Cutting edge: Fc receptor type I for IgG on macrophages and complement mediate the inflammatory response in immune complex peritonitis

The contributions of Fc receptors (FcRs) for IgG (FcgammaRs) and complement to immune complex (IC)-mediated peritonitis were evaluated in BALB/c-, C57BL/6-, FcRgamma chain-, and FcR type III for IgG (FcgammaRIII)-deficient mice, backcrossed to the C57BL/6 background. In BALB/c mice, but not in C57BL/6 mice, neutrophil migration was markedly attenuated after complement depletion. In mice lacking FcRgamma chain, neutrophil migration was abolished, whereas it was unaffected in FcgammaRIII-deficient mice. Huge amounts of TNF-alpha (TNF) were found in the peritoneal exudate of BALB/c and C57BL/6 mice but were absent in mice lacking FcRgamma chain or FcgammaRIII. Surprisingly, a functional inhibition of TNF in BALB/c and C57BL/6 mice had no effect on neutrophil infiltration. These data provide evidence that in IC peritonitis, the activation of FcR type I for IgG on peritoneal macrophages and the activation of the complement cascade, but not the interaction of ICs with FcgammaRIII and the subsequent release of TNF, initiate the inflammatory response in BALB/c and C57BL/6 mice.     

Murine cryoglobulinemia: pathogenic and protective IgG3 self-associating antibodies

A murine IgG3 mAb, 6-19, derived from autoimmune MRL-lpr/lpr mice, is a rheumatoid factor (RF) specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intra-peritoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces skin leukocytoclastic vasculitis and acute glomerulonephritis associated with cryoglobulinemia. Because IgG3 interact with each other, we have determined whether noncryoprecipitating IgG3 mAb were able to inhibit the cryoprecipitation of 6-19 mAb and the development of related tissue lesions. In vitro, the cryoprecipitation of 6-19 mAb was almost completely inhibited by a fourfold excess of a noncryoprecipitating non-RF IgG3 (9-106) mAb derived from MRL-lpr/lpr mice. Cryoprecipitation of five other IgG3 mAb was similarly inhibited by the 9-106 mAb, and two other noncryoprecipitating IgG3 mAb, including the 2-6D antinuclear autoantibody, inhibited the cryoprecipitation of 6-19 mAb. In vivo, pretreatment of BALB/c mice with 9-106 or 2-6D mAb prevented the development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was greatly diminished in 9-106 or 2-6D mAb-treated mice, although their serum levels of 6-19 mAb and RF activity were comparable to those of control mice. This indicated that pretreatment with non-cryoglobulin IgG3 inhibited the cryoglobulin generation and cryoglobulin-associated tissue lesions induced by an IgG3 RF cryoglobulin-generating mAb. These results suggest that the balance of formation of IgG3 autoantibodies with or without the cryoglobulin activity may be critical for the development of IgG3 cryoglobulin-mediated tissue lesions in murine lupus, particularly in MRL-lpr/lpr mice.     

Prevention of murine cryoglobulinemia and associated pathology by monoclonal anti-idiotypic antibody

A murine IgG3 mAb, clone 6-19, derived from non-manipulated autoimmune MRL/MpJ-lpr/lpr mice is a rheumatoid factor specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intraperitoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces, within 18 h, remarkable pathology characterized by skin vasculitis and acute glomerulonephritis associated with cryoglobulinemia. In order to evaluate the possibility of modulating the development of tissue lesions by an anti-Id antibody, we have raised an IgG2b anti-Id mAb specific to the 6-19 mAb. The cryoprecipitation of 6-19 mAb was completely inhibited in the presence of excess amounts of anti-Id mAb in vitro. In vivo, pretreatment of BALB/c mice with anti-6-19 anti-Id mAb inhibited development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was markedly diminished due to enhanced elimination of the 6-19 mAb from the circulation. In contrast, pretreatment with an IgM anti-IgG3 rheumatoid factor mAb neither protected nor aggravated the development of tissue lesions. These results suggest possible implications in the anti-Id treatment of similar vascular diseases in man.     

