BCG-induced enhancement of endotoxin sensitivity in C3H/HeJ mice. II. T cell modulation of macrophage sensitivity to LPS in vitro

BCG infection induces a marked increase in LPS sensitivity in vivo and will render genetically defective, LPS hyporesponsive, C3H/HeJ mice almost as sensitive to LPS as normal mice. In this study, we have examined the endotoxin sensitivity of lymphocytes and macrophages from BCG infected mice in order to determine the cellular basis of this effect. We have found that the alteration in endotoxin sensitivity is mediated by a primary effect of BCG infection on T lymphocytes rather than on macrophages. Macrophages from «LPS sensitive», BCG-infected C3H/HeJ mice remain unresponsive to LPS when tested in vitro. However, when peritoneal T lymphocytes from these LPS »corrected» mice were cocultured with LPS unresponsive C3H/HeJ macrophages, a conversion to the LPS-responsive state was observed as manifested by the ability of the macrophages to produce LAF (IL 1) upon LPS stimulation. T cells from normally LPS-responsive or BCG-infected C3H/HeN mice, but not from control C3H/HeJ mice, were also able to render C3H/HeJ macrophages sensitive to LPS. This activity was not affected by treatment of the column-purified T cells with anti-macrophage serum plus complement, indicating that the response was not due to residual LPS-responsive macrophages contaminating the T cell preparations. The ability of the T cell suspension to render C3H/HeJ macrophages capable of producing LAF (IL 1) in response to LPS was abrogated by treatment of the T cell preparations with anti-Thy 1.2 plus complement. These findings establish the importance of T lymphocytes in regulating the LPS sensitivity of macrophages in BCG infected C3H/HeJ mice and support the concept that macrophage LPS responsiveness is dependent upon a certain state of macrophage activation which is regulated by lymphocytes.     

Effects of BCG infection on the susceptibility of mouse macrophages to endotoxin.

Mice infected intravenously with Mycobacterium bovis (BCG) are 100 to 1,000 times more sensitive to the lethal effects of bacterial lipopolysaccharides (LPS). Since BCG infection results in macrophage activation and LPS may cause pathophysiological effects through interaction with this cell type, it was of interest to determine whether macrophages from BCG-infected animals were more susceptible to the toxic effects of LPS in vitro. When LPS-susceptible, C57BL/6 mice were infected with BCG, a significant reduction in the 50% lethal dose of LPS was first observed after 7 days and persisted for several weeks. Macrophages from these animals had greatly increased susceptibility to LPS in vitro, which correlated with the development of acquired cellular resistance as determined by their ability to inhibit the growth of Listeria monocytogenes. In contrast, BCG infection of C3H/HeJ mice, a strain resistant to LPS, did not alter the 50% lethal dose of LPS for these animals or increase the sensitivity of their peritoneal macrophages to LPS in vitro. These results indicate that susceptibility of BCG-infected mice to the lethal effects of LPS parallels the susceptibility of their macrophages in vitro; release of vasoactive substances from LPS-susceptible activated macrophages in vivo may be, in part, responsible for lethality.     

Comparison of gamma interferon, tumor necrosis factor, and direct cell contact in activation of antimycobacterial defense in murine macrophages.

We compared the abilities of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and sensitized murine lymph node lymphocytes to activate syngeneic murine peritoneal macrophages to inhibit the growth of intracellular Mycobacterium bovis BCG in vitro. IFN-gamma could activate antimycobacterial defense only when added to macrophage cultures prior to their infection with BCG. TNF-alpha was without any effect. In contrast, BCG-sensitized lymphocytes could induce antimycobacterial defenses when added after macrophages had been infected with BCG. The cell-mediated effect required direct contact between effector lymphocytes and the targets (BCG-infected macrophages), as revealed in studies in which these cell populations were separated by a semipermeable membrane. Cyclosporin A, which inhibits the production of relevant macrophage-activating lymphokines, did not abrogate the ability of sensitized lymphocytes to activate antimycobacterial effects in infected macrophages. Furthermore, only BCG-sensitized lymphocytes, and not Listeria-sensitized lymphocytes, could activate the antimycobacterial effects. These lymphocytes were not cytotoxic to the infected macrophages. The presence of anti-TNF-alpha antibody in cocultures reduced the antimicrobial effects. We propose that the activation of antimycobacterial defense in macrophages can occur by direct physical contact with sensitized lymphocytes. This process may be due to lymphocyte membrane-associated TNF-alpha, as we previously demonstrated in our studies of antileishmanial defense.     

