Substance P augments tumor necrosis factor release in human monocyte-derived macrophages.

Substance P (SP) is an undecapeptide that has the amino sequence Arg-Pro-Lys-Pro-Gin-Gln-Phe-Phe-Gly-Leu-Met-NH2 and that belongs to a family of structurally related peptides known as tachykinins, the latter are widely distributed in the central nervous system. SP is involved in the biological activities of cells in the immune system, including the induction of cytokines in immune cells. We have investigated the effects of SP on constitutive and/or lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF) in cultured blood monocyte-derived macrophages (MDM). Cells cultured in vitro for 14 days were treated with SP at various concentrations (10(-10) to 10(-6M) in the presence of LPS before culture supernatants were harvested. TNF bioactivity in culture supernatants was measured with L929 cell line MDM from 10 of 12 donors treated with a SP alone showed increased TNF production. SP and LPS also interacted in a synergistic fashion in upregulating TNF production in MDM from responders. The stimulatory effect of SP was inhibited by two SP antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-7-D-Trp-9-leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the SP receptor). In addition, an anti SP polyclonal antibody blocked the SP effect on TNF production in cultured MDM, further indicating the specificity of these effects. These results demonstrate that SP is an important regulator of monokine production by human monocytes/macrophages.     

Bacterial-lipopolysaccharide-induced release of lactoferrin from human polymorphonuclear leukocytes: role of monocyte-derived tumor necrosis factor alpha.

We have examined the role played by human peripheral blood monocytes in mediating responses of human polymorphonuclear leukocytes (PMN) to bacterial lipopolysaccharide (LPS) in vitro. When incubated with Salmonella typhimurium LPS at 37 degrees C, human PMN suspended in serum-free buffer released the specific granule constituent lactoferrin into the surrounding medium. Release of lactoferrin from PMN varied with the concentration of LPS (1 to 1,000 ng/ml) as well as with the duration of incubation (2 to 60 min) and was not accompanied by significant release of the cytoplasmic enzyme lactate dehydrogenase. LPS-induced release of lactoferrin from PMN was augmented significantly when cell suspensions were supplemented with additional monocytes and lymphocytes. Only monocytes, however, secreted significant amounts of lactoferrin-releasing activity (in a time- and concentration-dependent manner) when incubated separately with LPS. Lactoferrin-releasing activity was heat (80 degrees C for 15 min) labile, eluted after chromatography on Sephadex G-100 with an apparent molecular weight of approximately 60,000, and was inhibited by antibodies to tumor necrosis factor alpha. Thus, LPS-induced noncytotoxic release of lactoferrin from human PMN suspended in serum-free buffer is mediated, at least in part, by tumor necrosis factor alpha derived from contaminating monocytes.     

Human eosinophil peroxidase enhances tumor necrosis factor and hydrogen peroxide release by human monocyte-derived macrophages

Inhibition of growth or eradication of experimentally induced tumors has been shown to be accompanied by infiltration of eosinophils and macrophages into the tumor mass. Since macrophages are important mediators of host antitumor activity, the possibility arises that a collaboration may exist between these two cell types in the control of tumor growth. In this study, we report the effect of eosinophil peroxidase (EPO), a basic protein contained in eosinophils that binds to several cell types including macrophages, on tumor necrosis factor (TNF) production and hydrogen peroxide release by human monocyte-derived macrophages. After incubation with EPO, the macrophages produced large amounts of TNF and displayed an enhanced phorbol 12-myristate 13-acetate-triggered hydrogen peroxide release. These effects were accompanied by an increased cell protein content and by morphologic changes leading the large, round macrophages of the control cultures to become elongated, pear-like or spindle shaped cells after treatment with EPO. The stimulatory effect of EPO on hydrogen peroxide release was insensitive to addition of exogenous catalase, a H₂O₂-degrading enzyme, suggesting that an extracellular catalytic activity of EPO was not involved. In addition, myelo-peroxidase, the homologous peroxidase of neutrophils with a catalytic activity similar to that of EPO, was ineffective. The EPO-induced effects differed in several aspects from the effects of lipopolysaccaride and interferon-γ, two well-known macrophage activators. These findings provide supportive evidence for a functional interrelationship between eosinophils and macrophages that may be physiologically relevant in the tumoricidal activity of macrophages.     

