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.     

Stimulation of antimycobacterial activity in mouse peritoneal macrophages by priming with trehalose dimycolate (TDM).

We examined the potential of two bacterial immunomodulators, trehalose dimycolate (TDM) and lipopolysaccharide (LPS), to stimulate the capacity of mouse peritoneal macrophages to control the growth of the intracellular bacterium, Mycobacterium tuberculosis BCG. Macrophages were obtained from mice innately susceptible (Bcgs) or resistant (Bcgr) to BCG infection. In all mouse strains tested (Bcgr and Bcgs), with the exception of BALB/c (Bcgs), TDM was sufficient to elicit macrophages with strong antimycobacterial activity in vitro. In BALB/c mice, the induction of anti-BCG activity required two signals, TDM and LPS, given in sequence. Our data suggest that additional gene(s), besides the Bcg locus, control macrophage resistance to BCG.     

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.     

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.     

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.     

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.     

Genetic differences in BCG-induced resistance to Schistosoma mansoni are not controlled by genes within the major histocompatibility complex of the mouse.

Induction of nonspecific resistance to Schistosoma mansoni infection after the i.v. injection of viable BCG was investigated in outbred mice and a panel of inbred and H-2 congenic strains. Significant protection was induced in CF1, A/J, C57BL/6, C57BL/10, DBA/2, C57BR, and SJL mice. BALB/c mice were not protected whereas CBA and C3H mice expressed intermediate degrees of protection. Expression of the protective phenomenon is not controlled by genes within the MHC as shown by the marked differences in response between BALB/c and DBA/2 (H-2d) as well as between C57BR and C3H (H-2k) mice. H-2 congenic strains with C57BL/10 background (B10.A and B10.D2) were high responders. BALB.B10 mice carrying the high responder (B10) MHC on the nonresponder (BALB/c) background were not protected. The degree of splenic hypertrophy did not correlate with the expression of nonspecific resistance. These results demonstrate that, in addition to controlling specific immune responses, genetic differences influence the nonspecific protective phenomena related to BCG administration as well.     

Correlation between the inducible keratinocyte expression of Ia and the movement of Langerhans cells into the epidermis

A direct correlation between the induced expression of Ia by the host keratinocytes and the infiltration of donor Langerhans cells (LC) into the epidermis was demonstrated in athymic (nude) BALB/c mice that received an adoptive transfer of lymphoid cells from normal semi-syngeneic donors. Neither keratinocyte expression of Ia nor donor LC movement into the epidermis was observed in BALB/c recipients of lymphoid cells from allogeneic C3H nude mice. Further evidence for this relationship was provided by experiments in which the keratinocytes of BALB/c nude mice were induced to express Ia by the injection of normal mouse serum (NMS). By this procedure it was shown that LC precursors derived from allogeneic C3H nude donors were able to infiltrate the epidermis when adoptively transferred into BALB/c nude recipients whose keratinocytes had been induced to express Ia by the simultaneous injection of NMS. These findings suggest that keratinocytes through their expression of Ia may function to facilitate the movement of LC into the epidermis.     

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.     

Leishmania major: analysis of lymphocyte and macrophage cellular phenotypes during infection of susceptible and resistant mice

Genetically susceptible BALB/c and resistant C57BL/6 mice were infected with Leishmania major and the phenotypes of the responding cells in the draining lymph nodes and cutaneous lesions were analyzed. As early as 1 week, significantly increased numbers of L3T4+ cells as compared to Lyt-2+ cells were present in BALB/c mice lymph nodes (P less than 0.005). Increases in L3T4+ and Lyt-2+ cells were comparable in C57BL/6 mice, resulting in threefold lower L3T4/Lyt-2 ratio than in BALB/c mice. T cell subsets were activated in both strains to express interleukin-2 receptor (IL2R) above resting values, although greater numbers of activated L3T4+ cells were present in the draining lymph nodes from BALB/c at 1 and 3 weeks of infection than in C57BL/6 (P = 0.02). Despite the presence of activated L3T4+ cells in both strains, macrophages differed in the expression of immunologically important surface molecules during infection. Tissue macrophages from BALB/c mice were IgG1/G2b Fc receptor (FcR)+ and Ia- late in disease, whereas macrophages in C57BL/6 became FcR and Ia during healing. BALB/c mice, treated with monoclonal antibody GK1.5 to transiently deplete L3T4+ cells, became resistant to subsequent infection and developed a macrophage phenotype that was FcR- and Ia+. These differences in macrophage phenotype were closely linked to susceptibility during infection with L. major and may play a role in the pathophysiology of murine leishmaniasis.     

