Characteristics of immunosuppressive macrophages induced in host spleen cells by Mycobacterium avium complex and Mycobacterium tuberculosis infections in mice

The profile of generation and characteristics of immunosuppressive macrophages (M phi s), which suppress the ConA-mitogenic response of spleen cells (SPCs), in host CBA/JN mice during the course of Mycobacterium avium complex (MAC) and M. tuberculosis (MT) infections were investigated. In both infections, a marked reduction in ConA mitogenic response of splenic T cells was seen around 2 weeks after infection, and this was accompanied by generation of potent immunosuppressive M phi s in the SPCs of infected mice. The suppressive activity was much stronger in MT-infected mice than in MAC-infected ones. In both infections, the large part of the suppressive M phi s exhibited suppressor activity that depended on the arachidonic acid cascade, particularly mediated by prostaglandins (PGs), and the remainder showed the suppressor action independent from PGs. The unique finding of this study is that the generation of IL-2 reactive T cell populations in SPCs in response to ConA signal was markedly inhibited by the MAC- and MT-induced immunosuppressive M phi s, whereas the suppressive M phi s failed to reduce the IL-2-producing ability of splenic T cells. In any case, the present results indicate a close similarity in immunosuppressive M phi s induced by MAC and MT infections.     

Different correlations of drug susceptibilities to colonial morphology in Mycobacterium avium complex strains

In Mycobacterium avium and Mycobacterium intracellulare complex strains isolated from patients who were not treated previously by any antituberculosis drugs or from fowls, the colonial morphology, smooth, domed, opaque (SmD) or smooth, flat, transparent (SmT) colonial forms, significantly correlated with susceptibilities to rifampicin, minocycline, streptomycin, kanamycin, enviomycin, ethambutol, and sulfadimethoxine, whereas it did not correlate with susceptibilities to isoniazid, cycloserine, and ethionamide. Strains with the SmT colonial morphology were more resistant to the former seven drugs than strains with the SmD colonial morphology. Since the susceptibilities to antituberculosis drugs with large molecules correlated with the colonial morphology, it has been suggested that a permeability barrier that allows passage of small molecules but prevents passage of large molecules exists in the strains with the SmT colonial morphology.     

Extracellular-regulated kinase activation regulates replication of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> intracellularly in primary human monocytes

Mycobacterium avium-intracellulare (MAI) is a ubiquitous environmental pathogen that causes disseminated infection in immunocompromised patients, such as those with human immunodeficiency virus, interleukin-12 deficiency, or interferon-γ receptor mutation. Colony morphotypes are associated with MAI pathogenicity. Our previous studies have reported that smooth-transparent (SmT) morphotypes are more virulent and induce less cytokine (interleukin-1β and tumor necrosis factor-α) production by human monocytes than the smooth-domed (SmD) morphotypes. Mitogen-activated protein (MAP) kinases such as extracellular-regulated kinase (ERK) are activated by the phagocytosis of particle antigens in macrophages, and this ERK activation subsequently influences cytokine expression and the control of intracellular pathogen growth. The influence of MAP kinase activation on MAI replication in human monocytes was examined. Peripheral blood monocytes isolated from healthy subjects by Ficoll-Hypaque sedimentation were infected with virulent SmT or avirulent SmD MAI without or with MAP kinase inhibitors. MAP kinase activities were determined by in vitro kinase assay, intracellular MAI growth by CFU assay, and cytokines by enzyme-linked immunosorbent assay. MAI infection induced ERK and p38 activation. Inhibition of ERK by PD98059, but not p38, significantly increased intracellular MAI growth. Tumor necrosis factor-α release and interleukin-1β production in response to MAI were reduced by MAP kinase inhibition. p38 inhibition tended to reduce cytokine production more substantially. These data suggest that ERK activation limits intra-monocytic MAI replication and enhances monocytic cytokine release, whereas p38 activation influences only cytokine release. The effect of MAP kinases on MAI growth might thus be mediated by the modulation of cytokine production.     