Comparative analysis of clinics,pathologies and immune responses in BALB/c and C57BL/6 mice infected with Streptobacillus moniliformis

Streptobacillus (S.) moniliformis is a rat-associated zoonotic pathogen that occasionally causes disease in other species. We investigated the working hypothesis that intranasal infection might lead to different immune responses in C57BL/6 and BALB/c mice associated with distinct pathologies. This study confirmed with 75% mortality the known high susceptibility of C57BL/6 mice to Streptobacillus moniliformis infection in comparison to BALB/c mice which did not develop signs of disease. Main pathologies in C57BL/6 mice were purulent to necrotizing lymphadenitis and pneumonia. Significant seroconversion was recorded in surviving mice of both strains. Differentiation of IgG-subclasses revealed mean ratios of IgG2b to IgG1 below 0.5 in sera of all mice prior to infection and of BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 2.5 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice. Evaluation of different sentinel systems revealed that cultural and serological investigations of these animals might not be sufficient to detect infection. In summary, an intranasal S. moniliformis infection model in C57BL/6 mice leading to purulent to necrotizing inflammations in the lung, the lymph nodes and other organs associated with a Th1 immune response is described.     

Onchocerca volvulus keratitis (river blindness) is exacerbated in BALB/c IL-4 gene knockout mice

To determine the outcome of Onchocerca volvulus keratitis in IL-4(-/-) BALB/c mice, animals were immunized subcutaneously and injected into the corneal stroma with soluble O. volvulus antigens. IL-4(-/-) BALB/c mice had a deviated cellular response, with decreased serum IgE and IgG1 and elevated IgG2a compared with control BALB/c mice. In marked contrast to control BALB/c, C57BL/6, and IL-4(-/-) C57BL/6 mice, IL-4(-/-) BALB/c mice developed severe corneal opacification and neovascularization that was associated with a pronounced neutrophil infiltrate to the corneal stroma. STAT-6(-/-) BALB/c mice had the same phenotype as IL-4(-/-) BALB/c mice, and complement depletion had no effect on the severity of O. volvulus keratitis in these mice. These findings indicate that on a BALB/c background, IL-4 has a critical role in regulating neutrophil recruitment to the cornea and development of O. volvulus keratitis.     

Differences in interleukin-12 and -15 production by dendritic cells at the early stage of Listeria monocytogenes infection between BALB/c and C57 BL/6 mice

The mechanisms responsible for the resistance of C57BL/6 mice and for the susceptibility of BALB/c mice to infection with Listeria monocytogenes were studied by comparing early IL-12 and IL-15 production by dendritic cells (DC) after infection with L. monocytogenes. Splenic DC expressing CD11b(low) and CD11c(+) obtained from C57BL/6 mice at 3 and 6 h after L. monocytogenes infection expressed higher levels of IL-12 p40 mRNA and IL-12 p40 protein than did those from BALB/c mice. Concurrently, a larger amount of IFN-gamma was produced by the splenic T cells from C57BL/6 mice in response to immobilized anti-TCRalphabeta mAb than by those from BALB/c mice, while the splenic T cells from BALB/c mice produced a higher level of IL-4 upon TCR alphabeta stimulation than did those of C57BL/6 mice. IL-15 mRNA and intracellular IL-15 protein were detected more abundantly in the DC from C57BL/6 mice than in those from BALB/c mice on day 3 after infection. CD3(+) IL2Rbeta (+) cells in the spleen were increased in C57BL/6 mice but not in BALB/c mice at the early stage after infection. Furthermore, IL-12Rbeta2 gene expression was up-regulated in T cells from C57BL/6 mice but not in those from BALB/c mice at the early stage after listerial infection. These results suggest that the difference in early production of IL-12 and IL-15 by DC may at least partly underlie the difference in susceptibility to L. monocytogenes between C57BL/6 and BALB/c mice.     

