inhibit M phi responses to rIFN-gamma and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-alpha did not significantly affect NO2- production by IFN-gamma/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances M phi responses by a process more complex than the sole induction of TNF-alpha. Phagocytosis also increased M phi NO2- production elicited by IFN-gamma plus TNF-alpha in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating M phi microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.     

Leukotriene B(4) induces nitric oxide synthesis in Trypanosoma cruzi-infected murine macrophages and mediates resistance to infection

The production of nitric oxide (NO) by gamma interferon (IFN-gamma)-activated macrophages is a major effector mechanism during experimental Trypanosoma cruzi infection. In addition to IFN-gamma, chemoattractant molecules, such as platelet-activating factor (PAF) and CC chemokines, may also activate macrophages to induce NO and mediate the killing of T. cruzi in an NO-dependent manner. Here we investigated the ability of leukotriene B(4) (LTB(4)) to induce the production of NO by macrophages infected with T. cruzi in vitro and whether NO mediated LTB(4)-induced parasite killing. The activation of T. cruzi-infected but not naive murine peritoneal macrophages with LTB(4) induced the time- and concentration-dependent production of NO. In addition, low concentrations of LTB(4) acted in synergy with IFN-gamma to induce NO production. The NO produced mediated LTB(4)-induced microbicidal activity in macrophages, as demonstrated by the inhibitory effects of an inducible NO synthase inhibitor. LTB(4)-induced NO production and parasite killing were LTB(4) receptor dependent and were partially blocked by a PAF receptor antagonist. LTB(4) also induced significant tumor necrosis factor alpha (TNF-alpha) production, and blockade of TNF-alpha suppressed LTB(4)-induced NO release and parasite killing. A blockade of LTB(4) or PAF receptors partially inhibited IFN-gamma-induced NO and TNF-alpha production but not parasite killing. Finally, daily treatment of infected mice with CP-105,696 was accompanied by a significantly higher level of blood parasitemia, but not lethality, than that seen in vehicle-treated animals. In conclusion, our results suggest a role for LTB(4) during experimental T. cruzi infection. Chemoattractant molecules such as LTB(4) not only may play a major role in leukocyte migration into sites of inflammation in vivo but also, in the event of an infection, may play a relevant role in the activation of recruited leukocytes to kill the invading microorganism in an NO-dependent manner.     

Modulation of Forssman glycosphingolipid expression by murine macrophages: coinduction with class II MHC antigen by the lymphokines IL4 and IL6

In contrast to murine spleen M phi, resident peritoneal M phi from health mice express very little Forssman glycolipid antigen (Fo). The following experiments suggest that Fo expression by peritoneal M phi may be associated with inflammation. Balb/c and CBA/J mice were given inflammatory stimuli by i.p. injection of live BCG, thioglycollate (TG), Corynebacterium parvum (CP), proteose peptone (PP), or LPS. Control animals received pyrogen-free saline. Expression of Fo and Ia antigen by peritoneal M phi was determined by immunofluorescence after 4 d. Application of TG or CP led to an up to 30-fold increase in Fo+, Ia+ double positive M phi over that in control animals. LPS caused mainly an increase in the percentage of double-positive M phi, whereas no effects were seen in BCG or PP treated animals. To clarify the possible involvement of cytokines in this process and to identify these, the effects of LPS and various cytokines on in vitro induction of Fo and Ia expression were studied in further experiments. LPS, IL6, and IL4 caused induction of up to 15% Fo+ and Ia+ M phi after a 4 d culture period. M phi colony stimulating factor (M-CSF) from lung-conditioned medium was also moderately active. IL1, TNF, and IL2 had no influence, whereas IFN-gamma only induced Ia. For a successful in vitro induction of Fo and Ia, a prior priming of the mice with PP appeared mandatory. This suggests that only M phi of a certain developmental stage can acquire Fo under the influence of the appropriate cytokines. The data may provide the first evidence for cytokine-mediated modulation of a glycolipid antigen of known chemical structure.     

A study of the differential respiratory burst activity elicited by promastigotes and amastigotes of Leishmania donovani in murine resident peritoneal macrophages.