Normal and tumor-bearing host macrophage factor-mediated modulation of mixed-lymphocyte reaction responsiveness: Separation of T-lymphocyte subset susceptibility to enhancing and inhibitory factors

The mixed-lymphocyte reaction reactivity of normal and tumor-bearing host (TBH) T-cell subsets was examined in response to normal and TBH macrophage (Mø) supernatants. Both inhibiting and enhancing activities were identified in normal and TBH Mø supernatants. The present data suggest that TBH Mø supernatants contained more inhibitory activity than normal host Mø supernatants and that enhancing activity of Mø supernatants was restricted to the Lyt 2,3⁺ population of cells. TBH Lyt 2,3⁺ cells were more responsive to the enhancing molecule(s) than their normal counterparts. These data were consistent with studies which implicate Mø as being partially responsible for the immune dysfunction seen in TBH, and extends previous findings on the ability of Mø to regulate the immune response in an attempt to achieve homeostasis.     

Activation of the mitogen-activated protein kinase signaling pathway is instrumental in determining the ability of Mycobacterium avium to grow in murine macrophages.

Of the two common morphotypes of Mycobacterium avium, designated smooth transparent (SmT) or smooth opaque (SmO), the SmO morphotype is avirulent, whereas the SmT morphotype is virulent. The role of the host macrophage in determining these different virulence phenotypes was analyzed using an in vitro model of macrophage infection. Initial studies confirmed previous reports of the increased ability of the SmT bacteria to grow in macrophages; this increased virulence correlated with reduced induction of inflammatory cytokines. Examination of the response of the mitogen-activated protein kinase (MAPK) pathway following infection with either morphotype revealed that all three members of the MAPK pathway were activated. Pharmacologic inhibition of either the extracellular signal-regulated kinase (ERK) or p38(MAPK) pathways resulted in distinct consequences for the growth of the two morphotypes. In particular, inhibition of the p38(MAPK) resulted in attenuated growth of the SmT morphotype, which correlated with reduced PGE(2) production. Inhibition of cyclooxygenase 2 by indomethacin also inhibited growth of SmT, substantiating the role for PGE(2) in promoting the growth of SmT. In contrast, SmO induction of the ERK pathway was increased compared with the SmT morphotype, and inhibition of ERK resulted in decreased TNF-alpha synthesis and enhanced SmO growth. Pharmacologic inhibitors of the MAPK pathway were present for only the first 4 h of infection and yet had consequences for bacterial growth at 7 days. Therefore, the data suggest that induction of the MAPK pathway during uptake of bacteria is instrumental in determining the eventual fate of the bacteria.     

A SmD peptide induces better antibody responses to other proteins within the small nuclear ribonucleoprotein complex than to SmD protein via intermolecular epitope spreading

Autoantibody response against the small nuclear ribonucleoprotein (snRNP) complex is a characteristic feature of systemic lupus erythematosus. The current investigation was undertaken to determine whether activation of SmD-reactive T cells by synthetic peptides harboring T cell epitopes can initiate a B cell epitope spreading cascade within the snRNP complex. T cell epitopes on SmD were mapped in A/J mice and were localized to three regions on SmD, within aa 26-55, 52-69, and 86-115. Immunization with synthetic peptides SmD(31-45), SmD(52-66), and SmD(91-110) induced T and B cell responses to the peptides, with SmD(31-45) inducing the strongest response. However, only SmD(52-66) immunization induced T cells capable of reacting with SmD. Analysis of sera by immunoprecipitation assays showed that intermolecular B cell epitope spreading to U1RNA-associated A ribonucleoprotein and SmB was consistently observed only in the SmD(52-66)-immunized mice. Surprisingly, in these mice, Ab responses to SmD were at low levels and transient. In addition, the sera did not react with other regions on SmD, indicating a lack of intramolecular B cell epitope spreading within SmD. Our study demonstrates that T cell responses to dominant epitope on a protein within a multiantigenic complex are capable of inducing B cell responses to other proteins within the complex. This effect can happen without generating a good Ab response to the protein from which the T epitope was derived. Thus caution must be taken in the identification of Ags responsible for initiating autoimmune responses based solely on serological analysis of patients and animals with systemic autoimmune disorders.     

