Expression of I-A by macrophages from Bcgr and Bcgs mice. Transient expression of I-A is due to degradation of MHC class II glycoproteins

Murine peritoneal macrophages from mice that are resistant to Mycobacterium bovis (strain BCG) can be induced to continuously express MHC class II (Ia) glycoproteins. In contrast, macrophages from BCG susceptible mice will only transiently express Ia. The purpose of this investigation was to determine the biochemical basis of continuous or transient Ia expression. We therefore compared Ia biosynthesis by macrophages from C.D2Bcgr mice to that by macrophages from congenic BALB/c.Bcgs mice. We show that macrophages from both strains of mice synthesize Ia initially and that very little synthesis occurs after 5 days of in vitro culture. No differences in the amount of Ia synthesized by the macrophages was apparent as determined by quantitative immunoprecipitation and by ELISA. Despite the lack of synthesis, macrophages from C.D2Bcgr mice continue to express Ia. The results of pulse-chase experiments indicate that macrophages from BALB/c.Bcgs mice degrade newly synthesized MHC class II glycoproteins whereas the majority of Ia remains associated with macrophages from the C.D2Bcgr mice. The addition of chloroquine to cultures of macrophages from BALB/c.Bcgs mice prevented the degradation and prolonged the expression of Ia. The results of this investigation show that there are no differences in the synthesis of Ia by macrophages from the two congenic strains of mice. The transient expression of Ia by macrophages from BALB/c.Bcgs mice is due to its degradation.     

Regulation of I-A expression by murine peritoneal macrophages: differences linked to the Bcg gene

We previously reported that Mycobacterium bovis (strain BCG) induces continuous I-A expression when injected into BCG-resistant strains of mice. We have extended this observation by showing that Corynebacterium parvum also induces continuous I-A expression by macrophages from BCG-resistant but not BCG-susceptible mice. We have linked continuous expression to BCG resistance by using C.D2Ityr mice, which are congenic with BCG-susceptible BALB/c mice except for genes on a portion of chromosome 1, which contains the gene(s) for BCG resistance. Macrophages from C.D2Ityr mice continuously expressed I-A, whereas macrophages from BALB/c mice transiently expressed I-A. Continuous expression by macrophages from both Bcgr and Bcgs mice could be induced in vitro with rIFN-gamma. However, the continuous expression of I-A by macrophages from Bcgs mice required the continued presence of IFN-gamma, whereas that by Bcgr macrophages did not. The continuous expression of I-A by macrophages from Bcgs mice was also inhibited by hydrocortisone, cyclohexamide, tunicamycin, and monensin, whereas I-A expression by Bcgr macrophages was not affected. The continuous expression of I-A by macrophages from Bcgr mice did not require its continued synthesis. The significance of these findings to the induction of immunity and to antimicrobial resistance are discussed.     

Pleiotropic effects of the Bcg gene. I. Antigen presentation in genetically susceptible and resistant congenic mouse strains

The Ag-presentation ability of Bcgr and Bcgs spleen cells was studied in two sets of Bcg-congenic systems; namely, the BALB/c-BALB/c.Bcgr pair and the B10.A-B10.A-Bcgr pair, by using three sonicated soluble bacterial Ag (mycobacterium bovis bacillus Calmette-Guérin, Salmonella typhimurium, and Brucella abortus) as well as a particulate Ag (heat-killed Escherichia coli). Pulsed Bcgr spleen cells were shown to induce a stronger proliferation of the T cell-indicator system than their Bcgs counterparts. No difference in Ag-presenting ability could be shown between Bcgr and Bcgs peritoneal macrophages from normal animals. However, elicited peritoneal macrophages from immune Bcgr mice were superior in their Ag-presentation ability. Differences at the level of Ag presentation of Bcgr and Bcgs splenic cells were investigated further. Depletion of T cells and B cells did not alter the differences in Ag-presenting ability between Bcgr and Bcgs spleen cells. Furthermore, splenic dendritic cells of Bcgr or Bcgs allelic types were equally efficient in presenting bacillus Calmette-Guérin Ag to accessory cell-depleted T cells. In a final experiment, it was shown that spleen macrophages were the cell type involved in the superior Ag presentation by Bcgr splenic cells.     