In vitro killing of Entamoeba histolytica trophozoites by interferon-gamma-activated mouse macrophages

The effect of murine interferon gamma (IFN-gamma) on macrophage activation for amoebicidal activity was examined. Peritoneal macrophages were harvested from C57BL/6 mice and preincubated with IFN-gamma and/or lipopolysaccharide (LPS). In vitro amoebicidal activity of these macrophages was determined by trypan blue exclusion test against a virulent strain of E. histolytica (IP:0682:1). It was found that in vitro amoebicidal activity was evident in macrophage monolayers treated with both IFN-gamma and LPS. Macrophages treated with IFN-gamma alone did not develop cytotoxic activity unless they were exposed to LPS as a second triggering signal. The ability of IFN-gamma to prime macrophages to respond to trigger signals of LPS and develop cytotoxicity increased with time of incubation, the highest response being observed after 24 h. There was a dose-dependent relationship between the concentrations of both IFN-gamma and LPS used to activate macrophages and the number of dead trophozoites. These data suggest that macrophages are important in host defense against amoebiasis.     

Macrophage polykaryon formation in vitro by peritoneal cells from mice given injections of sodium periodate.

Peritoneal macrophages from mice that have received two separate intraperitoneal injections of the sterile, soluble oxidant NaIO4 form macrophage polykaryons (MPs) in vitro, but peritoneal macrophage from untreated, peptone-treated, or mice infected with bacille Calmette-Guerin (BCG) do not. The polykaryons are noted after 18-24 hours of culture and continue to form over a 60-72-hour period. The MPs do not form if the macrophage density is less than 4 x 10(3)/sq mm. The polykaryons appear in vitro only in cultures with less than or equal to 1-5 ng/ml lipopolysaccharide (LPS) (amounts of LPS that commonly contaminate culture medium and serum). Hydrocortisone hemisuccinate (2.6 x 10(-9) M) inhibits MP formation in vitro. Lymphocytes do not influence the polykaryon formation, and supernatants from MP cultures do not cause fusion of other macrophages. Microcinephotography demonstrates fusion of the macrophages to form the large polykaryons, which are less motile than uninuclear macrophages. The polykaryons assume different forms; generally, the nuclei (mean, 16.8 nuclei/MP; range, 2-137 nuclei/MP) are centrally located, and the nuclear chromatin of all nuclei appears similar. The MPs phagocytize polystyrene spheres and glutaraldehyde-treated erythrocytes to the same degree as do uninuclear macrophages when determined as particles per nucleus (phagocytic index), but their phagocytic index of IgG-coated erythrocytes is decreased. Peritoneal macrophages from mice given double injections of NaIO4 are nontumoricidal in the absence of LPS, but LPS, in amounts sufficient to inhibit polykaryon formation, renders the macrophages tumoricidal. Populations of macrophages containing MPs formed over 3 days of cultures also respond to LPS or macrophage activating factor (MAF) to demonstrate enhanced tumoricidal activity.     

