Human antigen-specific IgA responses in blood and secondary lymphoid tissue: an analysis of help and suppression

Immunization with a virulent Salmonella typhimurium, strain SL3235, has been found to provide high levels of protection against challenge with virulent Salmonella in hypersusceptible mouse strains in the C3H lineage. These mouse strains include the lipopolysaccharide-hyporesponsive C3/HeJ mouse and the closely related but lipopolysaccharide-responsive C3HeB/FeJ mouse. To assess the role of cellular immunity in the protection elicited by this attentuated organism, delayed-type hypersensitivity (DTH) was measured in these mouse strains and in inherently resistant mice. Of the mouse strains tested, only the inherently resistant CD-1 and C3H/HeNCrlBR mice developed significant DTH responses, as assessed by footpad swelling tested at various times after immunization with SL3235. The hypersusceptible C3H/HeJ and C3HeB/FeJ mice failed to exhibit significant DTH responses despite their high levels of immunity.     

Macrophage-mediated mitogenic suppression induced in mice of the C3H lineage by a vaccine strain of Salmonella typhimurium

Salmonella typhimurium, strain SL3235, an avirulent organism, has been used as a live vaccine in mice of the C3H lineage and has been found to confer high levels of protection. In the present study, it was found that intraperitoneal injection of approximately 5 X 10(5) live SL3235 induced potent suppression of spleen cell mitogenic responses to a panel of B- and T-cell mitogens in the Salmonella-hypersusceptible C3H/HeJ and C3HeB/FeJ, and the inherently resistant C3H/HeNCrlBR mice. Maximal suppression (greater than 99%) was seen at 1 week, and was still significant but waning (50%) at 3 weeks postimmunization. In contrast, cells of mice receiving acetone-killed cells were not suppressed. Removal of macrophages, but not T or B cells, restored responsiveness, indicating that suppression was macrophage mediated. Prostaglandins were not the major mediator of suppression, as in vitro administration of indomethacin failed to abrogate suppression. As mitogenic suppression occurred in mice with high levels of Salmonella immunity, the suppression is interpreted as a marker of a powerful immunomodulatory process induced by live cells, rather than as an indication of poor immune status of the host.     

Evidence for separate genetic defects in C3H/HeJ and C3HeB/FeJ mice, that affect susceptibility to gram-negative infections

Past studies have suggested a linkage between susceptibility to Salmonella typhimurium infection and the Lpsd genotype in C3H mice. Recently, this linkage was questioned by the finding that C3HeB/FeJ mice (Lpsn,Lpsn) were highly susceptible to systemic S. typhimurium infection. The present study shows a marked difference between C3H/HeJ and C3HeB/FeJ in their susceptibility to Gram-negative urinary tract infection. The number of E. coli and S. typhimurium recovered from the kidneys 24 hr after infection was 70 to 100 times higher in C3H/HeJ than in C3HeB/FeJ or C3H/HeN mice. Subsequently, in C3HeB/FeJ mice S. typhimurium multiplied to the level of C3H/HeJ mice, resulting in a shorter mean survival time of C3H/HeJ and C3HeB/FeJ compared with C3H/HeN mice. In contrast, E. coli remained localized to the urinary tract of C3H/HeJ mice but were eliminated from C3HeB/FeJ and C3H/HeN mice. Thus, experimental E. coli urinary tract infection appears to provide a method to differentiate the genetic defects of C3H/HeJ and C3HeB/FeJ mice. The results support an influence of the Lpsd genotype on clearance of Gram-negative bacteria from the kidneys of C3H mice.     

Cellular immunity induced by avirulent Salmonella in LPS-defective C3H/HeJ mice

An avirulent strain of Salmonella, SL3235, has been shown to confer high levels of immunity on lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice. Immunized mice were also protected against challenge with Listeria monocytogenes, indicating that the Salmonella vaccine activates macrophages. It was shown that protection and macrophage activation occurred without correction of the LPS defect, as assessed by in vivo endotoxin toxicity, in vitro spleen cell mitogenicity, and the ability of in vivo treatment with LPS to enhance in vitro macrophage ingestion of C3b-coated erythrocytes. It is concluded that LPS responsiveness is neither a necessary nor a sufficient condition for Salmonella immunity, and that macrophage activation can apparently occur in C3H/HeJ mice in the face of a sustained LPS defect.     

Genetic control of responses to bacterial lipopolysaccharides in mice. II. A gene that influences a membrane component involved in the activation of bone marrow-derived lymphocytes by lipipolysaccharides.