Cytokines regulate the pattern of rejection and susceptibility to cyclosporine therapy in different mouse recipient strains after cardiac allografting

We determined the role of cytokines in regulating the pattern of rejection and recipient susceptibility to cyclosporine (CsA) in a mouse cardiac allograft model. Hearts from C3H mice transplanted into untreated BALB/c (Th2-dominant) and C57BL/6 (Th1-dominant) mice showed different patterns of rejection. C3H allografts in BALB/c mice showed typical acute vascular rejection (AVR) with strong intragraft deposition and high serum levels of anti-donor IgG with predominant IgG1, while C3H allografts in C57BL/6 mice showed typical acute cellular rejection (ACR) with massive intragraft infiltration of CD4(+) and CD8(+) lymphocytes and low serum levels of anti-donor IgG with predominant IgG2a. Elevated intragraft mRNA expression of IL-2, IFN-gamma, and IL-12 mRNA was present in C57BL/6 recipients, whereas allografts in BALB/c mice displayed increased IL-4 and IL-10 mRNA levels. CsA therapy completely inhibited ACR and induced indefinite allograft survival in C57BL/6 recipients, while the same therapy failed to prevent AVR, and only marginally prolonged graft survival in BALB/c recipients. In contrast, rapamycin blocked AVR, achieving indefinite survival in BALB/c recipients, but was less effective at preventing ACR in C57BL/6 recipients. The disruption of the IL-12 or IFN-gamma genes in C57BL/6 mice shifted ACR to AVR, and resulted in concomitant recipient resistance to CsA therapy. Conversely, disruption of IL-4 gene in BALB/c mice markedly attenuated AVR and significantly prolonged allograft survival. These data suggest that the distinct cytokine profiles expressed by different mouse strains play an essential role in regulating the pattern of rejection and outcome of CsA/rapamycin therapy.     

Analyses of Idiotypes on Anti-α-1, 6-Dextran Antibodies in Mice

When mice of strains C57BL/6, C3H/He, and BALB/c were immunized with native dextran B512, only a small amount of IgM antibody was produced, but a substantial amount of anti-dextran antibody of IgG class was produced after immunization with a conjugate of dextran T10 and keyhole limpet hemocyanin regardless of the mouse strain used. Isoelectric focusing (IEF) spectra revealed limited heterogeneity of anti-dextran antibody of IgG class with strict consistency in all individual sera from C57BL/6 mice, even after secondary immunization, whereas antibodies from C3H/He and BALB/c mice showed more heterogeneous IEF spectra with some individual variations. Rabbit anti-idiotypic (Id) antibodies were raised by immunization with a subfraction of anti-dextran antibody of IgG class from C57BL/6 mice, which showed major bands focused at around pH 7.7 upon IEF. It was found by using the anti-Id antibodies that virtually all anti-dextran antibody molecules of both IgG and IgM classes from C57BL/6 mice possessed common Id determinants which can be classified into two specificities, one specific for antibody from C57BL/6 mice and the other cross-reactive with antibodies from BALB/c and C3H/He mice. About 80% of the antibody molecules from BALB/c and less than 20% of those from C3H/He mice were positive for the interstrain cross-reactive Id. Both Id determinants seemed to be closely related to the antigen binding sites, or at least to reside in the vicinity of the antigen binding sites of anti-dextran antibody.     

T cells from Leishmania major-susceptible BALB/c mice have a defect in efficiently up-regulating CXCR3 upon activation