Increased numbers and functional activity of CD56+ T cells in healthy cytomegalovirus positive subjects

Mycobacterium indicus pranii (MIP) is an atypical mycobacterial species possessing strong immunomodulatory properties. It is a potent vaccine candidate against tuberculosis, promotes Th1 immune response and protects mice from tumours. In previous studies, we demonstrated higher protective efficacy of MIP against experimental tuberculosis as compared with bacillus Calmette–Guérin (BCG). Since macrophages play an important role in the pathology of mycobacterial diseases and cancer, in the present study, we evaluated the MIP in live and killed form for macrophage activation potential, compared it with BCG and investigated the underlying mechanisms. High levels of tumour necrosis factor-α, interleukin-12p40 (IL-12p40), IL-6 and nitric oxide were produced by MIP-stimulated macrophages as compared with BCG-stimulated macrophages. Prominent up-regulation of co-stimulatory molecules CD40, CD80 and CD86 was also observed in response to MIP. Loss of response in MyD88-deficient macrophages showed that both MIP and BCG activate the macrophages in a MyD88-dependent manner. MyD88 signalling pathway culminates in nuclear factor-κB/activator protein-1 (NF-κB/AP-1) activation and higher activation of NF-κB/AP-1 was observed in response to MIP. With the help of pharmacological inhibitors and Toll-like receptor (TLR) -deficient macrophages, we observed the role of TLR2, TLR4 and intracellular TLRs in MIP-mediated macrophage activation. Stimulation of HEK293 cells expressing TLR2 in homodimeric or heterodimeric form showed that MIP has a distinctly higher level of TLR2 agonist activity compared with BCG. Further experiments suggested that TLR2 ligands are well exposed in MIP whereas they are obscured in BCG. Our findings establish the higher macrophage activation potential of MIP compared with BCG and delineate the underlying mechanism.     

Cytotoxic action of macrophages activated by BCG on tumor target cellsin vitro

The activating action of liquid BCG vaccine, lyophilized BCG vaccine, and killed BCG cells on macrophages was studied. Activation of mouse peritoneal macrophages was identified by their cytotoxic actionin vitro on syngeneic tumor target cells labeled with⁵¹Cr. Macrophages obtained from mice two to three weeks after a single injection of liquid BCG vaccine were shown to have a cytotoxic action on tumor cells. A single injection of lyophilized BCG vaccine or killed BCG cells into mice did not cause activation of their macrophages. Two injections of liquid or lyophilized BCG vaccine caused activation of macrophages much earlier — three to five days after the second injection. Intraperitoneal injection of BCG activated macrophages to a greater degree than subcutaneous injection.Laboratory of Experimental Tumor Therapy, P. A. Gertsen Moscow Oncologic Research Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Timofeevskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 1, pp. 36–39, January, 1979.     

In vitro NADH-oxidase,NADPH-oxidase and myeloperoxidase activity of macrophages after Tinospora cordifolia (guduchi) treatment

It is believed that the enhanced microbicidal and tumoricidal capability of activated macrophages is related to the remarkable increase in the production of oxygen metabolites. Both the production of H2O2 and the oxidation of NAD(P)H are directly dependent upon NAD(P)H-oxidase. It has been established that the respiratory burst is due to activation of NAD(P)H-oxidase localised in the plasmalemma. Myeloperoxidase is believed to be involved in augmenting the cytotoxic activity of H2O2. It was observed that the macrophage cell line J774A.1 when treated with Tinospora cordifolia (guduchi) and LPS showed enhanced NADH-oxidase, NADPH-oxidase and myeloperoxidase production as compared to macrophages treated with medium alone. The direct drug treatment to J774A cells showed activation as assessed by biochemical assays. These results suggest that high NADH-oxidase, NADPH-oxidase and myeloperoxidase activities may account for tumoricidal and microbicidal properties via macrophage activation.     

