Pristane诱导小鼠系统性红班狼疮动物模型的研究

目的:建立pristane诱导的系统性红斑狼疮(SLE)小鼠模型,并对该小鼠模型的发病机制进行初步的探讨。方法:6-8周龄雌性BALB/c小鼠单次腹腔注射pristane0.5mL,对照组单次腹腔注射PBS0.5mL,注射前及注射后每2周行流式细胞术(FCM)检测外周血中IFN-α分泌细胞(CD11b+Ly6Chigh)的比例及细胞活化状态B220+Aβ1dhigh),ELISA检测血清中自身抗体(anti-dsDNA,anti-smRNP,anti-ribosomalP0)的含量。至6个月处死动物,FCM检测腹腔细胞中IFN-α分泌细胞(CD11b+Ly6Chigh)的比例和脾脏中细胞的活化(B220,Aβ1d),采用直接免疫荧光法标记小鼠肾脏免疫球蛋白复合物及H&E染色评估小鼠肾脏免疫复合物的沉积及损伤情况。结果:小鼠腹腔注射pristane第2个月开始血清总IgG升高,第3个月起出现自身抗体阳性,到个6月时达到最高,并维持高水平至被处死;Pristane处理6个月后,Pristane处理组小鼠出现关节炎症状,肾脏免疫复合物的大量沉积和明显肾脏损伤。Pristane注射2周起,小鼠外周血中IFN-α分泌细胞(CD11b+Ly6Chigh)的比例明显高于PBS注射组,小鼠腹腔细胞中IFN-α分泌细胞的比例也明显升高;同时外周血和脾细胞中B细胞表面MHCII分子Aβ1d的平均荧光强度(MFI)均高于对照组,表明pristane处理组小鼠中B细胞发生了显著活化。结论:BALB/c小鼠腹腔注射pristane可诱导构建小鼠SLE模型,其SLE的发病可能与IFN-α的持续分泌导致B细胞的异常活化有关。该模型的建立为进一步研究SLE的发病机制提供了良好的动物模型。     

miR-150对降植烷诱导的狼疮鼠肺出血的影响

本研究旨在观察miR-150在降植烷诱导的狼疮鼠中对肺出血的发生和严重程度的影响并初步探讨其可能的机制。实验选用8周龄C57/B6小鼠和miR-150基因缺陷小鼠,腹腔注射降植烷(pristane),在注射后的第7天、第10天、第14天观察肺脏大体病理变化,苏木素-伊红(HE)染色观察肺组织病理变化并进行病理评分。发现注射降植烷第10天和(或)第14天后C57/B6组和miR-150缺陷小鼠组均发生不同程度的肺出血,后者的严重程度大于前者。肺组织病理评分显著升高;miR-150缺陷组与C57/B6组比较血浆TNF-α表达升高,右肺上叶组织湿干比值(W/D)也显著升高。提示在降植烷诱导的狼疮鼠模型中miR-150参与了肺出血的发病过程,miR-150有望成为预测狼疮肺出血的生物标记物。     

Distinct granuloma responses in C57BL/6J and BALB/cByJ mice in response to pristane

Granuloma formation is an inflammatory response of the host against invading pathogens or indigestible substances. We generated mesenteric oil granulomas by injecting pristane into the peritoneal cavity (PC) of mice, and compared oil granuloma formation in the C57BL/6J and BALB/cByJ strains of mice. The formation and kinetics of oil granulomas were distinct between the two strains. In C57BL/6J mice, injected pristane induced oil granuloma formation at both the mesenteric centers (MG) and margins (SG). MG was resolving by 11 weeks, and SG persisted. In BALB/cByJ mice, MG developed slower but persisted longer than in C57BL/6J mice, and SG resolved sooner than in C57BL/6J mice. Injection of India ink revealed that phagocytes were localised mainly to the SG in C57BL/6J mice, but were located diffusely in both MG and SG of BALB/cByJ mice. SG cells expressed more monocyte chemotactic protein‐1 (MCP‐1) mRNA than MG cells in C57BL/6J mice, but there was no difference in MCP‐1 expression between the MG and SG in BALB/cByJ mice. These observations suggest that the recruitment of inflammatory leucocytes under the direction of chemokines differentiates the patterns of granuloma responses to pristane in C57BL/6J and BALB/cByJ mice.     