C3H/HeJ mice contain a defect in a single autosomal locus which is not linked to the H-2 histocompatibility or the heavy chain allotype loci that restrict immune, mitogenic, and polyclonal responses to bacterial lipopolysaccharides (LPS). Adult thymectomized C3H/HeJ mice that have been irradiated and reconstituted with C3HeB/FeJ bone marrow cells respond well to LPS. Cell-mixing experiments using C3H/HEJ-C3HeB/FeJ spleen cultures show that the failure of C3H/HeJ spleen cells to support responses to LPS is not due to nonspecific or LPS-induced suppressive events, or the lack of accessory cell types. C3H/HeJ and C3HeB/FeJ spleen cells bind LPS and respond to other B cell mitogens equally well. We suggest that the B lymphocytes of C3H/HeJ mice have a defect in a membrane component that is activated via interaction with LPS, and initiates the intracellular events that lead to cell proliferation.     

Involvement of lipopolysaccharide in the pathogenicity of Treponema hyodysenteriae

Treponema hyodysenteriae, the etiologic agent of swine dysentery, caused gross and microscopic lesions in the large intestines of C3HeB/FeJ mice. No gross lesions were observed in the intestines of the closely related, but lipopolysaccharide-resistant, C3H/HeJ strain of mice, and microscopic lesions were mild, if present at all. In the presence of actinomycin D, 1 mg of T. hyodysenteriae lipopolysaccharide (LPS) was lethal for C3HeB/FeJ but not for C3H/HeJ mice. Also, the treponemal LPS was chemotactic for macrophages from C3H/HeJ mice but not for macrophages from C3HeB/FeJ mice. The difference between the two mouse strains in lesion development may be due to the nondestructive nature of LPS in C3H/HeJ mice, which suggests that the treponemal LPS is involved in the pathogenicity of T. hyodysenteriae. T. hyodysenteriae may prove to be a useful bacterium in the study of LPS-resistant C3H/HeJ mice, because resistance to the treponemal LPS and to the treponeme itself appear to correlate.     

Defective tumoricidal capacity of macrophages from C3H/HeJ mice

Peritoneal macrophages from C3H/HeN mice treated i.p. with T cell mitogens or viable BCG organisms were cytotoxic to syngeneic tumor cells in vitro. Macrophages from endotoxin-unresponsive C3H/HeJ mice treated with BCG or T cell mitogens, however, were not tumoricidal. Furthermore, unlike cells from C3H/HeN mice, macrophages from C3H/HeJ mice could not be activated for tumor cytotoxicity after in vitro treatment with bacterial endotoxins or with lymphokine-rich supernatants. The subnormal induction of cytotoxic macrophages after in vitro or in vivo treatments in C3H/HeJ mice appears to be a highly selective defect. Macrophage responses (yield, phagocytosis, or peroxidase staining) in inflammatory exudates induced by BCG, T cell mitogens, or heterologous serum in C3H/HeJ or C3H/HeN mice were identical. C3H/HeJ macrophages also responded normally in vitor to chemotactic lymphokines. Thus, C3H/HeJ macrophages possess a profound and selective defect in tumoricidal capacity. This defect was not dependent upon exogenous endotoxins. Defective macrophage cytotoxic responses may reflect non-LPS related functions regulated by the LPS gene.     

Intrinsic B lymphocyte and macrophage defects in C3H/HeJ mice

C3H/HeJ mice are genetically defective in their responsiveness to lipopolysaccharide (LPS). Their B cells also have a characteristically low cloning efficiency in semisolid agar cultures, where LPS does not seem to be required. Adherent macrophages facilitate clonal proliferation in such cultures via diffusible substances. C3H/HeJ macrophages functioned poorly in this respect, and addition of normal C3HeB/FeJ macrophages to cultures of C3H/HeJ B cells did not lead to normal colony numbers. Although immune interferon can stimulate normal resident peritoneal macrophages to function well in semisolid agar cultures, it did not improve the cloning efficiency of C3H/HeJ cells. Similarly, addition of indomethacin or interleukin 1 to the cultures did not reveal that abnormally elevated production of prostaglandins or a deficiency in interleukin 1 are responsible for poor C3H/HeJ colony formation. These results indicate that C3H/HeJ mice have defects intrinsic to both B cells and macrophages that are not overcome by interferon. Purified B cells from these mice cloned poorly and did not respond to stimulation in liquid cultures with anti-mu-coated beads plus factors. There was a tendency for the poor cloning of C3H/HeJ B cells to improve with age.     