Fungal beta-glucan interacts with vitronectin and stimulates tumor necrosis factor alpha release from macrophages.

beta-Glucans are polymers of D-glucose which represent major structural components of fungal cell walls. It was shown previously that fungi interact with macrophages through beta-glucan receptors, thereby inducing release of tumor necrosis factor alpha (TNF-alpha). Additional studies demonstrated that vitronectin, a host adhesive glycoprotein, binds to fungi and enhances macrophage recognition of these organisms. Since vitronectin contains a carbohydrate-binding region, we postulated that vitronectin binds fungal beta-glucans and subsequently augments macrophage TNF-alpha release in response to this fungal component. To study this, we first determined the release of TNF-alpha from alveolar macrophages stimulated with fungal beta-glucan. Maximal TNF-alpha release occurred with moderate concentrations of beta-glucan (100 to 200 micrograms/ml), whereas higher concentrations of beta-glucan (> or = 500 micrograms/ml) caused apparent suppression of the TNF-alpha activity released. This suppression of TNF-alpha activity by high concentrations of beta-glucan was mediated by the particulate beta-glucan binding soluble TNF-alpha, through the lectin-binding domain of the cytokine, rendering the TNF-alpha less available for measurement. Next, we assessed the interaction of vitronectin with beta-glucan. Binding of 125I-vitronectin to particulate fungal beta-glucan was dose dependent and specifically inhibitable by unlabeled vitronectin. Furthermore, treatment of beta-glucan with vitronectin substantially augmented macrophage TNF-alpha release in response to this fungal component. These findings demonstrate that fungal beta-glucan can directly modulate TNF-alpha release from macrophages. Further, these studies indicate that the host adhesive glycoprotein vitronectin specifically binds beta-glucan and augments macrophage cytokine release in response to this fungal element.     

Role of gamma interferon and tumor necrosis factor alpha in resistance to Salmonella typhimurium infection.

In mice infected with a sublethal dose of Salmonella typhimurium, the injection of an anti-gamma interferon (IFN-gamma) monoclonal antibody increased bacterial proliferation in the spleen and led to death on day 7 or 8. Depletion of both CD4+ and CD8+ T cells with monoclonal antibodies in vivo had a much less marked effect during the first week of infection than the administration of anti-IFN-gamma antibodies, suggesting that cells other than T lymphocytes participate in the production of IFN-gamma at this time. Administration of anti-tumor necrosis factor alpha (TNF-alpha) antibodies to mice infected with a sublethal dose of S. typhimurium induced the same effect as anti-IFN-gamma antibodies, while the administration of both antibodies resulted in a synergistic interaction. When mice were infected with an avirulent strain of S. typhimurium and challenged on day 7 either with a virulent strain of S. typhimurium or with Listeria monocytogenes, their resistance to reinfection was slightly depressed by anti-IFN-gamma or anti-TNF-alpha antibodies given 1 day before challenge and much more strongly depressed by the simultaneous administration of both antibodies. Taken together, these results indicate that IFN-gamma and TNF-alpha play an essential role in acquired resistance during the early phase of S. typhimurium infection.     

Simultaneous induction of interferon gamma and tumor necrosis factor alpha by different seleno-organic compounds in human peripheral blood leukocytes.

Ebselen is known as anti-inflammatory and anti-oxidant selenium containing drug. We have synthetized 13 seleno-organic compounds, analogs of ebselen. Seven of them were found to be inducers of interferon gamma (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) in human peripheral blood leukocytes (PBL) cultures. The most active cytokine inducers were: 2-phenyl-1,2-benzisoselenazol-3(2H)-one (1, ebselen), bis [2-(N-phenylcarbamoyl)]phenyl diselenide (7) and bis (2-[N-(2-pyridyl)carbamoyl])phenyl diselenide (8). The amounts of IFN and TNF produced by PBL cultures in response to the seleno-organic compounds were found to be similar to that induced by phytohemagglutinin (PHA). The activities of the seleno-organic compounds were dose-dependent and related to the chemical structure of the drugs suggesting involvement of the specific cytokine-inducer receptor. The simultaneous inductions of IFN-gamma and TNF-alpha were highly correlated, but independent on each other.     

Association between virulence of Yersinia pestis and suppression of gamma interferon and tumor necrosis factor alpha.