Anti‐interleukin‐5‐neutralizing antibody attenuates caradiac injury and cadiac dysfunction by aggravating the inflammatory response in doxorubicin‐treated mice

Previous studies have demonstrated that interleukins (ILs) are closely associated with doxorubicin (DOX)‐induced cardiac injury. IL‐5 is an important member of the IL family, and this study was performed to investigate whether IL‐5 affects DOX‐induced cardiac injury and its underlying mechanisms. The cardiac IL‐5 expression was first detected and the results showed that cardiac IL‐5 levels were significantly lower in DOX‐treated mice, and IL‐5 was mainly derived from cardiac macrophage (Mø). In addition, some DOX‐treated mice received an injection of anti‐IL‐5‐neutralizing antibody (nAb), and we found that treatment with a mouse anti‐IL‐5 nAb significantly upregulated the levels of myocardial injury markers, aggravated cardiac dysfunction, increased M1 macrophage (Mø1) and decreased M2 macrophage (Mø2) differentiation, and promoted apoptotic marker expression. Furthermore, the effect of mouse IL‐5 nAb on DOX‐induced Mø differentiation and its role on mouse cardiomyocyte (MCM) cells apoptosis were detected in vitro, and the results exhibited that mouse IL‐5 nAb promoted Mø1 differentiation but inhibited Mø2 differentiation in vitro and alleviated apoptosis in MCM cells. Our results found a mouse anti‐IL‐5 nAb‐aggravated DOX‐induced cardiac injury and dysfunction by alleviating the inflammatory response and myocardial cell apoptosis.     

Impaired innate immune response and enhanced pathology during Citrobacter rodentium infection in mice lacking functional P‐selectin

The selectin family of adhesion molecules mediates recruitment of immune cells to sites of inflammation which is critical for host resistance against infection. To characterize the role of selectins in host defence against Citrobacter rodentium infection, wild‐type (WT) mice and mice lacking P‐selectin glycoprotein ligand‐1 (PSGL‐1), P‐, E‐ and L‐selectin were infected using a Citrobacter‐induced colitis model. Infected mice lacking PSGL‐1 or P‐selectin showed a more pronounced morbidity associated with higher bacterial load, elevated IL‐12 p70, TNF‐α, IFN‐γ, MCP‐1 and IL‐6 production, more severe inflammation and surprisingly higher leucocyte infiltration in the guts than WT control. Recruitment of neutrophils and macrophages and caecal inflammation were drastically reduced in infected P‐selectin knockout mice receiving blocking monoclonal antibodies to ICAM‐1 or LFA‐1, indicating that these adhesion molecules may compensate for the loss of selectins in leucocyte recruitment. Furthermore, the adaptive immune response in mice lacking PSGL‐1 or P‐selectin remained functional since these infected mice were capable of eradicating the bacteria and being protected upon re‐challenge with C. rodentium. These data demonstrate a definitive phenotypic impairment of innate response in mice lacking PSGL‐1 or P‐selectin, and suggest that these adhesion molecules are important in host innate immune response against Citrobacter infection.     

Effect of melphalan in vitro on induction of murine suppressor T cells by ConA

Summary The effect of treatment with melphalan in vitro on the activity of spleen cells from BALB/c mice was investigated. Incubation of spleen cells with 1.5–5 g melphalan/1×10⁷ inhibited subsequent mitogenic stimulation by ConA or PHA and the allogeneic response of BALB/c spleen cells against C57B1 target spleen cells. Incubation of spleen cells with ConA led to induction of suppressor T cells which when added to fresh cultures inhibited the allogeneic response. Preincubation of spleen cells with melphalan even at low concentrations (0.15–0.5 g 1×10⁷ cells) which do not directly affect mitogenic stimulation or allogeneic response partially inhibited the generation of suppressor T cells by ConA. Treatment with melphalan had no effect on already induced suppressor T cells as shown by incubation of spleen cells with melphalan (0.15–5 g/1×10⁷ cells) after incubation with ConA. Addition of cells treated with melphalan alone (without ConA) to fresh cultures led to an increase in the allogeneic response.     