Tumor cell killing and cytostasis by C3H/HeJ macrophages activated in vitro by lipid A-associated protein and interferon gamma

Experiments have been carried out to assess the ability of purified protein-free lipopolysaccharide (LPS) and lipid A-associated protein (LAP) containing LPS to activate macrophages from C3H/HeJ endotoxin-unresponsive mice. Assays for in vitro activation have included cytotoxic and cytostatic effects on a simian virus 40 (SV40)-transformed fibroblast cell line. While neither preparation of LPS would effect C3H/HeJ macrophage activation for either cytostasis or cytolysis, the addition of murine interferon gamma to cultures of LAP-LPS stimulated C3H/HeJ macrophages resulted in cells which were both cytotoxic and cytostatic. These results suggest that LAP can provide one component of the triggering mechanism but, of itself, is insufficient to effect full activation. A second signal, which can be provided by murine interferon gamma, appears also to be required.     

Role of resident macrophages in canatoxin-induced in vivo neutrophil migration

Canatoxin (Cntx), a toxic protein purified fromCanavalia ensiformis seeds, was shown to have lipoxygenase-mediated effects either in vivo or in vitro. Data here show that Cntx induced a dose-dependent migration of neutrophils and mononuclear cells when injected into rat peritoneal cavities. Furthermore, Cntx was able to induce neutrophil migration into pleural cavities and into air pouches. These effects were inhibited by dexamethasone but not by inhibitors of arachidonic acid metabolism (indomethacin, NDGA, and BW-755c) or by a PAF antagonist (BN 52021). In the peritoneal cavity Cntx caused an increase in vascular permeability inhibited by dexamethasone and BW-755c. Neutrophil migration induced by this toxin was dependent on the number of resident macrophages, since the migratory effect was enhanced by increasing the peritoneal macrophage population with thioglycollate pretreatmen; and was diminished when this population was reduced by peritoneal wash. It was also observed that Cntx induced release of a chemotactic factor from macrophage monolayers in vitro. Dexamethasone blocked this release but did not affect in vivo neutrophil recruitment induced by that factor. These data suggest that Cntx-induced neutrophil migration may be mediated by the same macrophage-derived neutrophil chemotactic factor released by other stimuli such as LPS, IL-1, and INF-gamma.     

Prostaglandin E2 down-regulates viable Bacille Calmette-Guérin-induced macrophage cytotoxicity against murine bladder cancer cell MBT-2 in vitro

The regulatory effect of prostaglandin (PG) E2 and a cyclooxygenase (COX) inhibitor on Bacille Calmette-Guérin (BCG)-induced macrophage cytotoxicity in a bladder cancer cell, MBT-2, was studied in vitro. BCG stimulated thioglycollate-elicited murine peritoneal exudate cells (PEC) to induce cytotoxic activity and to produce cytokines such as interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and PGE2. NS398, a specific COX-2 inhibitor, and indomethacin (IM), a COX-1 and COX-2 inhibitor, enhanced viable BCG-induced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. However, NS398 and IM did not enhance these activities induced by killed BCG. Enhanced cytotoxicity was mediated by increased amounts of IFN-gamma and TNF-alpha. Exogenous PGE2 reduced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. These results suggest that PGE2 produced by BCG-activated macrophages has a negative regulatory effect on the cytotoxic activity of macrophages. Accordingly, a PG synthesis inhibitor may be a useful agent to enhance BCG-induced antitumour activity of macrophages.     

Human alveolar macrophage function: differences between smokers and nonsmokers

Human alveolar macrophages and peripheral blood monocytes were obtained from smoking and nonsmoking normal volunteers. The macrophages and monocytes were incubated in vitro with bacterial lipopolysaccharide (LPS). The oxidative metabolic response of these cells was measured by superoxide anion production. Macrophages from smokers were suppressed in their superoxide anion response to LPS activation as compared to macrophages from nonsmokers. Monocytes from smokers and nonsmokers were not different. The cytotoxic properties of these macrophages and monocytes were assessed by an in vitro 3H-thymidine release assay against various allogeneic target cells. Macrophages and monocytes exposed to LPS were rendered tumoricidal. Macrophages from nonsmokers appeared to generate greater cytotoxic activity than macrophages from smokers. Macrophages from both smokers and nonsmokers were cytotoxic for three different tumorigenic cell lines but not for a nontumorigenic cell line. Monocytes from smokers and nonsmokers were not different in cytotoxic activity. We conclude that macrophages from both smokers and nonsmokers can be activated after exposure to LPS; however, macrophages from smokers may be slightly suppressed in their responses.     