Pleiotropic effects of the Bcg gene. II. Genetic restriction of responses to mitogens and allogeneic targets

The response of Bcgr and Bcgs spleen cells to allogeneic Ag, mitogens, and in a system of oxidative mitogenesis using neuraminidase and galactose oxidase was investigated in two Bcg congenic systems. The Bcgr macrophages supported the MLR across H-2 barrier much better than the Bcgs macrophages. At sub-optimal or optimal doses of mitogens Bcgr mice were higher responders than their Bcgs counterparts. The superior response of Bcgr spleen cells to Con A was further investigated with the aim of identifying the population expressing this phenotype. T cells of either Bcgr or Bcgs type showed equal ability to respond to Con A in the presence of macrophages. Purified splenic macrophages from Bcgr mice contained a significantly greater percentage of Ia+-bearing macrophages compared to Bcgs mice. Splenic macrophages of the Bcgr type were more efficient than their Bcgs counterparts at restoring the Con A response of accessory cell-depleted spleen cells. Resident peritoneal macrophages as well as splenic dendritic cells from Bcgr and Bcgs mice were equally efficient at restoring this response. Glutaraldehyde-fixed Bcgr splenic macrophages were shown to be more efficient than the Bcgs cells at replenishing the response of Con A-unresponsive spleen cells when supplemented with IL-1.     

Phenotypic expression of genetically-controlled natural resistance to Mycobacterium bovis (BCG)

Mycobacterium bovis (BCG), when maintained in vitro, readily incorporates [3H]uracil, the RNA precursor. The rate of [3H]uracil incorporation into bacilli is sharply reduced when the BCG is phagocytized by murine adherent resident peritoneal macrophages and subsequently released by the lysis of monolayers. Macrophages derived from mouse strains that are innately resistant to BCG infection in vivo (Bcgr) are able to inhibit the [3H]uracil incorporation into the bacilli in a significantly more effective way than macrophages from BCG-susceptible (Bcgs) strains. This difference is best demonstrated with a low rate of infection (BCG: macrophage ratio between 1:1 and 2:1), and is most pronounced at 4 to 5 days after in vitro infection of macrophage monolayers. In vivo interaction of BCG with peritoneal macrophages in situ results in the same pattern of enhanced inhibition of [3H]uracil incorporation by Bcgr macrophages. The use of Bcg-congenic mouse strains has confirmed that the Chromosome 1 Bcg (Ity, Lsh) locus is regulating the antimycobacterial activity of macrophages. We conclude that the resident macrophage is the cell population that expresses the phenotype of genetically determined resistance to BCG infection.     

Regulation of class II MHC gene expression by macrophages from Bcgr and Bcgs mice

The presence of class II mRNA was determined following stimulation of macrophages from Bcgr and Bcgs mice with rIFN-gamma. Despite the continuous expression of surface I-A glycoprotein by macrophages from Bcgr mice, class II mRNA was no longer present. The transient expression of I-A by macrophages from Bcgs mice, however, was accompanied by the disappearance of class II mRNA from the cells. Restimulation of macrophages from Bcgs mice, with rIFN-gamma resulted in the reappearance of class II mRNA and surface I-A expression. The reappearance of class II mRNA and the surface expression of I-A glycoprotein was inhibited by PGE2. These results indicate that differences in I-A expression by macrophages from Bcgr and Bcgs are not at the level of class II gene expression.     