Acridine orange and ethidium bromide and a combination of fluorescent and transmitted light microscopy used in conjunction with the qualitative nitroblue tetrazolium assay for superoxide anion (O2-) release demonstrated dramatic differences in the binding of and respiratory burst (RB) activity elicited by promastigotes and amastigotes of Leishmania donovani in resident peritoneal macrophages (M phi) from C57BL/10ScSn mice. When amastigotes were incubated with M phi for 30 min the number of parasites per 100 M phi was 2-4-fold higher, a higher proportion of M phi became infected and the mean number of parasites per infected M phi was higher than in promastigote infections. RB activity was higher for promastigotes than amastigotes both in terms of the percentage of infected M phi containing formazan positive parasites and the percentage of individual formazan positive parasites. In an attempt to explain the differential response to promastigotes and amastigotes, RB activity was examined for sodium azide-treated, glutaraldehyde-fixed and heat-killed parasites and for various transformation intermediates between amastigotes and promastigotes. Binding and RB activity were also examined in conjunction with competitive binding assays designed to determine the specific receptors involved in ligand binding of both forms of the parasite to the M phi. The results indicate that, while amastigotes may possess an azide-sensitive mechanism which either competes for O2- produced or causes localized inactivation of RB activity, this cannot account for the full magnitude of the difference between the two forms of the parasite. The transformation and competitive binding studies suggest that the more likely explanation lies in both qualitative and quantitative differences in the distribution of surface ligands involved in binding the parasite to the M phi plasma membrane and that the well characterized mannose/fucose receptor may be important in promastigote, but not amastigote, binding and RB activity.     

Activation of human macrophages for the killing of intracellular Trypanosoma cruzi by TNF-alpha and IFN-gamma through a nitric oxide-dependent mechanism.

The protozoan parasite Trypanosoma cruzi is able to replicate in the cytoplasm of primary resident macrophages, but is killed by activated macrophages. Pretreatment of human macrophages with recombinant IFN-gamma and to a lesser extent with TNF-alpha, induced a significant trypanocidal activity. Furthermore, TNF-alpha had a synergistic effect with IFN-gamma on macrophage activation in T. cruzi killing. Similarly, IFN-gamma triggered the production of nitric oxide (NO) by macrophages, whereas TNF-alpha was less effective, although it was also synergistic with IFN-gamma. Both NO production and trypanocidal activity, but not superoxide (O2-) generation, induced in macrophages by TNF-alpha or IFN-gamma alone or in combination, were inhibited by N-monomethyl-L-arginine (N-MMLA), a competitive inhibitor of NO synthase activity. Furthermore, a strong correlation was found between the levels of NO production and trypanocidal activity induced by different lymphokine preparations. These results suggest that IFN-gamma and TNF-alpha are involved in the activation of the trypanocidal activity of human macrophages through a NO-dependent mechanism.     

Antigen presentation by dendritic cells provides optimal stimulation for the production of interleukin (IL) 2, IL 4 and interferon-gamma by allogeneic T cells.

Previous studies have shown that dendritic cells are the most potent inducers of T cell proliferation in vitro and that this is reflected in the release of interleukin (IL) 2 into culture supernatants during dendritic cell-T cell interaction. However, the role of the dendritic cells, and, indeed, of the antigen-presenting step, has not yet been explored with respect to other T cell-derived cytokines, in either a qualitative or relative fashion. In this study, therefore, we have examined the comparative role of different antigen-presenting cells (APC) as inducers of T cell cytokine release in allogeneic responses. We have confirmed that dendritic cells are the most effective inducers for IL2 and have shown that this is true not only in primary alloresponses, but also in alloresponder T cells maintained for extended periods and then rechallenged. Dendritic cells were also the most potent inducers of IL3 and interferon-gamma (IFN-gamma) in primary cultures. No IL4 was demonstrable irrespective of the type of presenting cells used, and both tissue macrophages and dendritic cells can induce synthesis of IL6. Likewise, in secondary alloresponses both dendritic cells and to a lesser extent tissue macrophages induce release of IL3, no IL4 is detectable, and activated macrophages and B cells raise IFN-gamma levels in the supernatants albeit to a lower concentration than that seen when dendritic cells are used as stimulators. The results were similar in the tertiary alloresponse except that (a) IL4 was now detectable in the supernatants but only where dendritic cells had been used as APC, and (b) both resting and activated macrophages induced IL2 and IFN-gamma. By the eighth cycle of allostimulation there is negligible IL2. Dendritic cells, tissue macrophages and activated B cells constitute a hierarchy of APC for IL3, IFN-gamma and IL4. These findings therefore demonstrate the role of dendritic cells as potent in vitro inducers of IL3, IL4 and IFN-gamma synthesis as well as of IL2.     