CD8+ T Lymphocytes Are the Major Cell Population Involved in the Early Gamma Interferon Response and Resistance to Acute Primary Toxoplasma gondii Infection in Mice

Gamma interferon (IFN-γ) is known to be a major mediator influencing host defense against Toxoplasma (T.) gondii. To evaluate lymphocyte populations involved in this cytokine-mediated early resistance to T. gondii, the effects of in vivo administration of monoclonal antibodies (MAbs) against T-cell subsets and anti-asialo GM1 antibody on the course of infection and IFN-γ response were investigated in mice infected acutely with this parasitic protozoan. A single injection of anti-CD8 MAb on day ?1 or day 4 severely exacerbated the infection, in accordance with a marked suppression of endogenous IFN-γ production. Moreover, the administration of anti-IFN-γ MAb on day 0 but not later than day 4 resulted in a total abrogation of resistance to T. gondii, suggesting that endogenous IFN-γ produced during the first several days of infection is critical for the generation of antitoxoplasmal resistance in mice. In contrast, no significant increase in mortality was observed when injected with either anti-CD4 MAb or anti-asialo GM1 antibody on day ? 1, while these antibodies reduced significantly the ability of mice to produce IFN-γ. Indeed, simultaneous depletion of CD4⁺ and CD8⁺ cells had no greater suppressive effect on host defense and endogenous IFN-γ production than depletion of CD8⁺ cells alone. Together, these results suggest that CD8⁺ T cells play a central role for resolution of acute toxoplasmosis by participating in endogenous IFN-γ production. The possible role of early produced IFN-γ in the development of protective immune response to T. gondii is also discussed.     

Suppression of IgM responses to type III pneumococcal polysaccharide by lipopolysaccharide (LPS).

Lipopolysaccharide (LPS) suppressed the primary IgM response of mice to Type III pneumococcal polysaccharide (S3). LPS and S3 had to be given together in order for maximum suppression to occur and suppression was not due to a change in the time of the peak response in mice which received LPS. Suppression was not due to an effect of LPS on T cells since S3 responses of nude mice were suppressed by LPS. LPS did not suppress S3 responses of C₃H/HeJ mice and base hyrolysis of LPS destroyed the suppressive effect, i.e., suppression was dependent of B cell mitogenic activity of the LPS. The suppressive effect of LPS was presumably directed against virgin B cells since LPS did not suppress the S3 response of primed mice and did not suppress the development of IgG memory responses to S3.     

In vivo modulation of atypical mycobacterial infection: adjuvant therapy increases resistance to Mycobacterium avium by enhancing macrophage effector functions

Susceptible BALB/c mice were infected iv with a strain of Mycobacterium avium and infused with different biological response modifiers (BRM) in a gel delivery system so as to modify the progression of the infection in a beneficial fashion. Infusion of IL-2 or IL-4 in hydrophobic gels led to no significant enhancement of resistance. Infusion of muramyl dipeptide in hypromellose led to a significant enhancement of resistance against the M. avium, as seen by a significant reduction of colony-forming units (CFU) in the spleens of infected mice. Similarly, infusion of interleukin-1 beta in hypromellose in infected mice led to a significant reduction in CFU counts in the organs of mice. The mechanism(s) responsible for this enhanced resistance was studied further. It was found that infected mice developed profound immunosuppression, as judged by mitogenic and antigenic stimulation. Mice infused with MDP/hypromellose developed a similar immuno-suppression, suggesting that this adjuvant immunotherapy did not act by stimulating a T-cell response or by abrogating a putative suppressive phenomenon. Macrophages from mice infused with MDP alone were no more bacteriostatic for a virulent M. avium than control cells. However, macrophages from infected mice infused with MDP/hypromellose were more bacteriostatic for M. avium than cells from mice infected with M. avium and infused with the hydrophobic gel only. Overall, these results suggest that adjuvant immunotherapy is beneficial in M. avium infections.     