Genetic background influences the outcome of Leishmania major infection. C57BL/6 mice mount a Th1 response and resolve infection. In contrast, BALB/c mice mount a Th2 response and develop chronic lesions. This susceptible phenotype is seen even though BALB/c mice generate IFN-gamma-producing T cells at proportions similar to C57BL/6 mice in their lymph nodes (LN) early after infection. We had previously shown that chemokine receptor CXCR3 mediates immunity against L. major by recruiting IFN-gamma-producing T cells to the lesions of C57BL/6 mice. Therefore, we hypothesized that IFN-gamma-secreting T cells in BALB/c mice are unable to confer protection because they may be defective in up-regulating CXCR3. To test this hypothesis, we analyzed kinetics of CXCR3-expressing T cells in the LN and lesions of BALB/c and C57BL/6 mice during L. major infection. Additionally, we compared the ability of T cells from BALB/c and C57BL/6 mice to up-regulate CXCR3 upon activation. We found that resolution of L. major infection in C57BL/6 mice was associated with an increase in the proportion of CXCR3(+) T cells in regional LN and lesions, whereas disease progression in BALB/c mice was associated with a decrease in these populations. Anti-CD3/CD28-activated T cells from naive BALB/c but not C57BL/6 mice were defective in up-regulating CXCR3. Impaired induction of CXCR3 on BALB/c T cells was not due to lack of IFN-gamma and was mediated partially by IL-10 but not IL-4 or IL-13. We propose that defective CXCR3 up-regulation on T cells in BALB/c mice may contribute to L. major susceptibility.     

Leishmania amazonensis: humoral response to amastigote excreted-antigens in murine leishmaniasis

With the purpose of studying the antigenic role that factors excreted by Leishmania amastigotes might have during murine infection, immunoblots were carried out with sera from C57BL/6 and BALB/c mice infected with two strains of Leishmania (L.) amazonensis, NR and IFLA/BR. Both strains differ widely in virulence in BALB/c mice. BALB/c but not C57BL/6 sera recognized several excretion products. The excreted antigens showed a strong response towards IgG1 and IgG2a isotypes whilst they reacted only weakly against IgG2b and IgG3. A low-molecular weight antigen (about 20 kDa) excreted by both Leishmania strains was strongly recognized by IgG1 from BALB/c mice sera infected with IFLA/BR, the most virulent strain. Sera from NR infected mice were incapable of recognizing this antigen in spite of its presence in NR excreted products. The results indicate that the humoral immune response to excreted antigens of amastigotes depends on both the host genetic background and the parasite strain.     

The influence of Mesocestoides corti on subsequent Angiostrongylus cantonensis infections in mice.

The influence of Mesocestoides corti on subsequent Angiostrongylus cantonensis infection in mice (C57BL/6 and BALB/c) was assessed. Both strains of mice infected with M. corti demonstrated a peak blood eosinophilia at around 3 weeks post-infection (p.i.). C57BL/6 and BALB/c mice primarily infected with M. corti were given A. cantonensis infection 18 days later, but pre-existing M. corti infection did not affect the recovery of intracranial worms of A. cantonensis at day 21 p.i. BALB/c mice with mixed parasite infections showed low morbidity and mortality as compared with mice singly infected with A. cantonensis and some mice demonstrated a pulmonary migration of intracranial worms. In C57BL/6 mice, intracranial worms were killed and thus all mice survived. C57BL/6 mice with mixed parasite infections failed to resist A. cantonensis reinfection. The blastogenic responses of spleen cells against A. cantonensis antigen were lower in BALB/c than in C57BL/6 mice and mixed parasite infections also resulted in less blastogenic responses against both concanavalin A and A. cantonensis antigen than monoinfection. The recovery of M. corti biomass was significantly higher in mice with mixed parasite infections than mice with monoinfection with M. corti. These data suggest a distinct difference in response to A. cantonensis infection between C57BL/6 and BALB/c mice, and the induction of immunosuppression in both mouse strains following M. corti infection. Blood eosinophilia provoked by M. corti infection is not directly associated with the killing of worms in subsequent A. cantonensis infection.     