Immunosuppression induced by nitric oxide and its inhibition by interleukin-4.

Mice immunized with attenuated Salmonella typhimurium, strain SL3235, while protected against virulent challenge, are unable to mount in vivo and in vitro antibody responses to non-Salmonella antigens, such as tetanus toxoid and sheep red blood cells, and exhibit profoundly suppressed responses to B and T cell mitogens. Suppression of antibody responses is mediated by macrophage (M phi)-released soluble factors, and is completely reversed by treatment with interleukin (IL)-4. The present report identifies the suppressor factor as nitric oxide (NO), and provides evidence for a mechanism by which IL-4 abrogates suppression. Suppressed antibody responses correlated with high levels of NO secretion by splenocytes of SL3235-immunized mice. NO production was observed only in cultures consisting of the adherent cell fraction of immune splenocytes. Further, immunosuppression was reversed by NG-monomethyl-L-arginine (NMLA), a competitive inhibitor of NO synthesis, and was completely blocked by the addition of excess L-arginine. Treatment with IL-4, or anti-interferon (IFN)-gamma monoclonal antibody (mAb), also abrogated suppression. Optimal reversal of suppression was observed only when NMLA, IL-4, or anti-IFN-gamma mAb, was added at day 0 of the 5-day plaque-forming cell assay. Treatment with either IL-4 or anti-IFN-gamma mAb also lead to a sharp inhibition of NO production by immune spleen cells. Moreover, the addition of IL-4 to splenic adherent M phi inhibited their ability to generate NO. Our data characterize an immunoregulatory pathway, involving IFN-gamma and NO, by which M phi mediate immunosuppression and identify IL-4 as a potent inhibitor of this pathway.     

Synergism between tumor necrosis factor-alpha and interferon-gamma on macrophage activation for the killing of intracellular Trypanosoma cruzi through a nitric oxide-dependent mechanism.

Intracellular replication of the protozoan parasite Trypanosoma cruzi inside macrophages is essential for the production of the disease and the development of the parasite. Two CD4+ T cell lines, A10 and A28, were established from T. cruzi-infected BALB/c mice which specifically proliferated to parasite antigens. The trypanocidal activity of BALB/c macrophages was induced upon culture with the A10, but not with the A28 T cell line. The cell-free supernatant from this A10 line, as well as from immune spleen cells stimulated with specific antigen or concanavalin A, but not from the A28 T cell line also activated the trypanocidal activity of peritoneal macrophages or of the J774 macrophage-like cell line. when the lymphokine content of the supernatants from both cell lines was analyzed, it was found that the A10 T cell line secreted interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin 2, whereas the A28 line did not secrete IFN-gamma upon stimulation. Furthermore, the trypanocidal-inducing ability of A10 supernatant was completely abrogated by neutralizing anti-IFN-gamma antibodies and partially abrogated by neutralizing anti-TNF-alpha antibodies. When recombinant cytokines were added to J774 cells, IFN-gamma was able to induce significant trypanocidal activity whereas TNF-alpha was almost ineffective. However, TNF-alpha or lipopolysaccharide (LPS) showed a synergistic effect with IFN-gamma on macrophage activation. IFN-gamma triggered nitric oxide (NO) synthesis by J774 cells whereas TNF-alpha was almost ineffective. TNF-alpha and LPS were also synergistic with IFN-gamma in the NO production. Both the NO production and the trypanocidal activity in J774 cells induced by T cell supernatants or lymphokine combinations were inhibited by N-monomethyl-L-arginine, a competitive inhibitor of NO synthase activity. A good correlation between the levels of NO production and trypanocidal activity induced by different lymphokine preparations was found. Those results suggest that IFN-gamma and TNF-alpha, secreted by T. cruzi-immune T cells, are involved in the activation of the trypanocidal activity of mouse macrophages through an NO-dependent mechanism.     