Factors mediating lipopolysaccharide-induced ganglioside expression in murine peritoneal macrophages

The ganglioside composition of endotoxin-responsive C3H/HeN murine peritoneal macrophages is known to undergo dramatic changes in vivo in response to intraperitoneal lipopolysaccharides (LPS), unlike endotoxin-hyporesponsive C3H/HeJ macrophages. To better investigate the mechanism behind LPS-induced macrophage ganglioside changes, resident C3H/HeN peritoneal macrophages were treated in vitro with 0.1-1.0 micrograms/ml LPS for 6-96 hr, but showed no differences in membrane ganglioside patterns. Coincubation of macrophages with lymphocytes and treating with LPS again elicited no ganglioside changes. In contrast, interferon gamma (IFN-gamma)-primed macrophages showed a dramatic shift in intensity of one ganglioside when treated with LPS in vitro; an additional macrophage ganglioside appeared when IFN-gamma-primed, LPS-treated macrophages were coincubated with lymphocytes. Ganglioside expression induced in vitro still did not approach the complex changes seen in vivo. However, transplanting C3H/HeN macrophages intraperitoneally into C3H/HeJ mice, followed by administration of intraperitoneal LPS, did reveal striking changes in ganglioside expression that resembled the pattern seen in vivo. Thus, LPS alone does not provide the necessary direct signal to promote macrophage ganglioside change even though it alters macrophage function. IFN-gamma appears to be one important mediator; however, complex interactions involving other cytokines or migration of independent populations of mononuclear cells may be required for the full manifestation of LPS-induced ganglioside expression in macrophages.     

Macrophages as a source of tumoricidal activity (tumor-necrotizing factor).

Macrophage-enriched peritoneal exudate cells from mice infected with Mycobacterium bovis BCG, macrophage-like tumor cells (PU 5-1.8), and peritoneal macrophages propagated in vitro with macrophage growth factor released tumoricidal activity into the culture medium within 2 to 3 h after stimulation with nanogram quantities of bacterial lipopolysaccharide. The cytotoxic activities from each of the macrophage culture supernatants eluted from diethylaminoethyl-Sephacel columns at a sodium chloride concentration of 200 mM exhibited a molecular weight of 50,000 to 60,000 as estimated by gel filtration, were stable at 56 degrees C for 30 min, and were active at a pH range of 6 to 10. A rabbit antiserum directed against serum-derived cytotoxic activity (tumor-necrotizing factor) from BCG-infected and lipopolysaccharide-challenged mice inhibited all of the cytotoxic activities generated in vitro. This suggests that the macrophage-derived cytotoxins are identical with serum-derived cytotoxic factor, which further implies that the macrophage is the cellular source of tumor-necrotizing factor.     