It is established that Yersinia pestis, the causative agent of bubonic plague, and enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica share a ca. 70-kb low-calcium response or Lcr plasmid (Lcr+). The latter is known to encode regulatory functions that restrict growth at 37 degrees C in Ca(2+)-deficient medium and virulence factors that are expressed only in vitro within this environment (e.g., certain Yops and V antigen). In this study, gamma interferon (IFN-gamma) was never detected in mice infected with 10 minimum lethal doses (MLD) of Lcr+ cells of Y. pestis, and significant levels of tumor necrosis factor alpha (TNF-alpha) arose only prior to death. Prompt and marked synthesis of these cytokines was observed upon infection with avirulent Lcr- mutants. Treatment of mice with exogenous IFN-gamma plus TNF-alpha inhibited multiplication of Lcr+ yersiniae in vivo, thereby providing protection against challenge with 10 MLD. Administration of both cytokines was required for absolute survival, suggesting a synergistic rather than cumulative interaction. This protective effect entailed cytokine priming as judged by subsequent detection of substantial levels of endogenous IFN-gamma and TNF-alpha. Monospecific anti-V-antigen, known to provide passive immunity against 10 MLD of Lcr+ Y. pestis, permitted significant synthesis of endogenous IFN-gamma and TNF-alpha. These findings demonstrate that Lcr+ yersiniae suppress synthesis of cytokines and suggest that this effect is mediated by one or more Lcr plasmid-encoded virulence factors.     

Interleukin-12-mediated resistance to Trypanosoma cruzi is dependent on tumor necrosis factor alpha and gamma interferon.

The aim of this study was to determine if interleukin-12 (IL-12) has a role in the immune response to Trypanosoma cruzi. Infection of BALB/c mice with the virulent Tulahuen strain of T. cruzi is characterized by a high-level parasitemia, pathology in the heart associated with the presence of amastigotes, and death during the acute phase of the disease. Administration of IL-12 to BALB/c mice infected with T. cruzi resulted in a reduced parasitemia and a significant delay in the time to death compared with those for infected controls. This protective effect was correlated with increased levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in serum. To determine if these cytokines were involved in the protective effects of IL-12, we treated infected mice with IL-12 alone or in combination with monoclonal antibodies specific for IFN-gamma or TNF-alpha. These antibodies antagonized the protective effect of exogenous IL-12. Treatment of infected mice with a polygonal antibody specific for IL-12 resulted in a significant increase in parasitemia but did not affect the time to death. These latter studies demonstrate a role for endogenous IL-12 in resistance to T. cruzi. Together, our data identify an IL-12-mediated mechanism of resistance to T. cruzi, which is dependent on IFN-gamma and TNF-alpha.     

Inhibition of type 2,5'-deiodinase by tumor necrosis factor alpha, interleukin-6 and interferon gamma in human thyroid tissue.

The effects of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6) and interferon gamma (IFNgamma) were studied on the activity of type 2,5'-deiodinase and on the binding of [125I] T(4) to proteins in human thyroid cytosolic (supernatant) and membrane (pellet) fractions. The activity of thyroid type 2,5'-deiodinase was measured by iodothyronine outer ring deiodinase assay. The binding of [125I] T(4) to the proteins of thyroid cytosolic and membrane fractions was determined by autoradiography. The results showed that thyroid type 2,5'-deiodinase activity could be detected also in the cytosolic fraction, not only in the membrane. TNFalpha, IL-6 and IFNgamma could inhibit the type 2 deiodinase activity in vitro. The dose-dependent binding of [125I] T(4) to the proteins of 29, 66 and 200 kDa could be observed both in the cytosolic and membrane fractions. TNFalpha and IFNgamma inhibited the binding of [125I] T(4) to the two characteristic proteins of type 2,5'-deiodinase, to proteins of 29 and/or 200 kDa, both in the cytosolic and membrane fractions. We conclude that TNFalpha, IL-6 and IFNgamma can inhibit the activity of type 2,5'-deiodinase. Furthermore, TNFalpha and IFNgamma can still also decrease the binding of [125I] T(4) to the enzyme in vitro. It can be suggested that the increased level of these cytokines can be one of the reasons in the induction of the clinical symptoms in nonthyroidal illness associated with low levels of T(3).     

Effects of recombinant gamma interferon and tumor necrosis factor on in vitro interactions of human mononuclear phagocytes with Coccidioides immitis.

Human peripheral blood monocytes readily phagocytized Coccidioides immitis endospores (2 to 5 microns) in vitro. Within 24 to 30 h at 37 degrees C, the phagocytized endospores started developing into immature spherules. However, when the monocytes were incubated with recombinant human gamma interferon (rIFN-gamma) or recombinant human tumor necrosis factor alpha (rTNF-alpha) and then infected, fewer endospores developed into spherules. Treatment with rIFN-gamma or rTNF-alpha activated the fungicidal capabilities of the monocytes as evidenced by the significant reduction in CFU that could be recovered from rIFN-gamma- or rTNF-alpha-activated monocytes compared with nontreated controls.     