Correlation between levels of immunoglobulins and immune complexes in plasma of C57BL/6 and C57L/J mice infected with MAIDS retrovirus.

Infection with helper-free, defective MAIDS murine leukemia virus (MuLV) caused a rapid polyclonal activation of B cells in 0.75-, 2-, and 6-month-old C57L/J mice (H-2b, Fv-1n/n), similar to that in C57BL/6 mice (H-2b, Fv-1b/b), which was recognized by elevated plasma immunoglobulin concentrations. However, changes in plasma immunoglobulin levels differed in C57BL/J and C57BL/6 mice. In C57L/J mice, infection resulted in a rapid increase in plasma IgM and IgG2a, and the elevation of IgG2a persisted undiminished for 21 weeks. Levels of IgG2b also became slightly elevated, but those of IgG1 and IgG3 were not significantly affected. Plasma of 6 to 7-month-old C57BL/6 mice contained already high levels of IgM (30-40 mg/ml), which persisted undiminished in uninfected mice but decreased progressively in infected mice to 10% of the original concentration during 25 weeks of observation. In C57BL/6 mice, plasma IgG1 and IgG2b as well as IgG2a became similarly elevated after infection but also only transiently. Their levels began to decrease progressively about 10 weeks after infection and fell to far below the maximum concentration observed. The drastic loss of plasma IgM and IgGs observed in C57BL/6 mice during the later stages of MAIDS MuLV infection did not seem to be a consequence of the polyclonal activation of B cells per se but seemed to reflect additional immunological abnormalities arising in infected C57BL/6 but not C57L/J mice. In both mouse strains these changes in plasma Ig levels correlated with the formation of Ig-containing immune complexes that bound to high-affinity, protein-binding ELISA plates in the absence of antigen coating, which may represent unusual forms of self-antigen-antibody complexes.     

FcgammaRIIB deficiency with Fas mutation is sufficient for the development of systemic autoimmune disease

MRL.Fas(lpr/lpr) mice, a model for systemic lupus erythematosus (SLE) and arthritis in humans, have a Fas mutation that results in spontaneous development of systemic autoimmune diseases and a short life span. Half of them die by 5-6 months of age due to massive progression of systemic autoimmune diseases, such as lupus nephritis. However, C57BL/6 (B6).Fas(lpr/lpr) strain does not develop such disorders within the normal life span, indicating that suppressor gene(s) in B6 mice may control the onset and exacerbation of disease. Here, we show that the gene for a unique inhibitory Fc receptor for IgG (Fc gamma RIIB) is a critical SLE suppressor. Fc gamma RIIB-deficient B6.Fas(lpr/lpr) (B6.IIB(-/-)Fas(lpr/lpr)) mice developed systemic autoimmune diseases, including anti-DNA and anti-type II collagen autoantibodies and cryoglobulin production, immune complex glomerulonephritis and arthritis. They were short-lived, due to enhanced autoantibody production by B cells culminating in fatal lupus nephritis. Thus, Fc gamma RIIB deletion with Fas mutation is sufficient for the development of systemic autoimmunity in B6 mice. The inhibitory signaling cascade via Fc gamma RIIB may be critical for suppressing SLE in humans.     

Histological characteristics of lupus nephritis in F1 mice with chronic graft-versus-host reaction across MHC class II difference.