Suppressor cell activity of intestinal mucosal leucocytes from buffalo (Bubalus bubalis arni)

Suppressor activity of buffalo intestinal intraepithelial leucocytes and lamina propria leucocytes was induced by Concanavalin A, and was assayed against the mitogenic response of autologous and allogenic leucocytes to mitogens. Appreciable suppression was observed with 25 micrograms ConA/ml on the proliferative activity of the responder cells cocultured at a ratio of < 2:1 (suppressor:responder cell). Mitomycin C treatment of intestinal leucocytes did not totally vanish the viability and functionality of leucocytes.     

Brucella abortus lipopolysaccharide is mitogenic for spleen cells of endotoxin-resistant C3H/HeJ mice.

Preparations of lipopolysaccharide (LPS) from rough and smooth strains of Brucella abortus were mitogenic for spleen cells of athymic nude mice, C3H/HeAU mice, and the endotoxin-resistant C3H/Hej mice. The mitogenic response induced by crude smooth-LPS (f5) was greater than that produced by purified smooth-LPS (f5p); however, the dose-response curves were similar for both preparations. The mitogenic activity of mouse spleen cells to both f5 and f5p was higher than that produced by stimulation with purified rough-LPS. The dose-response curves with rough-LPS were also qualitatively different from those produced with the preparations of smooth-LPS.     

Single-epitope DNA vaccination prevents exhaustion and facilitates a broad antiviral CD8+ T cell response during chronic viral infection

Induction of a monospecific antiviral CD8+ T cell response may pose a risk to the host due to the narrow T cell response induced. At the individual level, this may result in selection of CD8+ T cell escape variants, particularly during chronic viral infection. Second, prior immunization toward a single dominant epitope may suppress the response to other viral epitopes, and this may lead to increased susceptibility to reinfection with escape variants circulating in the host population. To address these issues, we induced a memory response consisting solely of monospecific, CD8+ T cells by use of DNA vaccines encoding immunodominant epitopes of lymphocytic choriomeningitis virus (LCMV). We analyzed the spectrum of the CD8+ T cell response and the susceptibility to infection in H-2(b) and H-2(d) mice. Priming for a monospecific, CD8+ T cell response did not render mice susceptible to viral variants. Thus, vaccinated mice were protected against chronic infection with LCMV, and no evidence indicating biologically relevant viral escape was obtained. In parallel, a broad and sustained CD8+ T cell response was generated upon infection, and in H-2(d) mice epitope spreading was observed. Even after acute LCMV infection, DNA vaccination did not significantly impair naturally induced immunity. Thus, the response to the other immunogenic epitopes was not dramatically suppressed in DNA-immunized mice undergoing normal immunizing infection, and the majority of mice were protected against rechallenge with escape variants. These findings underscore that a monospecific vaccine may induce efficient protective immunity given the right set of circumstances.     

Taenia crassiceps infection abrogates experimental autoimmune encephalomyelitis

Helminth infections induce strong immunoregulation that can modulate subsequent pathogenic challenges. Taenia crassiceps causes a chronic infection that induces a Th2-biased response and modulates the host cellular immune response, including reduced lymphoproliferation in response to mitogens, impaired antigen presentation and the recruitment of suppressive alternatively activated macrophages (AAMФ). In this study, we aimed to evaluate the ability of T. crassiceps to reduce the severity of experimental autoimmune encephalomyelitis (EAE). Only 50% of T. crassiceps-infected mice displayed EAE symptoms, which were significantly less severe than uninfected mice. This effect was associated with both decreased MOG-specific splenocyte proliferation and IL-17 production and limited leukocyte infiltration into the spinal cord. Infection with T. crassiceps induced an anti-inflammatory cytokine microenvironment, including decreased TNF-α production and high MOG-specific production of IL-4 and IL-10. While the mRNA expression of TNF-α and iNOS was lower in the brain of T. crassiceps-infected mice with EAE, markers for AAMФ were highly expressed. Furthermore, in these mice, there was reduced entry of CD3⁺Foxp3⁻ cells into the brain. The T. crassiceps-induced immune regulation decreased EAE severity by dampening T cell activation, proliferation and migration to the CNS.     