Adaptive differentiation of H-2- and Igh-restricted B lymphocyte in tetraparental bone marrow chimera

Immunization of BALB/c mice with MOPC-104E myeloma protein induced idiotype-specific enhancing B cells that acted on anti-dextran antibody producing B cells. The enhancing cells have the surface phenotype of B cells. With the use of several H-2 or Igh congenic mice, it was found that the cooperation among B cells was controlled by both the major histocompatibility complex (MHC) and Igh. The capability to generate enhancing B cell activity was analyzed by using tetraparental bone marrow chimeras. (C57BL/6 X BALB/c)F1 mice, for example, were lethally irradiated and were reconstituted with C57BL/6 and BALB/c bone marrow cells. Nine to 12 wk after the reconstitution, the chimeras were immunized with the myeloma protein and were tested for their enhancing B cell activity. After the removal of C57BL/6 origin cells by treatment with anti-H-2b + complement, residual cells exhibited enhancing B cell activity on BALB.B, as well as BALB/c antidextran antibody response. This indicates that the generation of H-2-restricted, idiotype-specific enhancing B cell activity differentiated adaptively so as to recognize foreign MHC as self under chimeric conditions. On the other hand, splenic B cells treated with anti-H-2d + complement did not enhance the responses of BALB/c or BALB.B. Even in a chimeric environment, the B cells of C57BL/6 origin could not obtain the ability to generate enhancing B cell activity upon immunization of the idiotype. The results described here, taken in conjunction with our previous studies, suggest that the Ig heavy chain gene(s) predominantly control the Igh restriction properties of enhancing B cells, and the capability of MHC recognition by B cells is selected under chimeric conditions.     

Contribution of classic and alternative effector pathways in peanut-induced anaphylactic responses

Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. However, the pathways of anaphylaxis in food allergy are still relatively unknown. We investigated the effector pathways of allergic and anaphylactic responses of different strains of mice in a clinical relevant model of peanut allergy. C3H/HeOuJ, C57BL/6 and BALB/c mice were sensitized by intragastric peanut extract and challenged by intragastric or intraperitoneal injection of peanut. Peanut-specific T cell responses, IgE, IgG1 and IgG2a and mucosal mast cell degranulation were induced to different extent in C3H/HeOuJ, C57BL/6 and BALB/c mice. Interestingly, anaphylactic symptoms after systemic challenge were highest in C3H/HeOuJ followed by C57BL/6 but were absent in BALB/c mice. Mechanistic studies showed that the food allergic systemic anaphylaxis was dependent on platelets, FcRγ and mast cells, and partially dependent on platelet activating factor and monocytes/macrophages, depending on mouse strain. These data demonstrate that in three mouse strains, components of the classic and alternative anaphylactic cascade are differently expressed, leading to differential outcomes in parameters of allergic disease and food induced systemic anaphylaxis.     

A lupus-suppressor BALB/c locus restricts IgG2 autoantibodies without altering intrinsic B cell-tolerance mechanisms

FcgammaR2B-deficient mice develop autoantibodies and glomerulonephritis with a pathology closely resembling human lupus when on the C57BL/6 (B6) background. The same mutation on the BALB/c background does not lead to spontaneous disease, suggesting differences in lupus susceptibility between the BALB/c and B6 strains. An F2 genetic analysis from a B6/BALB cross identified regions from the B6 chromosomes 12 and 17 with positive linkage for IgG autoantibodies. We have generated a congenic strain that contains the suppressor allele from the BALB/c chromosome 12 centromeric region (sbb2(a)) in an otherwise B6.FcgammaR2B(-/-) background. None of the B6.FcgammaR2B(-/-)sbb2(a/a) mice tested have developed IgG autoantibodies in the serum or autoimmune pathology. Mixed bone marrow reconstitution experiments indicate that sbb2(a) is expressed in non-B bone marrow-derived cells and acts in trans. sbb2(a) does not alter L chain editing frequencies of DNA Abs in the 3H9H/56R H chain transgenic mice, but the level of IgG2a anti-DNA Abs in the serum is reduced. Thus, sbb2(a) provides an example of a non-MHC lupus-suppressor locus that protects from disease by restricting the production of pathogenic IgG isotypes even in backgrounds with inefficient Ab editing checkpoints.     

Role of Toxoplasma gondii HSP70 and Toxoplasma gondii HSP30/bag1 in antibody formation and prophylactic immunity in mice experimentally infected with Toxoplasma gondii.