髓样细胞表达的触发受体1抑制LPS应激下巨噬细胞的自噬

目的　观察髓样细胞表达的触发受体1（TREM-1）对脂多糖（LPS）应激下巨噬细胞自噬相关基因表达的影响。 方法　观察LPS应激下的巨噬细胞TREM-1蛋白的表达。分别选用TREM-1的激动剂（MAB1187）和TREM-1的拮抗剂（LR12）作用于巨噬细胞,利用qPCR检测巨噬细胞自噬相关基因ATG7、ATG5、ATG12及自噬标志蛋白微管相关蛋白1轻链3（LC3）mRNA的表达;采用Western Blot检测巨噬细胞TREM-1、LC3Ⅱ/Ⅰ、ATG7蛋白的表达;采用免疫荧光检测在LPS应激与TREM-1激活的情况下,巨噬细胞的自噬标志蛋白LC3表达。 结果　LPS（400 ng/mL、1000 ng/mL）应激下巨噬细胞TREM-1蛋白表达明显增加;LPS（1000 ng/mL）作用巨噬细胞24 h,巨噬细胞TREM-1蛋白表达达到峰值。LPS应激的巨噬细胞自噬基因ATG7、ATG5、ATG12及自噬标志分子LC3 mRNA表达均降低;当给予TREM-1拮抗剂后,巨噬细胞的ATG7、ATG5、ATG12、LC3 mRNA表达均升高,而采用TREM-1激动剂后,自噬基因表达被抑制。Western Blot检测结果显示,TREM-1可抑制LPS应激下巨噬细胞LC3 Ⅱ/Ⅰ、ATG7蛋白的表达。免疫荧光检测表明TREM-1激动剂可使巨噬细胞胞内的LC3蛋白表达量减少。 结论　巨噬细胞胞膜上TREM-1激活后,可使巨噬细胞自噬减弱,提示LPS应激下的巨噬细胞TREM-1可能通过抑制巨噬细胞的正常自噬从而发挥炎症放大作用。     

Inactivation of TMEM106A promotes lipopolysaccharide‐induced inflammation via the MAPK and NF‐κB signaling pathways in macrophages

Pattern recognition receptors, such as Toll‐like receptors (TLRs), play an important role in the host defense against invading microbial pathogens. Their activation must be precisely regulated, as inappropriate activation or overactivation of TLR signaling pathways may result in inflammatory disorders, such as septic shock or autoimmune diseases. TMEM106A is a type II transmembrane protein constitutively expressed in macrophages. Our current study demonstrated that TMEM106A levels were increased in macrophages upon lipopolysaccharide (LPS) stimulation, as well as in the peripheral monocytes of patients with sepsis. Tmem106a knockout mice were more sensitive to lipopolysaccharide (LPS)‐induced septic shock than wild‐type mice. Further experiments indicated that Tmem106a ablation enhanced the expression of CD80, CD86 and major histocompatibility complex (MHC)‐II in mouse macrophages upon LPS stimulation, accompanied with up‐regulation of tumor necrosis factor (TNF)‐α, interleukin (IL)‐6, interferon (IFN)‐β and inducible nitric oxide synthase (iNOS), indicating the activation of macrophages and polarization towards the M1 inflammatory phenotype. Moreover, elevated mitogen‐activated protein kinase (MAPK) and nuclear factor kappa B (NF‐κB) signaling were found to be involved in the LPS‐induced inflammatory response in Tmem106a^?/? macrophages. However, this effect was largely abrogated by macrophage deletion in Tmem106a^?/? mice. Therefore, deficiency of Tmem106a in macrophages may enhance the M1 polarization in mice, resulting in inflammation. This suggests that TMEM106A plays an important regulatory role in maintaining macrophage homeostasis.     

The induction of lymphocytes with the capacity to render macrophages cytotoxic in an allogeneic murine system.

Sensitized spleen and peripheral lymph node lymphocytes were tested after different types of immunization with allogeneic tumour cells for their capacity to induce macrophage cytotoxicity in vitro. The macrophages were rendered cytotoxic either by direct contact with lymphocytes and tumour cells (activation of macrophages) or by a factor (macrophage arming factor, MAF), released by the sensitized lymphocytes incubated with tumour cells (arming of macrophages). Both types of reactions are T-cell dependent. Macrophage activation is a more sensitive way to detect lymphocytes with the capacity to render macrophages cytotoxic than arming of macrophages. The route of immunization subcutaneously (s.c.) or intraperitoneally (i.p.) with allogeneic cells did not influence the induction of lymphocytes with the capacity to render macrophages cytotoxic. However, the tumour cells had to be intact as disrupted cells (suspended in Freund's complete adjuvant, FCA) did not induce macrophages activating lymphocytes. The adjuvant dimethyl dioctadecyl ammonium bromide (DDA) did not increase the lymphocyte response. Intact allogeneic tumour cells were needed in vitro when used for secondary antigenic stimulation. This secondary stimulation was independent of antigen presentation by macrophages. This suggests that also in vivo the primary response is independent of macrophage antigen presentation. Delayed-type hypersensitivity and antibody responses against the allogeneic tumour cells were comparable after s.c. and i.p. immunization and after immunization with FCA and DDA.     