Somatic cell hybrids between macrophages from Bcgr and Bcgs mice: characterization of MHC class II expression

In order to gain a better understanding of the regulation of MHC class II expression related to the Bcg gene, we have produced macrophage-macrophage somatic cell hybrids by fusing the RAW 309 macrophage cell line derived from BALB/c.Bcgs mice with peritoneal macrophages from Bcgr C3H/HeN mice. The differential screening of the hybrids was based on the differential sensitivity of Ia expression to suppression with cycloheximide. We found that most of the hybrids expressed Ia without further stimulation with rIFN-gamma. Cycloheximide suppressed the expression of Ia by some of the hybrids. Treatment of these cells with rIFN-gamma resulted in a cycloheximide resistant Ia expression of both parental haplotypes. The macrophage hybrids produced IL-1 beta, IL-1 alpha, and TNF-alpha when stimulated with LPS. There was no correlation between the levels of monokines produced and the persistence of Ia expression. The results of this investigation indicate that the product of the Bcg gene contributed by macrophages from C3H/HeN mice will affect the expression of the I-Ad glycoprotein that is normally transiently expressed by the RAW 309 cell line.     

Mechanisms of enhanced macrophage-mediated prostaglandin E2 production and its suppressive role in Th1 activation in Th2-dominant BALB/c mice

PGE(2) has been known to suppress Th1 responses. We studied the difference in strains of mice in PGE(2) production by macrophages and its relation to Th1 activation. Macrophages from BALB/c mice produced greater amounts of PGE(2) than those from any other strains of mice, including C57BL/6, after LPS stimulation. In accordance with the amount of PGE(2) produced, macrophage-derived IL-12 and T cell-derived IFN-gamma production were more strongly suppressed in BALB/c macrophages than in C57BL/6 macrophages. When macrophages were treated with indomethacin or EP4 antagonist, Th1 cytokines were more markedly increased in cells from BALB/c mice than in those from C57BL/6 mice. Although cyclooxygenase-2 was expressed similarly after LPS stimulation in these mouse strains, the release of arachidonic acid and the expression of type V secretory phospholipase A(2) mRNA were greater in BALB/c macrophages. However, exogenous addition of arachidonic acid did not reverse the lower production of PGE(2) by C57BL/6 macrophages. The expression of microsomal PGE synthase, a final enzyme of PGE(2) synthesis, was also greater in BALB/c macrophages. These results indicate that the greater production of PGE(2) by macrophages, which is regulated by secretory phospholipase A(2) and microsomal PGE synthase but not by cyclooxygenase-2, is related to the suppression of Th1 cytokine production in BALB/c mice.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.     

Mouse strain susceptibility to trypanosome infection: an arginase-dependent effect

We previously reported that macrophage arginase inhibits NO-dependent trypanosome killing in vitro and in vivo. BALB/c and C57BL/6 mice are known to be susceptible and resistant to trypanosome infection, respectively. Hence, we assessed the expression and the role of inducible NO synthase (iNOS) and arginase in these two mouse strains infected with Trypanosoma brucei brucei. Arginase I and arginase II mRNA expression was higher in macrophages from infected BALB/c compared with those from C57BL/6 mice, whereas iNOS mRNA was up-regulated at the same level in both phenotypes. Similarly, arginase activity was more important in macrophages from infected BALB/c vs infected C57BL/6 mice. Moreover, increase of arginase I and arginase II mRNA levels and of macrophage arginase activity was directly induced by trypanosomes, with a higher level in BALB/c compared with C57BL/6 mice. Neither iNOS expression nor NO production was stimulated by trypanosomes in vitro. The high level of arginase activity in T. brucei brucei-infected BALB/c macrophages strongly inhibited macrophage NO production, which in turn resulted in less trypanosome killing compared with C57BL/6 macrophages. NO generation and parasite killing were restored to the same level in BALB/c and C57BL/6 macrophages when arginase was specifically inhibited with N(omega)-hydroxy-nor-L-arginine. In conclusion, host arginase represents a marker of resistance/susceptibility to trypanosome infections.     