Renal lesions at the chronic phase of MHC class-II-disparate graft-versus-host reaction (GVHR) were examined. To induce GVHR, C57BL/6 (B6) spleen cells were injected twice into either (B6 x bm12)F1 (class-II-disparate), (B6 x bm1)F1 (class-I-disparate) or (bm1 x bm12)F1 mice (class-I + II-disparate). For comparison, (C57BL/10 x DBA/2)F1 (BDF1) mice injected with DBA/2 spleen cells were also used. (B6 x bm12)F1 and BDF1 recipients showed marked elevation of anti-DNA antibodies, circulating immune complexes (CIC) and the number of immunoglobulin producing cells (IgPC). At 20 weeks after cell injection, severe immune complex glomerulonephritis (ICGN) was observed in (B6 x bm12)F1 recipients, but was far less severe in (bm1 x bm12)F1 recipients and was not observed in (B6 x bm1)F1 recipients. ICGN was also observed in BDF1 recipients at 12 weeks after cell injection. By immunofluorescent microscopy, IC deposition was detected along the capillary loops and also in the mesangial area in (B6 x bm12)F1 recipients, while BDF1 recipients showed only a capillary pattern. By light microscopy, the renal lesion of (B6 x bm12)F1 recipients appeared similar to those of BDF1 recipients. Histologically, (B6 x bm12)F1 recipients serve as a good model for lupus glomerulonephritis induced by class-II-disparate GVHR.     

Age-related changes in dermal mast cell prevalence in BALB/c mice: functional importance and correlation with dermal mast cell expression of Kit

Differences in dermal mast cell prevalence for adult mice of different strains have been reported previously. In this study, the dermal mast cell prevalence for BALB/c and C57BL/6 mice at 6 weeks of age was similar but as BALB/c mice matured from 6 to 10 weeks of age, their dermal mast cell prevalence halved. In contrast, there was no significant difference in the dermal mast cell prevalence of 6- and 10-week-old C57BL/6 mice. These differences determined the degree of susceptibility of BALB/c and C57BL/6 mice of different ages to UVB (UV radiation of wavelength 280-320 nm)-induced systemic immunosuppression. Expression of the receptor for stem cell factor, Kit protein, was examined on mast cells under conditions in which the dermal mast cell prevalence varied. A significant correlation was observed between Kit expression by mast cells from adult BALB/c, DBA/2 and C57BL/6 mice and dermal mast cell prevalence. In BALB/c mice, mast cell Kit expression decreased as the mice matured from 6 to 10 weeks of age and correlated with the reduction in dermal mast cell numbers. Kit levels on dermal mast cells from C57BL/6 mice were consistently higher than on mast cells from BALB/c mice although significant reductions in Kit were also measured with ageing from 6 to 10 weeks. We hypothesize that regardless of the extent of Kit expression, the dermal mast cell populations were maximally expanded in C57BL/6 mice. We suggest that BALB/c mice of 6 and 10 weeks of age are useful hosts in which to quantitatively evaluate mast cell involvement in a range of functional assays involving skin.     

Endogenous interleukin (IL)‐17A promotes pristane‐induced systemic autoimmunity and lupus nephritis induced by pristane

Interleukin (IL)-17A is increased both in serum and in kidney biopsies from patients with lupus nephritis, but direct evidence of pathogenicity is less well established. Administration of pristane to genetically intact mice results in the production of autoantibodies and proliferative glomerulonephritis, resembling human lupus nephritis. These studies sought to define the role of IL-17A in experimental lupus induced by pristane administration. Pristane was administered to wild-type (WT) and IL-17A^−/− mice. Local and systemic immune responses were assessed after 6 days and 8 weeks, and autoimmunity, glomerular inflammation and renal injury were measured at 7 months. IL-17A production increased significantly 6 days after pristane injection, with innate immune cells, neutrophils (Ly6G⁺) and macrophages (F4/80⁺) being the predominant source of IL-17A. After 8 weeks, while systemic IL-17A was still readily detected in WT mice, the levels of proinflammatory cytokines, interferon (IFN)-γ and tumour necrosis factor (TNF) were diminished in the absence of endogenous IL-17A. Seven months after pristane treatment humoral autoimmunity was diminished in the absence of IL-17A, with decreased levels of immunoglobulin (Ig)G and anti-dsDNA antibodies. Renal inflammation and injury was less in the absence of IL-17A. Compared to WT mice, glomerular IgG, complement deposition, glomerular CD4⁺ T cells and intrarenal expression of T helper type 1 (Th1)-associated proinflammatory mediators were decreased in IL-17A^−/− mice. WT mice developed progressive proteinuria, but functional and histological renal injury was attenuated in the absence of IL-17A. Therefore, IL-17A is required for the full development of autoimmunity and lupus nephritis in experimental SLE, and early in the development of autoimmunity, innate immune cells produce IL-17A.     