Immunosuppression induced by attenuated Salmonella. Evidence for mediation by macrophage precursors

An attenuated aro A- strain of Salmonella typhimurium, SL3235, was previously shown to afford excellent protection in C3HeB/FeJ mice against challenge by virulent Salmonella and cross-protection against Listeria monocytogenes. In the present study, the immunologic status of immunized mice was evaluated by studying their ability to mount in vivo and in vitro plaque-forming cell responses to SRBC. SL3235-immunized mice exhibited marked suppression in their ability to generate anti-SRBC plaque-forming cell responses. Suppression was active and mediated by soluble factors as demonstrated by the capacity of immune cells to suppress the responses of normal cells in co-culture and across a membrane in transwell plates. Depletion of T cells from immune spleens did not alleviate the suppressive activity of the remaining cells. Depletion of splenic adherent cells resulted in partial alleviation of suppressive activity, demonstrating that mature macrophages are partly responsible for mediating the observed suppression. A sequential multi-step depletion procedure resulted in marked enrichment of a second suppressor cell population that was Mac1+, Thy-1.2-, surface Ig-, J11d-, non-adherent, non-phagocytic, and esterase negative. When cultured in vitro in the presence of L cell-conditioned medium, but not in the presence of Con A supernatant, these cells matured into typical macrophages within 72 h of culture. The cell population enriched for macrophage precursors (75%) retained the suppressive capacity of unfractionated splenocytes. Our data indicate that, in addition to the well-described involvement of mature macrophages in suppressing immune responses, bacterial infection may induce appearance of macrophage precursors that may also play an important regulatory role in the immune system.     

Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.     

Macrophage stimulation by bacterial lipopolysaccharides. III. Selective unresponsiveness of C3H/HeJ macrophages to the lipid A differentiation signal.

Peritoneal macrophages from the endotoxin-unresponsive C3H/HeJ substrain of mice were entirely refractory to activation in vitro by protein-free LPS, a defect that was not overcome by co-culture of spleen cells from the responder C3H/St substrain with LPS resistant C3H/HeJ macrophages. The defect in responsiveness appears confined to the lipid A activation signal since C3H/HeJ macrophages were fully activated after in vitro treatment by lipid A protein (LAP)--LPS complex, isolated LAP, and BCG. Moreover, after exposure to allogeneic tumor cells in vivo, C3H/HeJ macrophages were cytotoxic for tumor target cells in vitro. By contrast, macrophages from the responder C3H/St strain were fully activated by protein-free LPS to become cytolytic for tumor cells in vitro. C3H/HeJ macrophages, therefore, exhibit a highly selective defect characterized by unresponsiveness to the lipid A activation signal of protein-free LPS and resistance to the toxic effects of high concentrations of LPS that were lethal to the responder C3H/St strain.     

The correlation between specific protein synthesis and tumoricidal function in murine peritoneal macrophages

Macrophages from the lipopolysaccharide (LPS)-responsive C3H/HeN mouse strain and the closely related LPS-nonresponsive C3H/HeJ strain were compared for tumoricidal activation and protein synthetic changes following in vivo and in vitro stimulation, utilizing two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Peritoneal macrophages elicited from C3H/HeN mice with heat-killed Propionibacterium acnes exhibited tumoricidal activity in a 16-hr cytolytic assay and expressed cytoplasmic levels of a 23.5-kDa protein during 48 hr of culture. The inability to detect persistent expression of p23.5 in P. acnes-stimulated C3H/HeJ macrophages correlated with the cytolytic impotence of those cells in the 16-hr chromium release assay. C3H/HeN macrophage populations lacking tumoricidal capacity could be rendered lytic, as could P. acnes-elicited C3H/HeJ macrophages, following in vitro stimulation with bacterial lipopolysaccharide. Concomitant with the LPS-induced expression of new functional activity was the appearance of augmented levels of several macrophage-specific proteins, including p23.5. This effect was dependent upon the lipid A moiety of LPS as the effects of LPS could be blocked by inclusion of polymyxin B sulfate in the culture medium. However, neither tumoricidal function nor protein modulation could be readily induced in C3H/HeJ proteose peptone-elicited or resident macrophages. These results identify biochemical responses to stimuli which may be requisite to acquisition or execution of cytolytic activity.     

Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibility with in vitro defective macrophage microbicidal activities

Eleven mouse strains were inoculated in footpads with amastigotes of Leishmania tropica and observed for 12 weeks. Liver and spleen impression smears from infected mice were examined for the presence of intracellular parasites. Four strains (BALB/cJ, C57L/J, NZW/N, and P/J) failed to heal the subcutaneous lesion and showed evidence of systemic infection; the remaining seven strains (A/J, C3H/HeJ, C3H/HeN, C3HeB/FeJ, C57BL/6J, C57BL/10J, and C57BL/10ScN) were each resistant to infection and resolved their lesions by Week 10. Macrophages from the four susceptible strains could not be activated to kill L. tropica amastigotes by treatment with soluble lymphocyte products in vitro. In contrast, macrophages from all seven resistant strains responded to lymphokine treatment and eliminated 80-90% of intracellular parasites. These results suggest that in vitro macrophage microbicidal activities predict the course of systemic leishmanial disease.     

Rejection of murine ovarian cancer following treatment with regional immunotherapy: correlations with a neutrophil-mediated activation of cytostatic macrophages

Rejection of the murine ovarian teratocarcinoma (MOT) in C3HeB/FeJ mice, following intraperitoneal (ip) treatment with Corynebacterium parvum (C. parvum), is abrogated by injections of silica. We, therefore, investigated whether C. parvum-elicited macrophages affect MOT targets in vitro. Tumor-cytostatic, but not cytolytic, macrophages were detected in normal and tumor-challenged mice treated with C. parvum. The dose responsiveness and kinetics of macrophage activation strongly correlated with tumor rejection. A pyridine extract of C. parvum, possessing greatly diminished tumor rejection properties, was significantly less effective in activating macrophages. Cytostatic macrophage activation and prevention of tumor outgrowth also followed treatment in C3H/HEJ mice, a strain with a known deficiency in cytolytic macrophage function. Peritoneal neutrophils, obtained 6 hr after treatment with C. parvum, were capable of activating cytostatic macrophages when reinjected ip into normal mice. These results indicate a critical role for tumor cytostatic macrophages in this immunotherapy model and suggest their activation is mediated by inflammatory neutrophils.     

Subchronic endotoxin inhalation causes persistent airway disease

The endotoxin component of organic dusts causes acute reversible airflow obstruction and airway inflammation. To test the hypothesis that endotoxin alone causes airway remodeling, we have compared the response of two inbred mouse strains to subchronic endotoxin exposure. Physiological and biological parameters were evaluated after 1 day, 5 days, or 8 wk of exposure to endotoxin [lipopolysaccharide (LPS)] in endotoxin-sensitive (C3HeB/FeJ) and endotoxin-resistant (C3H/HeJ) mice. After 5 days or 8 wk of LPS exposure, only C3HeB/FeJ had elevated airway hyperreactivity to inhaled methacholine. Only the C3HeB/FeJ mice had significant inflammation of the lower respiratory tract after 1 day, 5 days, or 8 wk of LPS exposure. Stereological measurements of small, medium, and large airways indicated that an 8-wk exposure to LPS resulted in expansion of the submucosal area only in the C3HeB/FeJ mice. Cell proliferation as measured by bromodeoxyuridine incorporation contributed to the expansion of the submucosa and was only significantly elevated in C3HeB/FeJ mice actively exposed to LPS. C3HeB/FeJ mice had significantly elevated levels of interleukin-1beta protein in whole lung lavage after 1 day and 5 days of LPS exposure and significantly elevated protein levels of total and active transforming growth factor-beta1 in whole lung lavage fluid after 5 days of LPS exposure. Our findings demonstrate that subchronic inhalation of LPS results in the development of persistent airway disease in endotoxin-responsive mice.     

Lipopolysaccharide receptors on lymphocytes. I. Lack of immunologic recognition of a putative LPS receptor on LPS-responder lymphocytes by LPS-nonresponder mice

We have examined the potential immunogenicity of viable lymphocytes from C3HeB/FeJ responder mice adoptively transferred into congenic nonirradiated C3H/HeJ nonresponder mice. Immunologic rejection or acceptance of donor cells was employed as indirect evidence for the presence or absence of an antigenically distinct "LPS receptor" present on donor lymphocytes. Immunogenicity was evaluated by in vitro assessment of the subsequent proliferative response of recipient splenocytes to protein-free LPS after multiple i.p. injections of responder lymphocytes. Control experiments have made use of syngeneic donor lymphocytes differing immunologically by the presence of the H-Y minor histocompatibility antigen. The results of these experiments provide evidence for the concept that if the phenotypic difference between LPS responder and nonresponder mice is also expressed antigenically in the form of an LPS receptor, that antigenic difference is significantly less immunogenic than a minor histocompatibility antigen.     