Interleukin 1alpha activity of peritoneal and bone marrow macrophages infected with Leishmania major and Leishmania donovani in vitro

In this study, the pattern of interleukin-1alpha (IL-1alpha) production by both peritoneal (PM) and bone marrow macrophages (BMM) from resistant (C3H/HeJ) and susceptible (BALB/c) mice was investigated, using a bioassay and an IL-1alpha-specific ELISA kit. PM from normal uninfected mice showed either an initial high (C3H/HeJ) or a neglected (BALB/c) level of IL-1alpha activity, respectively, probably due to thioglycollate stimulation. Infection with Leishmania major induced only a marginal effect on IL-1 production by both cells. Normal, uninfected and unstimulated BMM from both mice did not produce IL-1alpha over a 7-day period of cultivation in vitro. Upon stimulation with either lipopolysaccharide (LPS) (BALB/c) or concanavalin A (Con A) (C3H/HeJ), both cell types produced IL-1alpha that peaked within the first 12-24 h following stimulation. BMM from C3H/HeJ and BALB/c mice failed to produce IL-1alpha when infected in vitro with L. major or L. donovani promastigotes. However, infection with these two parasites did not interfere with the capability of the host cell to produce IL-1alpha when stimulated with LPS or Con A. The level of IL-1alpha production was independent of the degree of parasitization of the macrophages. Similar results were observed with IL-1beta and IL-6 production by BMM, even though their levels were generally slightly higher than those obtained with IL-1alpha.     

Innate resistance to experimental African trypanosomiasis: differences in cytokine (TNF-alpha, IL-6, IL-10 and IL-12) production by bone marrow-derived macrophages from resistant and susceptible mice

Resistance to African trypanosomiasis is under multigenic control. BALB/c mice are highly susceptible while C57Bl/6 mice are relatively resistant. Macrophages eliminate opsonized trypanosomes from the bloodstream and are involved in immunosuppression. We therefore investigated the production of a number of cytokines (IL-10, IL-6, TNF-alpha and IL-12) by bone marrow-derived macrophages (BMDM) from C57Bl/6 and BALB/c mice following challenge with either Trypanosoma congolense or Trypanosoma brucei. BMDM from C57Bl/6 mice, upon challenge with whole cell extracts (WCE) of T. congolense or T. brucei, produced significantly more TNF-alpha and IL-12 than those from BALB/c mice. The production of these cytokines was significantly enhanced by pretreatment of the cells with IFN-gamma. BMDM from BALB/c mice, however, produced significantly more IL-6 and IL-10 than those from C57Bl/6 mice. In contrast to LPS stimulation, simultaneous treatment of cells with WCE and IFN-gamma enhanced IL-10 synthesis by BMDM from BALB/c mice. These results indicate that cytokine genes are differentially regulated in macrophages from trypanosome-susceptible and -resistant mice and are consistent with our previous findings wherein retrovirus-immortalized macrophage cell lines from BALB/c and C57Bl/6 mice produce differential amounts of cytokines after phagocytosis of trypanosomes.     

Mitogen induction of murine C-type viruses. I. Analysis of lymphoid cell subpopulations.

We reported previously in vitro induction of endogenous C-type viruses from normal mouse spleen cells by lipopolysaccharide (LPS) as well as by combination treatment with concanabalin A and 5-bromo-2'-deoxyuridine (Con A/BrdU). To identify the cell types sensitive to virus induction and to study the relationship of mitogenicity to virus induction we have compared T cell populations (BALB/c thymus cells and cortisone-resistant thymus cells), B cell populations (nu/nu spleen cells and lymph node cells), adherent BALB/c peritoneal cells and mixed populations (BALB/c spleen cells, macrophage-depleted BALB/c spleen cells, and lymph node cells). LPS-induction occurred only in B cell-containing populations. In contrast, induction by Con A/BrdU depended on the presence of both T and B cells. In both instances, neither macrophages nor hemopoietic cells appeared to be a major source of virus. Treatment with anti-Ig serum and complement reduced virus induction by LPS/BrdU but not by Con A/BrdU suggesting that different cell populations produce virus after stimulation with these two different mitogens.     