Importance of endogenous tumor necrosis factor alpha and gamma interferon in host resistance against Pneumocystis carinii infection.

C.B-17 scid/scid (SCID) mice that have acquired natural pulmonary infection with Pneumocystis carinii clear these organisms by 19 days after reconstitution with spleen cells from immunocompetent mice and therefore serve as a model for studying the pathogenesis of and immunity to P. carinii pneumonia. The present study examined the importance of endogenous tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) in the clearance of P. carinii by treatment of reconstituted SCID mice with anti-TNF-alpha and anti-IFN-gamma immunoglobulin G (IgG). Treatment of reconstituted mice with monospecific rabbit anti-TNF-alpha IgG almost completely inhibited the clearance of P. carinii from the lungs. In contrast, treatment with either anti-IFN-gamma antibody (polyclonal or monoclonal) or control IgG had no detectable effect on the clearance of P. carinii. The importance of endogenous TNF-alpha in the clearance of P. carinii was further supported by the finding of TNF-alpha but not IFN-gamma in lung homogenate supernatants from reconstituted SCID mice. Further study revealed that for the complete clearance of P. carinii, TNF-alpha must be present at the early stage of reconstitution, since clearance could be blocked by a single injection of anti-TNF-alpha IgG into SCID mice at day 0 but not at day 6 and/or day 12 after reconstitution. These results strongly suggest that, in reconstituted SCID mice, endogenous TNF-alpha is important in host resistance against P. carinii infection, whereas IFN-gamma appears not to play a significant role.     

Two soluble antigens of Plasmodium falciparum induce tumor necrosis factor release from macrophages.

The production of cytokines such as tumor necrosis factor (TNF) may contribute to the pathology of malaria. We showed previously that crude preparations of heat-stable exoantigens from parasite cultures induce the release of TNF in vitro and in vivo. When separated from the culture medium by affinity chromatography, in which immune immunoglobulin G was used as ligand, the mixture of exoantigens of Plasmodium falciparum retained the capacity to induce the secretion of TNF, both by human monocytes from Gambian children and by mouse macrophages. Two individual antigens, Ag1 and Ag7, further purified by affinity chromatography and identified by crossed immunoelectrophoresis, also stimulated TNF production by both types of cell but differed in other functional properties. Thus, the activity of Ag7, but not that of Ag1, was inhibited by polymyxin B, and antisera made against boiled exoantigens of the rodent parasite Plasmodium yoelii which blocked the ability of these antigens to induce the production of TNF also inhibited the activity of Ag7 without affecting Ag1. Since the prevalence of antibody against Ag7 in sera from children in endemic areas appears to correlate with the development of immunity against the manifestations of the disease, this antigen may be one cause of pathology, perhaps through its ability to induce the production of TNF. Its serological relationship with rodent exoantigens suggests that it might be a candidate for an anti-disease vaccine which has the advantage that its active moiety is not subject to significant antigen polymorphism.     

Inhibition of Rickettsia conorii growth by recombinant tumor necrosis factor alpha: enhancement of inhibition by gamma interferon.

Purified human recombinant tumor necrosis factor alpha (rTNF-alpha) inhibited the growth of Rickettsia conorii (Casablanca strain) in HEp-2 cell culture. The effect was observed when the cells were pretreated with rTNF-alpha or when rTNF-alpha was added after adsorption of the rickettsiae. The inhibitory effect of rTNF-alpha on rickettsial growth was enhanced by gamma interferon. Cycloheximide had no effect on inhibition of the rickettsial yield, suggesting that de novo protein synthesis is not required for the inhibitory effect of rTNF-alpha. The addition of tryptophan partially abolished the inhibitory effect of rTNF-alpha and rTNF-alpha plus gamma interferon.     

Role of gamma interferon and tumor necrosis factor alpha during T-cell-independent and -dependent phases of Mycobacterium avium infection.

To design an effective immunotherapy for Mycobacterium avium infections, the protective host response to the infection must be known. Here we analyzed the role of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in the innate and acquired responses to M. avium infections in mice. T-cell depletion studies showed that CD4+ T cells were required for control of the infection. CD(4+)-depleted mice showed enhanced bacterial proliferation and at the same time showed a reduction in the level of expression of both IFN-gamma and TNF-alpha mRNAs in spleen cells. In contrast, M. bovis BCG immunization restricted M. avium proliferation and at the same time promoted expression of the mRNAs for the two cytokines. In vivo depletion studies using specific monoclonal antibodies showed that both IFN-gamma and TNF-alpha are involved in an early protection possibly involving NK cells, and furthermore, IFN-gamma is involved in the later T-cell-protective response to infection. In vivo neutralization of IFN-gamma during M. avium infection also blocked the priming for enhanced TNF-alpha secretion triggered by endotoxin. Both cytokines were found to be involved in the resistance expressed in BCG-immunized animals and exhibited additive bacteriostatic effects in vitro on bone marrow-derived macrophages infected with different strains of M. avium. These data suggest that both cytokines act in an additive or synergistic fashion in the induction of bacteriostasis and that IFN-gamma is also involved in priming TNF-alpha secretion.     