Trypanosoma cruzi: aberrant expression of class II major histocompatibility complex molecules in skeletal and heart muscle cells of chronically infected mice

In isolated skeletal, heart, and smooth muscle cells from BALB/c and C3H/HeJ mice infected with different strains of Trypanosoma cruzi the presence of class II MHC molecules was investigated by immunocytochemical techniques. We employed single muscle fibers instead of conventional cryostat sections to obtain a more accurate antigen localization. Approximately half of the skeletal muscle cells isolated from the rectus femoris expressed Ia antigens on their surface, irrespective of the mouse or parasite strain combination. Ia expression was apparent only at 30 days postinfection and thereafter. The heart muscle cells expressed class II molecules only at 1 and 3 months postinfection. In no case did the smooth muscle cells from infected mice express Ia antigens. Studies of the same molecules in the noninfected animals gave constantly negative results. We conclude that in the course of the chronic infection of mice with T. cruzi, ectopic expression of class II MHC molecules occurs at the surface of skeletal and heart muscle cells, providing a possible mechanism for explaining the anti-striped muscle autoreactivity present in Chagas' disease.     

Soluble factors produced during an immune response regulate Ia antigen expression by murine adenocarcinoma and fibrosarcoma cells

LT-85 is an alveologenic adenocarcinoma of C3Hf/HeN mice. Comparisons of the in vitro and in vivo surface properties of these cells revealed that under normal conditions, they expressed I-A and I-E antigens iv vivo only. By using clonally derived cells, it was established that this phenomenon was not due to the selection of an Ia antigen-positive tumor cell subpopulation, but resulted from phenotypic conversion of Ia antigen-negative tumor cells. These tumor cells and 1053 cells (a fibrosarcoma of C3H/HeN MTV- mice) could, however, be induced to express I-A, I-E, and much higher levels of H-2 antigens in vitro by co-culturing them with spleen cells from LT-85 tumor-bearing C3H/HeN MTV- mice. In vitro induction of Ia and H-2 antigens did not result from contaminating splenocytes or from antigen transfer, because splenocytes from BALB/c (H-2d) mice immunized with A/J (H-2k/d) cells were able to induce the expression of Iak antigens by both tumor cell lines. It was found that this phenomenon was neither H-2-restricted nor antigen-specific. The results clearly indicated, however, that an immune response was required to generate phenotypic conversion of the tumor cells, both in vivo and in vitro. It was further found that soluble, rather than cellular, factors produced during an immune response induced the expression of Ia antigens by LT-85 and 1053 tumor cells. In contrast to what has been reported about the induction of Ia antigens on macrophages and normal epithelial and endothelial cells, the induction of Ia antigens on LT-85 and 1053 cells did not appear to require T cells, and did not involve gamma-interferon. These findings demonstrate that some tumor cells are capable of altering their MHC antigen phenotype in response to factors produced during an immune response in vivo or in vitro. Because of the involvement of Ia antigens in several aspects of immune phenomena, the ability of tumor cells to differentially express Ia antigens in response to environmental factors may have profound effects on host-tumor interactions. Furthermore, the differences seen in the phenotypes of tumor cells grown in vitro and in vivo suggest that in vitro methodologies of tumor cell characterization may not present a complete picture of the natural state of the tumor cell surface.     

A new erythrocytic antigen of C57BL/10 (B10) mice

A new antigen, detectable on murine erythrocytes by hemagglutination assay with a (BALB/cCrl X SWR/J)F1 anti-B10.D2n/Sn alloantiserum, is described. Among the inbred and congenic mouse strains tested for reactivity with the antiserum, only the immunizing strain, B10.D2, and its congenic resistant partner, C57BL/10 (B10), reacted. Three other C57 strains, C57BL/6J, C57BL/6By, and C57L, were negative for the antigen. F1 hybrids between B10 and BALB/c, an antigen-negative strain, were positive for the antigen indicating that its expression is dominant. Typing of 39 (BALB/c X (BALB/c X B10)F1) and 62 [BALB/c X B10)F1 X BALB/c) backcross mice revealed that a single gene controls expression of the antigen. The gene is autosomal and not linked to H-2, Ly-4, or the c (albino) or b coat color genes.     