The role of tumour necrosis factor-alpha in combination with interferon-gamma or interleukin-1 in the induction of immunosuppressive macrophages because of Mycobacterium avium complex infection.

The role of some cytokines including tumour necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta) and interleukin-6 (IL-6) in the generation of immunosuppressive macrophages (M phi s) in host spleen cells of Mycobacterium avium complex (MAC)-infected mice was studied. M phi populations with potent suppressor activity against concanavalin A (Con A)-induced mitogenesis of splenocytes (SPCs) were elicited not only in euthymic but also in athymic nude mice during MAC infection. The suppressor M phi s are, therefore, inducible not only through a T-cell-dependent mechanism but also through T-cell-independent mechanism. However, MAC-induced M phi s of athymic mice displayed about four times lower suppressor activity than those of euthymic mice, indicating that mature T cells are important for M phi activation to the highly immunosuppressive state. Anti-TNF, anti-IFN-gamma, and anti-TGF-beta antibodies (Abs) but not anti-IL-6 Ab inhibited in vivo generation of MAC-induced immunosuppressive M phi s, and the neutralizing efficacy was in the order of anti-IFN-gamma Ab > anti-TNF Ab > anti-TGF-beta Ab. The effects of TNF-alpha, IL-1 alpha, IL-6, and IFN-gamma alone or combinations of them upon the acquisition of the suppressor activity by cultured splenic M phi s were studied. When normal splenic M phi s were treated with each cytokine for 3 days, TNF-alpha, IFN-gamma, and IL-1 alpha alone caused a slight elevation of their suppressive activity. Treatment of the normal M phi s with the combination of either TNF-alpha+IL-1 alpha or TNF-alpha+IFN-gamma yielded a marked increase in the suppressor activity, followed by IL-1 alpha+IFN-gamma. These findings indicate the important roles of TNF-alpha, IFN-gamma, and IL-1 alpha in the generation of MAC-induced suppressor M phi s.     

Relationship of tumor necrosis factor alpha, the nitric oxide synthase pathway, and lipopolysaccharide to the killing of gamma interferon-treated macrophage-like RAW264.7 cells by Rickettsia prowazekii.

Macrophage-like RAW264.7 cells are killed by the combination of gamma interferon (IFN-gamma) treatment and infection with Rickettsia prowazekii. The roles of tumor necrosis factor alpha (TNF-alpha), the nitric oxide synthase pathway, and lipopolysaccharide (LPS) in this killing were investigated. R. prowazekii, both the Breinl and Madrid E strains, induced RAW264.7 cells to produce TNF-alpha. However, dead rickettsiae (which cannot kill the IFN-gamma-treated RAW264.7 cells) induced the production of as much TNF-alpha as viable rickettsiae. Inhibition of the production of TNF-alpha (by the addition of actinomycin D or emetine during the rickettsial infection) or neutralization of TNF-alpha (by the addition of polyclonal rabbit anti-mouse TNF-alpha serum both during the IFN-gamma treatment and during the rickettsial infection) did not inhibit the killing of the RAW264.7 cells. Addition of polymyxin B (which inhibits many effects of LPS) during the IFN-gamma treatment did not inhibit the ability of IFN-gamma to prepare the RAW264.7 cells to be killed by R. prowazekii. Suppression of nitrite production by addition of the nitric oxide synthase inhibitor aminoguanidine both during the IFN-gamma treatment and during the rickettsial infection also did not inhibit the killing of the RAW264.7 cells. R. prowazekii-mediated killing of the RAW264.7 cells was dramatically suppressed in cultures treated with IFN-gamma plus LPS compared with that in cultures treated with IFN-gamma alone, and inhibition of nitric oxide synthase restored the rickettsia-induced killing of the RAW264.7 cells in cultures treated with IFN-gamma plus LPS. These data indicate that (i) TNF-alpha, LPS, and the nitric oxide synthase pathway are not required in order for IFN-gamma to prepare RAW264.7 cells to be killed by R. prowazekii; (ii) neither TNF-alpha nor the nitric oxide synthase pathway is responsible for the killing of the IFN-gamma-treated RAW264.7 cells by R. prowazekii; and (iii) in cultures treated with IFN-gamma plus LPS and then incubated with rickettsiae, a nitric oxide synthase pathway-dependent mechanism inhibits the killing of the RAW264.7 cells.     