Concurrent infections with Trypanosoma brucei and Nippostrongylus brasiliensis in mice deficient in inducible nitric oxide

Concurrent infection with Trypanosoma brucei (Tb) delays the normal protective responses of mice to the gastrointestinal parasite Nippostrongylus brasiliensis (Nb). The course of such infections was followed in mice genetically deficient in inducible nitric oxide synthase (INOS) to assess the role of nitric oxide (NO) in this effect. The time course of trypanosome infection in INOS deficient (INOS-/-) mice was similar to that in wild type (WT) and heterozygote (INOS+/-) mice but did not result in NO production. Although concurrent infection with Tb increased initial susceptibility to Nb in INOS-/- mice, the immune-mediated loss of N. brasiliensis and the associated decline in faecal egg output occurred more rapidly then in WT and INOS+/- littermates. Concurrent infection with trypanosomes markedly suppressed Concanavalin A (ConA)-induced in vitro proliferation of splenic lymphocytes in all groups, but had little effect on the responses of mesenteric node lymphocytes. Trypanosome infection was also associated with increased early release of interferon-gamma and reduced IL-5 from lymphocytes stimulated in vitro with ConA, but did not affect later release of IL-5. The overall similarity of proliferative and cytokine responses in WT, INOS+/- and INOS-/- mice suggest that the suppressive effects of T. brucei on N. brasiliensis infection do not simply reflect depressed lymphocyte responsiveness or altered cytokine profiles. NO appears to be involved in suppression only of the later phases of the host responses to Nb.     

Effect of E-64, thiol protease inhibitor, on the secondary anti-SRBC response in vitro

E-64, L-trans-epoxysuccinyl- leucylamido (4-guanidino) butane, a specific inhibitor of thiol proteases originally isolated from the culture of a fungus, was examined in connection with the immune responses to the splenocytes of mice. In cultures of C3H/He mouse splenocytes, E-64 and its analogues showed mitogenic activity, and some of them enhanced the lymphocyte blast transformation induced by a suboptimal concentration of concanavalin A. E-64 caused a significant suppressive effect on the secondary anti-SRBC responses when 7- or 14-day-primed BDF1 mouse splenocytes were cultured with SRBC, while it induced no effect on cultured splenocytes either from mice treated with cyclophosphamide, from mice sensitized with dinitrophenyl-Ficoll. The results with E-64 and its close analogues revealed that their effects on the immune response roughly correlated with their inhibitory activity against thiol protease. These results suggest that a thiol protease might be involved in the process of secondary immune response in mouse splenocytes.     

The role of antibody in recovery from experimental rabies. I. Effect of depletion of B and T cells.

The avirulent high egg passage (HEP) strain of rabies virus produces an inapparent infection limited to the central nervous system (CNS) in intracerebrally inoculated adult mice. Heavy chain isotype (anti-mu antiserum) immunosuppression potentiates the infection, with a mortality of about 60% and with elevated virus titers in the brain. Anti-mu-treated mice fail to raise antibody responses to rabies virus although their T cell function is normal when measured by the concanavalin A response of splenic lymphocytes. This indicates that the B cell response plays an important role in clearance of rabies virus from the neuroparenchyma. Treatment with cyclophosphamide or by adult thymectomy, x-irradiation, and bone marrow reconstitution potentiates HEP infection to a greater extent than does isotype suppression. Since these suppressive techniques impair both T and B lymphocyte responses, the data suggest that cellular immune mechanisms may also contribute to host defenses against this central nervous system (CNS) virus infection.