Macrophage activation to kill Leishmania tropica: characterization of P/J mouse macrophage defects for lymphokine-induced antimicrobial activities against Leishmania tropica amastigotes

Macrophages from P/J mice demonstrated both quantitative and qualitative defects in lymphokine (LK)-induced activated macrophage antileishmanial effector reactions: a) these cells recognized the same LK signals that generated resistance to infection in responsive C3H/HeN macrophages, but more signal was required to observe maximal activity; b) LK-induced intracellular destruction of Leishmania tropica by P/J macrophages was minimal (less than 20%), and was induced by only one of three LK signals that regulate antimicrobial activities in C3H/HeN macrophages. The defective microbicidal activity of P/J macrophages observed with LK activation in vitro could also be demonstrated in vivo. Macrophages from P/J mice exposed to the macrophage-activating agent Mycobacterium bovis strain BCG in vivo were capable of restricting the intracellular replication of L. tropica but could not eliminate intracellular parasites, even with further incubation with LK during the 72-hr culture period. The defect of P/J macrophages for intracellular destruction of L. tropica, then, occurred in the activation sequence before the triggering stage that characterizes the macrophage defect of C3H/HeJ mice. Genetic regulation of the P/J macrophage defect appears to be by a single autosomal gene, with defective microbicidal activity as a recessive trait in these animals.     

A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis

Infection of C3HeB/FeJ and C57BL/6 mice with Leishmania major stimulates a healing cell-mediated immune response, while Leishmania amazonensis infection leads to chronic disease. Here we show C3HeB/FeJ mice co-infected with both species of Leishmania heal, while co-infected C57BL/6 mice do not. Using an in vitro killing assay we determined B cells from infected C57BL/6 mice are ineffective in promoting parasite killing compared with B cells from infected C3HeB/FeJ mice. Furthermore, infected C57BL/6 mice produce less antigen-specific antibodies compared with infected C3HeB/FeJ mice. These findings suggest B cells play a required role in the cell-mediated immune response against L. amazonensis.     

Activation of C3H/HeJ macrophage tumoricidal activity and cytokine release by R-chemotype lipopolysaccharide preparations. Differential effects of IFN-gamma

We have investigated the relative immunostimulatory activities of S-chemotype LPS and R-chemotype LPS preparations on C3H/HeJ peritoneal macrophages in vitro. As assessed by either secretion of TNF-alpha or IL-1, some of the R-chemotype LPS manifest significant activity on these normally LPS-unresponsive cells. The expression of IL-1 activity by R-LPS-stimulated C3H/HeJ macrophages was unaffected by IFN-gamma; however, this cytokine significantly enhanced TNF-alpha production by the same cells. The R-chemotype LPS preparations alone were not able to activate C3H/HeJ macrophages to become tumoricidal but activity could readily be demonstrated in the presence of IFN-gamma. Of potential importance is the observation that the profile of relative activity of the various R-chemotype LPS preparations for macrophage activation does not parallel that previously obtained by us for the C3H/HeJ B-lymphocyte activation.     

Defective tumoricidal capacity of macrophages from A/J mice. I. Characterization of the macrophage cytotoxic defect after in vivo and in vitro activation stimuli.

Macrophages from A/J mice fail to develop tumoricidal activity after any of several in vivo or in vitro treatments that activate cells from C3H/HeN mice. Peritoneal macrophages from A/J mice treated i.p. with viable Mycobacterium bovis, strain BCG, killed Corynebacterium parvum, or pyran copolymer fail to develop in vitro tumoricidal activity; varying the numbers of macrophages from treated mice added to target cells, or the dose and time of treatment, or the treatment schedule of these in vivo activation stimuli did not evoke cytotoxic activity. Moreover, cytotoxic activity by macrophages from A/J mice was not observed with any of four target cell lines derived from three different mouse strains. In vitro treatment of peritoneal exudate macrophages from A/J mice with lymphokine-rich supernatants, bacterial endotoxins, or T cell mitogens was also ineffective; varying the numbers of treated macrophages added to target cells, the dose of in vitro activation stimuli, or the time of treatment did not evoke cytotoxic activity. Thus, A/J mice exhibit a profound defect in macrophage tumoricidal capacity to both in vivo and in vitro activation stimuli over a wide range of experimental conditions.