Engagement of human monocyte-derived dendritic cells into interleukin (IL)-12 producers by IL-1beta + interferon (IFN)-gamma

Dendritic cells (DCs) are potent antigen-presenting cells and can induce tumour- or pathogen-specific T cell responses. For adoptive immunotherapy purposes, immature DCs can be generated from adherent monocytes using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4, and further maturation is usually achieved by incubation with tumour necrosis factor (TNF)-alpha. However, TNF-alpha-stimulated DCs produce low levels of IL-12. In this study, we compared the effects of TNF-alpha, interferon (IFN)-gamma, IL-1beta or IFN-gamma + IL-1beta on the phenotypic and functional maturation of DCs. Our results show that IFN-gamma, but not IL-1beta, augmented the surface expression of CD80, CD83 and CD86 molecules without inducing IL-12 production from DCs. However, IL-1beta, but not IFN-gamma, induced IL-12 p40 production by DCs without enhancing phenotypic maturation. When combined, IFN-gamma + IL-1beta treatment profoundly up-regulated the expression of CD80, CD83, CD86 and major histocompatibility complex (MHC) class II antigens. Furthermore, IFN-gamma + IL-1beta-treated DCs produced larger amounts of IL-12 and induced stronger T cell proliferation and IFN-gamma secretion in primary allogeneic mixed lymphocyte reaction (MLR) than did TNF-alpha-treated DCs. Our results show that IFN-gamma + IL-1beta induced human monocyte-derived DCs to differentiate into Th1-prone mature DCs.     

Tumor necrosis factor alpha mediates resistance to Trypanosoma cruzi infection in mice by inducing nitric oxide production in infected gamma interferon-activated macrophages.

Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for continuation of the parasite life cycle and for production of Chagas' disease. T. cruzi is able to replicate in nucleated cells and can be killed by activated macrophages. Gamma interferon (IFN-gamma) is one of the major stimuli for the activation of macrophages and has been shown to be a key activation factor for the killing of intracellular parasites through a mechanism dependent upon nitric oxide (NO) biosynthesis. We show that although the addition of exogenous tumor necrosis factor alpha (TNF-alpha) does not potentiate the trypanocidal activity of IFN-gamma in vitro, treatment of resistant C57BI/6 mice with an anti-TNF-alpha monoclonal antibody increased parasitemia and mortality. In addition, the anti-TNF-alpha-treated animals had decreased NO production, both in vivo and in vitro, suggesting an important role for TNF-alpha in controlling infection. In order to better understand the role of TNF-alpha in the macrophage-mediating killing of parasites, cultures of T. cruzi-infected macrophages were treated with an anti-TNF-alpha monoclonal antibody. IFN-gamma-activated macrophages failed to kill intracellular parasites following treatment with 100 micrograms of anti-TNF-alpha. In these cultures, the number of parasites released at various time points after infection was significantly increased while NO production was significantly reduced. We conclude that IFN-gamma-activated macrophages produce TNF-alpha after infection by T. cruzi and suggest that this cytokine plays a role in amplifying NO production and parasite killing.     

Temporal relationship of cytokine release by peripheral blood mononuclear cells stimulated by the streptococcal superantigen pep M5.