Modulation of murine macrophage function by N-acetylcysteine in a model of endotoxic shock

In previous studies we have observed changes in several functions of peritoneal macrophages from BALB/c mice with irreversible endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. Since antioxidants, such as N-acetylcysteine (NAC), are free radical scavengers that improve the immune response, in the present work we have studied different functions of peritoneal macrophages from BALB/c mice suffering the endotoxic shock above indicated and administered N-acetylcysteine (150 mg/kg i.p.) at 30 minutes after LPS injection. In the peritoneal macrophages obtained at 2, 4, 12 and 24 h after LPS injection, the following functions were studied: adherence to substrate, mobility, ingestion of particles, and production of superoxide anion and tumour necrosis factor (TNF alpha). The increase in adherence, ingestion and superoxide anion and TNF alpha production shown by macrophages from animals with endotoxic shock was counteracted by NAC injection. Moreover, the survival time of mice with endotoxic shock was increased in the presence of NAC. These data suggest that NAC, administered intraperitoneally, may be useful for the treatment of irreversible endotoxic shock by modulation of the function of macrophages with decreased superoxide anion and TNF alpha production and concomitant increase of survival time.     

Immunobiological properties of lipopolysaccharides isolated from Fusobacterium nucleatum and F. necrophorum

Lipopolysaccharides (LPSs) were isolated from Fusobacterium nucleatum ATCC 10953 and F. necrophorum ATCC 25286 by the hot phenol/water procedure. F. nucleatum LPS was composed of 16% (w/w) carbohydrate, 10% (w/w) hexosamine and 40% (w/w) fatty acid, while F. necrophorum LPS was composed of 26% (w/w) carbohydrate, 12% (w/w) hexosamine and 28% (w/w) fatty acid. These LPS preparations induced mitogenic responses in spleen cells of BALB/c, BALB/c (nu/nu) and C3H/HeN mice, and these responses were suppressed by the addition of polymyxin B. The preparations also induced the polyclonal responses of C3H/HeN spleen cells. In addition, enhanced glucose utilization and interleukin-1 production by murine peritoneal macrophages were demonstrated. Neither spleen cells nor macrophages from the 'LPS-nonresponsive' C3H/HeJ mouse were activated by LPSs from the Fusobacterium species.     

Regulatory interactions between macrophages and T cells in Mycobacterium lepraemurium-specific T-cell activation

The antigen-specific proliferative response of draining lymph node cells was found to follow a similar pattern in both C57BL and BALB/c mice following subcutaneous infection with Mycobacterium lepraemurium (MLM), although the two strains differed in their ability to control bacterial growth at the site of infection. The proliferative response, which was maximal 1-2 weeks postinfection, was T-cell dependent as it was abrogated with anti-Thy 1.2 + C treatment. The response was also abrogated by pretreatment with anti-Lyt 1.2 + C and slightly reduced by treatment with anti-Lyt 2.2 + C. The decline in T-cell responsiveness, at least from 4 to 8 weeks postinfection, may have been associated with prostaglandin production by inflammatory macrophages, as it was partially restored by addition of indomethacin. Also highly purified T lymphocytes from lymph nodes taken 6-8 weeks postinfection gave a strong antigen-specific proliferative response when reconstituted with optimal numbers of syngeneic antigen-presenting cells from uninfected mice. Proliferation was inhibited by peritoneal macrophages from Corynebacterium parvum-pretreated mice and macrophages from C57BL but not BALB/c mice infected with M. lepraemurium which had been elicited with heat-killed (HK) MLM and thioglycollate. Resident peritoneal macrophages from both C57BL and BALB/c mice infected subcutaneously with M. lepraemurium were slightly more inhibitory than normal macrophages but not as inhibitory as macrophages from C. parvum-pretreated mice. Macrophage-dependent inhibition of T-cell proliferation was partially reversed by addition of indomethacin, suggesting these cells were not defective in processing and presentation of HK-MLM antigens, and that the inhibitory effects were associated with prostaglandin production. Resident peritoneal macrophages from both C57BL and BALB/c mice infected subcutaneously with M. lepraemurium produced comparable or slightly elevated levels of IL-1 on stimulation with LPS or HK-MLM.     