Production of antibodies against Toxoplasma gondii (T. gondii)-derived stress proteins, T. gondii HSP70 (T.g.HSP70) and T.g.HSP30/bagl, in C57BL/6 and BALB/c mice perorally infected with cysts of the avirulent Fukaya strain of T. gondii was analyzed. Production of anti-T.g.HSP70 IgG antibodies was transient, whereas production of anti-T.g.HSP30/bag1 IgG antibodies persisted after infection in both C57BL/6 and BALB/c mice. C57BL/6 mice, a susceptible strain, predominantly produced IgG antibodies specific for T.g.HSP70, whereas BALB/c mice, a resistant strain, predominantly produced IgG antibodies specific for T.g.HSP30/bag1, after T. gondii infection. Immunization with rT.g.HSP30/bag1 enhanced, whereas immunization with rT.g.HSP70 reduced host protective immunity against T. gondii infection with a cyst-forming avirulent strain, Fukaya, and a virulent strain, RH.     

Enalapril protects mice from pulmonary hypertension by inhibiting TNF-mediated activation of NF-kappaB and AP-1

The present study was undertaken to investigate the effects of treatment with the angiotensin-converting enzyme (ACE) inhibitor enalapril in a mouse model of pulmonary hypertension induced by bleomycin. Bleomycin-induced lung injury in mice is mediated by enhanced tumor necrosis factor-alpha (TNF) expression in the lung, which determines the murine strain sensitivity to bleomycin, and murine strains are sensitive (C57BL/6) or resistant (BALB/c). Bleomycin induced significant pulmonary hypertension in C57BL/6, but not in BALB/c, mice; average pulmonary arterial pressure (PAP) was 26.4 +/- 2.5 mmHg (P < 0.05) vs. 15.2 +/- 3 mmHg, respectively. Bleomycin treatment induced activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 and enhanced collagen and TNF mRNA expression in the lung of C57BL/6 but not in BALB/c mice. Double TNF receptor-deficient mice (in a C57BL/6 background) that do not activate NF-kappaB or AP-1 in response to bleomycin did not develop bleomycin-induced pulmonary hypertension (PAP 14 +/- 3 mmHg). Treatment of C57BL/6 mice with enalapril significantly (P < 0.05) inhibited the development of pulmonary hypertension after bleomycin exposure. Enalapril treatment inhibited NF-kappaB and AP-1 activation, the enhanced TNF and collagen mRNA expression, and the deposition of collagen in bleomycin-exposed C57BL/6 mice. These results suggest that ACE inhibitor treatment decreases lung injury and the development of pulmonary hypertension in bleomycin-treated mice.     

Repeated respiratory Mycoplasma pneumoniae infections in mice: effect of host genetic background

Respiratory Mycoplasma pneumoniae (Mp) infection is involved in several acute and chronic lung diseases including community-acquired pneumonia, asthma and chronic obstructive pulmonary disease. In the chronic disease process, recurrent respiratory bacterial infections could occur, which may result in varying degrees of symptoms and lung inflammation among patients. However, the lung immunologic differences of host responses to repeated bacterial (i.e., Mp) infections remain to be determined. In the present study, we examined cellular and humoral responses to multiple (up to 3) Mp infections in two genetically different strains of mice (BALB/c and C57BL/6). Mice were intranasally inoculated with one Mp infection, two or three Mp infections (4 weeks apart), and sacrificed on days 3, 7 and 14 after the last Mp infection. Overall, compared to C57BL/6 mice, BALB/c mice demonstrated a significantly higher degree of lung tissue inflammatory cell infiltrate, BAL cellularity, and release of pro-inflammatory cytokines (TNF-alpha, keratinocyte-derived chemokine (KC, a mouse homolog of human chemokine Gro-alpha [CXCL1], and IFN-gamma). In addition, BALB/c mice presented higher levels of serum Mp-specific IgG and IgM, but not IgA. Consistently with lung and serum data, Mp load in BAL and lung specimens was significantly higher in BALB/c mice than C57BL/6 mice. Moreover, repeated Mp infections in BALB/c, but not C57BL/6 mice, produced a greater inflammatory response than did a single Mp infection. Our results suggest that hosts with different genetic background may have different susceptibility to repeated respiratory Mp infections along with inflammatory responses.     