We undertook this study to determine the quality, quantity, and temporal relationship of pep M5-induced cytokine release. The ability of pep M5 to stimulate interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) production by a T-cell-depleted, monocyte- and B-cell-enriched cell population was dependent on the presence of T cells. The requirement for T cells could be met by addition of exogenous gamma interferon (IFN-gamma). In the presence of IFN-gamma, pep M5 induced the release of TNF-alpha, IL-1, and IL-6, TNF-alpha levels peaked at 24 h, while IL-1 and IL-6 levels peaked at 48 h. pep M5 induced T cells to produce IFN-gamma, which may have accounted for the ability of the super antigen to induce the production of IL-1, IL-6, TNF-alpha, and TNF-beta by peripheral blood mononuclear cells (PBMC). The addition of excess IFN-gamma to cultures of pep M5 and PBMC did not further increase the release of these cytokines at 24 and 48 h but resulted in sustained higher levels at 72 h. Interestingly, TNF-beta production occurred only in the presence of pep M5 and exogenous IFN-gamma. The ability of pep M5 to induce cytokine production was compared with that of a potent super antigen, staphylococcal enterotoxin B (SEB). SEB was a 2- to 14-fold-more-potent inducer of IFN-gamma production. Furthermore, the profile of cytokine released by PBMC in response to this super antigen mimicked that seen with pep M5 in the presence of exogenous IFN-gamma. In conclusion, pep M5 induces the production of cytokines that are involved in immune regulation and inflammation. These cytokines also play a major role in human T-cell responses to this super antigen.     

Activation of macrophage tumor cytotoxicity by the synergism of two T cell-derived lymphokines: immune interferon (IFN-gamma) and macrophage cytotoxicity-inducing factor 2 (MCIF2)

This report describes the role of T cell-derived lymphokines in induction of macrophage (M phi) tumor cytotoxicity. M phi tumor cytotoxicity was tested with the murine M phi-like tumor PU5-1.8. This M phi-like tumor reacts to stimulation with T cell-derived lymphokines and shows tumor cytotoxicity comparable to resident peritoneal M phi. The experiments demonstrate that immune interferon (IFN-gamma) secreted by T cell clones in limiting dilution microcultures was insufficient to induce M phi tumor cytotoxicity. Induction of M phi tumor cytotoxicity by T cell-derived supernatants with quantities of IFN-gamma undetectable in the IFN assay, however, was completely inhibited by an antiserum raised against recombinant IFN-gamma. Taken together, these results could be thus explained: (a) a T cell-derived lymphokine distinct from but serologically cross-reactive with IFN-gamma induces M phi tumor cytotoxicity, and (b) IFN-gamma activity in supernatants positive for induction of M phi tumor cytotoxicity escapes detection in the IFN assay but can still be inhibited by the anti-IFN-gamma antiserum in the M phi tumor cytotoxicity assay. The latter explanation requires positive evidence for another T cell-derived lymphokine inducing M phi tumor cytotoxicity together with IFN-gamma. This lymphokine was found in the supernatant of T cells in limiting dilution cultures and a long-term T cell clone and was called M phi cytotoxicity-inducing factor 2 (MCIF2). MCIF2 synergizes with IFN-gamma in induction of M phi tumor cytotoxicity; IFN-gamma and MCIF2 alone were ineffective.     

Production of tumor necrosis factor alpha by resident and activated murine macrophages.

Alveolar macrophages (Am phi s), resident peritoneal macrophages (RPm phi s), and thioglycolate-elicited peritoneal macrophages (TGPm phi s) were isolated from C57BL/6 mice and incubated with lipopolysaccharide (LPS), stimulated cell supernatant, or recombinant interferon gamma (IFN-gamma) for 24 h. Tumor necrosis factor (TNF) in cell-free supernatants was measured by enzyme-linked immunosorbent assay. Amo phi s incubated with 10(3) ng/ml LPS produced 50 times more TNF than RPm phi s and 5 times more than TGPm phi s, and LPS alone induced maximum TNF production by Am phi s. Stimulated cell supernatant or recombinant IFN-gamma alone did not induce TNF production. A combination of LPS with stimulated cell supernatant or IFN-gamma had only a limited synergistic effect on TNF production by Am phi s. However, both LPS and stimulated cell supernatant or recombinant IFN-gamma induced maximum TNF production by RPm phi s and TGPm phi s. TGPm phi s showed greater sensitivity to LPS and stimulated cell supernatant or IFN-gamma with regard to TNF production than the other macrophage populations investigated.     

Interleukin (IL)-4 production by human T cells: differential regulation of IL-4 vs. IL-2 production.