Inhibition of Chlamydia pneumoniae growth in HEp-2 cells pretreated with gamma interferon and tumor necrosis factor alpha.

An in vitro culture system was used to study the effects of increasing concentrations of human cytokines on the intracellular replication of Chlamydia pneumoniae. HEp-2 cell monolayers, pretreated for 24 h with 200 U of human recombinant gamma interferon (IFN-gamma) per ml restricted the intracellular replication of C. pneumoniae. Tumor necrosis factor alpha (TNF-alpha; 25 ng/ml) exhibited a synergistic effect with IFN-gamma by reducing the concentration of IFN-gamma necessary to restrict intracellular growth to 100 U/ml. The addition of 200 micrograms of tryptophan per ml significantly reversed the inhibitory effects of IFN-gamma and TNF-alpha, suggesting involvement of the indoleamine-2,3-dioxygenase pathway in the restriction process.     

Bikunin inhibits lipopolysaccharide-induced tumor necrosis factor alpha induction in macrophages

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-alpha) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-alpha expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-alpha protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 microM). (iii) Inhibition by bikunin of TNF-alpha induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-alpha release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-alpha production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.     

In situ production of gamma interferon, interleukin-4, and tumor necrosis factor alpha mRNA in human lung tuberculous granulomas

Human tuberculous granulomas from five adults undergoing surgery for hemoptysis were analyzed by nonradioactive in situ hybridization for tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), and interleukin-4 (IL-4) gene expression. All of the patients produced TNF-alpha mRNA. Three patients stained positive for both IFN-gamma and IL-4 mRNA; the other two stained positive for IFN-gamma but not IL-4 mRNA. Heterogeneity between the granulomas was observed in those patients staining positive for both IFN-gamma and IL-4 mRNA; these patients exhibited granulomas having IFN-gamma and not IL-4 mRNA as well as granulomas positive for both cytokine mRNAs. There was no evidence of caseation in these granulomas, and the cytokine patterns may represent events in the evolution of the granuloma. However, in those granulomas exhibiting caseous necrosis, very little IFN-gamma or IL-4 mRNA was observed, implying that progression of the granuloma is accompanied by a down regulation of T-cell responses. TNF-alpha mRNA expression was highest in patients with both IFN-gamma and IL-4 mRNA. Populations of CD68 positive macrophage-like cells within the granulomas produce mRNA for TNF-alpha, IFN-gamma, and IL-4. This implies that macrophages within the tuberculous granuloma may not be dependent on T-cell cytokines for modulation of their function but may be able to regulate their own activation state and that of the surrounding T cells. These findings have implications on the delivery of immunotherapies to patients with tuberculosis.     

Inhibition by delta-9-tetrahydrocannabinol of tumor necrosis factor alpha production by mouse and human macrophages.

Suppression by delta-9-tetrahydrocannabinol (THC) of tumor necrosis factor (TNF) production by macrophages has not been reported previously. The present study evaluated the effect in vitro of THC on soluble TNF-alpha production by cultured murine peritoneal macrophages. THC at 5 or 10 micrograms/ml added to medium [RPMI 1640 containing 10 ng LPS/ml, mouse IFN-gamma (100 u/ml), and 0.5% bovine serum albumin (BSA)] used to induce TNF significantly decreased TNF-alpha production by BALB/c mouse macrophages. Macrophages pretreated with THC at 0.1, 0.5, or 1.0 micrograms/ml in protein-free medium for 3 h at 37 degrees C, prior to TNF induction, also showed a decreased ability to produce TNF-alpha in a dose-dependent manner. Increasing the protein concentration from 0.5 to 5% BSA in the medium which was used to induce TNF prevented the inhibitory activity of THC. Human peripheral blood adherent cells treated with THC-containing medium produced less TNF-alpha than controls that were not exposed to THC. Thus, our data provide evidence that THC can inhibit TNF production by mouse and human macrophages. The drug's activity is concentration dependent and is related to the amount of serum protein in the medium used to induce this cytokine.     

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.