Legionella pneumophila growth in macrophages from susceptible mice is genetically controlled

Growth of the intracellular opportunistic bacterium Legionella pneumophila in macrophages from A/J mice is a vigorous as growth in macrophages from susceptible guinea pigs and human monocytes, whereas growth is inhibited in macrophages from other mouse strains, such as nonpermissive BALB/c mice. Permissiveness versus nonpermissiveness of macrophages from A/J versus BALB/c mice appeared to be controlled by a genetic mechanism dependent upon a single gene or a closely clustered family of genes. Susceptibility versus resistance of macrophages from F1 offspring of these two strains of mice and macrophages from backcrossed mice prepared from F1 hybrids and the original parental strain showed a segregation of permissiveness for growth of Legionella in vitro, consistent with genetic control.     

Age-dependent macrophage functions in New Zealand black mice.

Various functions of macrophage derived from young (2-month-old) and old (14- to 17-month-old) New Zealand Black (NZB) mice with autoimmune disease were studied and compared with macrophage functions of age-matched BALB/c mice. Macrophages from young and old NZB mice demonstrated elevated levels of β-glucuronidase, cathepsin D, lysozyme, and DNase compared with those from age-matched BALB/c. DNase activity in the macrophages of NZB mice significantly increased with age. Macrophages from young and old NZB mice had greater phagocytic capacity for both ¹²⁵I-labeled Shigella flexneri and Staphylococcus albus than did BALB/c macrophages. NZB macrophages from both young and old mice had higher bactericidal activity against S. albus than those from age-matched BALB/c mice. The number of macrophage/granulocyte colony-forming cells (CFC) in both bone marrow and spleen was markedly higher in young and old NZB mice than in BALB/c mice. Colony-stimulating factor (CSF) released by macrophages derived from NZB mice had higher CFC activity than that released from macrophages of age-matched BALB/c mice. In NZB mice, the CSF activity significantly increased with age. It is suggested that potentiation of macrophage number and activity compensates for the deficiency of T cell functions in NZB mice with autoimmune disease.     

Modulation of natural killer cytotoxicity by muramyl dipeptide and trehalose dimycolate incorporated in squalane droplets

Summary The effect on natural killer (NK) cytotoxicity of splenic cells from BALB/c mice pretreated i. v. with squalane-in-water preparations of muramyl dipeptide (MDP), trehalose dimycolate (TDM), or the combination of MDP-plus-TDM was investigated. MDP or TDM augmented the NK cytotoxicity which peaked 48 h after the pretreatment whereas the combination of MDP and TDM induced an inhibition of the NK activity. Infection with influenza virus, a potent stimulator of NK cells, after the pretreatment with biological response modifiers resulted in a markedly enhanced NK activity on day 2 in MDP and control groups. Mice pretreated with TDM or the combination of MDP and TDM showed only moderate NK activity which peaked on day 3 after influenza infection. The NK activity was susceptible to asialo GM₁ and complement treatment. The cytotoxicity of MDP-plus-TDM cells could be significantly enhanced after treatment with anti-macrophage monoclonal antibody and complement. NK activity induced by MDP or TDM was reduced by mixing MDP-plus-TDM cells. Addition of adherent cell-depleted MDP-plus-TDM suspension to MDP or TDM cells had a NK restorative effect. Splenic cells from mice pretreated 2 days earlier with MDP or TDM, but not MDP-plus-TDM, generated enhanced levels of luminol-dependent chemiluminescence.