We have examined the regulation of interleukin (IL)-4 production by human peripheral blood T cells. Production of IL-4 was shown to be regulated differently from IL-2 and interferon(IFN)-gamma production. Stimulation of peripheral blood lymphocytes with anti-CD3, anti-CD2, anti-CD28, Phorbol 12-myristate 13-acetate (PMA) or IL-2 as a single stimulant did not induce IL-4 production. However, combinations of anti-CD2 with either anti-CD28 or IL-2 resulted in IL-4 production, peaking at days 3-4. Stimulation with anti-CD3 instead of anti-CD2 gave similar results, but was less potent. After days 3-4, IL-4 levels decreased, most likely due to consumption of IL-4. PMA profoundly affected cytokine production, it enhanced IL-2 production by at least tenfold, whereas, in the same cell population, IL-4 production was almost completely inhibited. This was observed at the protein as well as at the mRNA level. In contrast, agents that increase intracellular cAMP levels inhibited IL-2 production but left IL-4 production unaffected. IFN-gamma production behaved similar to IL-2 production but the effects were less outspoken.     

Role of inorganic nitrogen oxides and tumor necrosis factor alpha in killing Leishmania donovani amastigotes in gamma interferon-lipopolysaccharide-activated macrophages from Lshs and Lshr congenic mouse strains.

The capacity of mature bone-marrow-derived macrophages and resident peritoneal macrophages from Lshr versus Lshs congenic mice to kill intracellular Leishmania donovani amastigotes when activated by recombinant gamma interferon-lipopolysaccharide (rIFN-gamma-LPS) was examined. IFN-gamma alone in doses up to 100 U/ml was unable to activate macrophages to kill L. donovani amastigotes in vitro; LPS was a necessary secondary stimulus. Similarly, LPS alone in doses up to 100 ng/ml produced no leishmanicidal activity. In bone marrow macrophages, a dose-dependent increase in leishmanicidal activity was observed as increasing rIFN-gamma-LPS dose combinations were introduced, with Lshr macrophages maintaining a significant but not dramatic advantage within any particular dose combination. For peritoneal macrophages, the reverse was true, with macrophages from Lshs mice being more efficient at killing for doses of LPS up to 10 ng/ml with doses of rIFN-gamma in the range of 11 to 33 U/ml. The degree of killing in both bone marrow and peritoneal macrophages correlated well with the levels of nitrites measured in the supernatants at 72 h, and a highly significant correlation was observed between 4-, 24-, or 72-h tumor necrosis factor alpha (TNF-alpha) release and nitrite production measured at 72 h. Inclusion of 200 microM NG-monomethyl-L-arginine, a competitive inhibitor of the L-arginine-dependent pathway for the synthesis of inorganic nitrogen oxides, inhibited the killing, as did the addition of neutralizing anti-TNF-alpha antibody. These results are consistent with previous data showing an important autocrine role for TNF-alpha in enhancing production of inorganic nitrogen oxides by primed or activated macrophages. In addition, our results suggest that production of TNF-alpha and nitrites after priming or activation signals may be under a different regulatory control in mature bone marrow macrophages than in the resident peritoneal macrophage population.     

Ontogeny of 'macrophage' function. VI. Down-regulation for Ia-expression of newborn mouse macrophages by endogenous beta-interferon

Peritoneal exudate macrophages (M phi) of newborn mice (NB-M phi) were apparently almost incapable of expressing Ia antigen even if stimulated by IFN-gamma. No significant difference was observed in the number and the affinity of receptors for IFN-gamma between NB-M phi and M phi of adult mice (Ad-M phi). Addition of indomethacin, a prostaglandin synthesis inhibitor, was ineffective in enhancing the Ia-expression of NB-M phi. Responsiveness of NB-M phi to IFN-gamma, however, was disclosed by the addition to the culture of anti-IFN-beta or anti-IFN-alpha/beta, but not anti-IFN-alpha antibody. Responsiveness of NB-M phi to IFN-gamma was not improved by the depletion of fibroblasts from NB-M phi populations. These results strongly argue that Ia-expression of NB-M phi, which is otherwise to be induced by IFN-gamma, is suppressed by IFN-beta derived from NB-M phi themselves.