Influence of the immunoglobulin heavy chain locus on expression of the VK1GAC light chain

The VK1GAC light chain represents the dominant V kappa structure employed in the antibody response of A/J mice to streptococcal group A carbohydrate ( GAC ). Two anti-idiotypic antisera, anti- Id5 and anti- Id20 , with specificity for the VK1GAC light chain were used to examine anti- GAC antibody responses in a series of inbred mouse strains that differ at the heavy chain constant region ( IgCH ) allotype locus. Both idiotypes were expressed in normal and immune sera from mice of most IgCH allotypes, except IgCHb (C57BL/6J) and IgCHf (CE/J). C57BL/6J mice expressed Id5 , but not Id20 , whereas CE/J mice did not express either idiotype. Testing of recombinant inbred strains between BALB/c and C57BL/6 indicated that the pattern of idiotype expression did not correlate with IgCH allotype. The C X B recombinants expressed all three idiotype patterns that were observed in the panel of inbred strains. Testing of allotype congenic mice between BALB/c and C57BL/6 showed that CB.20 and BC.8 mice were Id20 -, whereas BAB-14 mice were Id20 +, indicating that both VH and background (V kappa or regulatory) loci must be derived from BALB/c to obtain Id20 expression. The difference in the frequency of idiotype expression observed between BALB/c and BAB-14 mice indicates that the IgCH locus may exert a quantitative influence on the expression of this light chain. To examine the Id20 -, Id5 + antibodies of C57BL/6 mice, anti- GAC hybridomas were prepared. Of 16 C57BL/6-derived anti- GAC monoclonal antibodies, six were reactive with anti- Id5 and not with anti- Id20 . Isoelectric focusing of the purified kappa light chains from three of these antibodies revealed two distinct spectrotypes that co-migrated with the two known VK1GAC spectrotypes observed with A/J anti- GAC light chains. Idiotypic analysis of in vitro recombinants between the heavy and light chains of A/J and C57BL/6 monoclonal antibodies demonstrated that the C57BL/6 light chains were idiotypically similar to A/J light chains when they were free in solution or paired with A/J heavy chains. These results demonstrate that C57BL/6 mice can express a light chain that is very similar, if not identical, to the VK1GAC light chain, although the light chain is expressed in lower frequency and is paired with a distinct VH structure, which can mask expression of one of the VK1GAC idiotypes. These effects on V kappa expression map to at least three genetic loci: VH, CH, and an unlinked locus.     

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.     

Quantitative measurement of mouse IgG subclasses with the use of heteroantisera: the importance of allotype considerations.

Double antibody radioimmunoassays have been used to determine the quantities of IgG1, IgG2a and IgG2b in samples of normal serum IgG from BALB/cJ, AKR/J and C57BL/6J inbred mice. The assays employed subclass-specific goat antisera which had been prepared with BALB/c myeloma proteins as immunogens and as immunoabsorbents. 125I-labeled BALB/c myeloma proteins were used as probes. Results indicate that partial resolution of mouse IgG subclasses was achieved by ion exchange chromatography on DEAE-Sephadex. Nearly all of the protein in BALB/cJ and AKR/J IgG fractions could be accounted for as IgG1, IgG2a and IgG2b, and IgG2a was the predominant species observed. However, considerably less protein in C57BL/6J IgG fractions of purity similar to the BALB/cJ fractions could be accounted for as these three subclasses, and virtually no IgG2a was detected. Furthermore, an IgG2a myeloma protein bearing the C57BL/6 allotype failed to inhibit the IgG2a-specific assay significantly. Thus the IgG2a-specific antibody in the goat heteroantiserum employed appeared to consist nearly exclusively of antibody to BALB/c Ig-1a allotypic determinants. These findings point to the importance of allotype considerations in the use of heteroantisera to quantitate IgG subclasses.