Liver sinusoidal endothelial cells that endocytose allogeneic cells suppress T cells with indirect allospecificity

A portal venous injection of allogeneic donor cells is known to prolong the survival of subsequently transplanted allografts. In this study, we investigated the role of liver sinusoidal endothelial cells (LSECs) in immunosuppressive effects induced by a portal injection of allogeneic cells on T cells with indirect allospecificity. To eliminate the direct CD4+ T cell response, C57BL/6 (B6) MHC class II-deficient C2tatm1Ccum (C2D) mice were used as donors. After portal injection of irradiated B6 C2D splenocytes into BALB/c mice, the host LSECs that endocytosed the irradiated allogeneic splenocytes showed enhanced expression of MHC class II molecules, CD80, and Fas ligand (FasL). Due to transmigration across the LSECs from BALB/c mice treated with a portal injection of B6 C2D splenocytes, the naive BALB/c CD4+ T cells lost their responsiveness to stimulus of BALB/c splenic APCs that endocytose donor-type B6 C2D alloantigens, while maintaining a normal response to stimulus of BALB/c splenic APCs that endocytose third-party C3H alloantigens. Similar results were not observed for naive BALB/c CD4+ T cells that transmigrated across the LSECs from BALB/c FasL-deficient mice treated with a portal injection of B6 C2D splenocytes. Adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice via the portal vein prolonged the survival of subsequently transplanted B6 C2D hearts; however, a similar effect was not observed for BALB/c FasL-deficient LSECs. These findings indicate that LSECs that had endocytosed allogeneic splenocytes have immunosuppressive effects on T cells with indirect allospecificity, at least partially via the Fas/FasL pathway.     

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.     

Attenuation of MHC class II expression in macrophages infected with Mycobacterium bovis bacillus Calmette-Guérin involves class II transactivator and depends on the Nramp1 gene.

The natural resistance associated macrophage protein 1 (Nramp1) gene determines the ability of murine macrophages to control infection with a group of intracellular pathogens, including Salmonella typhimurium, Leishmania donovani, and Mycobacterium bovis bacillus Calmette-Guérin (BCG). The expression of the resistant allele of the Nramp1 gene in murine macrophages is associated with a more efficient expression of several macrophage activation-associated genes, including class II MHC loci. In this study, we investigated the molecular mechanisms involved in IFN-gamma-induced MHC class II expression in three types of macrophages: those expressing a wild-type allele of the Nramp1 gene (B10R and 129/Mphi), those carrying a susceptible form of the Nramp1 gene (B10S), and those derived from 129-Nramp1-knockout mice (129/Nramp1-KO). Previously, we published results showing that Ia protein expression is significantly higher in the IFN-gamma-induced B10R macrophages, compared with its susceptible counterpart. In this paper, we also show that the higher expression of Ia protein in B10R cells is associated with higher I-Abeta mRNA expression, which correlates with a higher level of IFN-gamma-induced phosphorylation of the STAT1-alpha protein and subsequently with elevated expression of class II transactivator (CIITA) mRNA, compared with B10S. Furthermore, we demonstrate that the infection of macrophages with M. bovis BCG results in a down-regulation of CIITA mRNA expression and, consequently, in the inhibition of Ia induction. Therefore, our data explain, at least in part, the molecular mechanism involved in the inhibition of I-Abeta gene expression in M. bovis BCG-infected macrophages activated with IFN-gamma.     

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.     

IL-10 Acts As a Developmental Switch Guiding Monocyte Differentiation to Macrophages during a Murine Peritoneal Infection

The peritoneal wash of BALB/c or C57BL/6 mice contains two populations of macrophages that differ in their level of expression of MHC class II (MHC II). Although both populations efficiently phagocytose bacteria in vivo, only the MHC II(lo) population is effective at phagocytosing apoptotic cells in vivo and only the MHC II(hi) population is effective at presenting Ag to T cells in vitro. Soon after induction of a peritoneal infection both of these macrophage populations are lost from the peritoneal wash fraction. Blood monocytes then enter the inflamed peritoneum and develop into new peritoneal macrophages. Whether these monocytes develop into MHC II(lo) or into MHC II(hi) macrophages is crucially dependent on the cytokine IL-10, which is transiently elevated in the peritoneal wash during the early phase of infection. Monocytes from CD45.1 animals transferred early in infection when the IL-10 concentration is high into congenic CD45.2 recipients develop into the MHC II(lo) macrophage population. Monocytes transferred later, when the IL-10 concentration has fallen, develop into the MHC II(hi) population. In infected IL-10-deficient animals monocytes fail to develop into the MHC II(lo) population but can be induced to do so by exogenous application of IL-10. Finally, high numbers of wild-type monocytes injected into IL-10R1-deficient animals develop into MHC II(lo) macrophages and were able by a bystander effect to induce the differentiation of the endogenous monocytes to the same fate.     

Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection

CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes. In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L. monocytogenes infection. The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6. C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L. monocytogenes were fully protected against a subsequent lethal infection. Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response. A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays. Adoptive transfer of this T cell line alone resulted in significant protection against L. monocytogenes challenge. These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L. monocytogenes.