Functional comparison of bone marrow-derived macrophages obtained by cultivation in serum-free or serum-supplemented medium

Bone marrow-derived macrophages obtained by cultivation in a serum-free or in a serum-supplemented medium were compared in terms of the activation of the respiratory burst and the activation of tumor cytotoxicity. Serum-free-cultured macrophages responded to interferon-gamma (IFN-gamma) and to lipopolysaccharide (LPS) by an enhancement of the respiratory burst. Macrophages obtained in a serum-supplemented medium are characterized by a diminished capacity to release O2-. These cells did not respond to IFN-gamma unless the stimulation was performed in a serum-containing medium. In terms of activation of tumor cell cytotoxicity, serum-supplemented macrophage cultures seem to be primed by unknown serum constituents because they only need one signal (IFN-gamma or LPS) to become fully cytotoxic. Serum-free cultivated macrophages can be rendered cytotoxic only after exposure to combinations of IFN-gamma and LPS.     

Macrophage functions in Biozzi mice.

The faster degradation of antigen by macrophages in Biozzi low (L) responder mice, compared to Biozzi high (H) responder mice, is thought to be responsible for their lower antibody response. We have measured four functions associated with macrophages to see whether macrophages from L mice were generally more active than those from H mice. Peritoneal macrophages obtained from normal mice were compared with those from groups of mice given Mycobacterium bovis BCG or Propionibacterium acnes. Cells from normal H mice gave a stronger oxidative burst when triggered with phorbol myristate acetate, and were more cytotoxic for tumour cells than cells from L mice. Cells from all mice injected with BCG or P. acnes gave a stronger oxidative burst, and were more cytotoxic for tumour cells; again, both responses were higher in H mice than in L mice. By contrast, when groups of mice that had received P. acnes were given endotoxin and bled, higher titres of tumour necrosis factor were found in the sera of L mice. Spleen cells from both lines of mice released similar levels of interleukin-1, both spontaneously and in response to lipopolysaccharide. Our results suggest that these various macrophage responses are expressed independently in H and L mice.     

Effect of lipoarabinomannan and mycobacteria on tumour necrosis factor production by different populations of murine macrophages.

Tumour necrosis factor (TNF) production is an important pathological mediator in mycobacterial infections, and yet little is known of the factors which influence its production. We have studied the influence of murine macrophage heterogeneity and activation state on TNF production following mycobacterial stimulation in vitro. Lipoarabinomannan (LAM) from strains of Mycobacterium tuberculosis and Myco. avium differentially stimulated TNF production in thioglycollate-elicited macrophages in a dose-dependent manner. In comparison, resident peritoneal macrophages produced much less TNF when stimulated with LAM, dead mycobacteria or lipopolysaccharide (LPS). In contrast, zymosan stimulated resident macrophages to a higher degree than thioglycollate-elicited cells. Another comparison between bone marrow and thioglycollate-elicited macrophages showed that both responded to LPS, but only the latter was stimulated significantly by H37Rv LAM. This may indicate that LAM stimulation of macrophages takes place through a different pathway than both zymosan- and LPS-stimulated TNF production. Also, in vitro activation of peritoneal macrophages with interferon-gamma (IFN-gamma), increased TNF response to several stimuli. Our studies indicate that the pathology of mycobacterial infections through TNF production may be influenced by the type and activation state of the macrophage which responds to that infection.