Unique B cell responses in B cell-dependent and B cell-independent EAE

Previous studies characterized B cell-dependent and B cell-independent models of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. To further characterize the B cell response generated in these two models, the serum antibody response and the B cell surface immunoglobulin (Ig) repertoire were analyzed following immunization of wild-type C57BL/6 mice with either recombinant myelin oligodendrocyte glycoprotein (MOG; B cell-dependent EAE) or the encephalitogenic MOG(35-55) peptide (B cell-independent EAE). Plasma ELISA revealed responses to unique linear epitopes of MOG following immunization with recombinant MOG that were absent in MOG(35-55)-immunized animals. B cell repertoire analysis by RT-PCR identified a unique response restricted to 7183 Ig heavy chain variable gene family in mice immunized with recombinant MOG that was not observed in MOG(35-55)-immunized mice. These insights could aid in the identification of the relevant B cell populations important to the pathogenesis of B cell-dependent EAE and in the mechanisms by which these B cell populations contribute to disease.     

Differential susceptibility of C57BL/6 and DBA/2 mice to ovalbumin-induced pulmonary eosinophilia regulated by Th1/Th2-type cytokines

To test the effect of genotype on immune response, C57BL/6 and DBA/2 mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) and challenged with aerosolized OVA. The serum immunoglobulin (Ig) E and IgG1 levels in C57BL/6 mice were higher than those in DBA/2 mice. In contrast, IgG2a levels in C57BL/6 mice were lower than that in DBA/2 mice. C57BL/6 mice were also much more susceptible than DBA/2 mice to OVA-induced pulmonary eosinophilia. Furthermore, patterns of cytokine generation in lung tissue were different between C57BL/6 and DBA/2 mice after OVA challenge. Th2-type cytokine interleukin (IL-) 4 and IL-5 generation in C57BL/6 mice was higher than that in DBA/2 mice, while Thl-type cytokine interferon-gamma (IFN-gamma) generation in C57BL/6 mice was lower than that in DBA/2 mice. Similar patterns of IL-4 and IL-5, and IFN-gamma production in splenocytes from both strains after OVA stimulation in vitro were also observed. The participation of IL-4 and IL-5, and IFN-gamma in the regulation of eosinophil infiltration into the lung was confirmed by injection of anti-IL-5, -IL-4 and -IFN-gamma monoclonal antibodies. These results indicate that C57BL/6 mice preferentially induce IL-4 and IL-5-mediated Th2-type response, while DBA/2 mice induce IFN-gamma-mediated Thl-type response. Thus, the genotype of laboratory strains partially determines whether Th1- or Th2-type immune responses are elicited.     

C57BL/6 mice are more susceptible to antigen-induced pulmonary eosinophilia than BALB/c mice, irrespective of systemic T helper 1/T helper 2 responses

Inflammatory response differences between C57BL/6 and BALB/c mice following ovalbumin (OVA) sensitization and a single challenge were investigated. Serum immunoglobulin (Ig)E and IgG1 levels were higher in C57BL/6 mice than in BALB/c mice. In contrast, IgG2a levels in C57BL/6 mice were lower than in BALB/c mice. Furthermore, the number of eosinophils infiltrating into lungs in C57BL/6 mice was significantly higher than in BALB/c mice after OVA challenge. The levels of the T helper 2 (Th2)-type cytokines interleukin (IL)-4 and IL-5, generated in challenged C57BL/6 lung tissue, were also higher than in BALB/c lung tissue. The participation of IL-4 and IL-5 in the induction of eosinophil infiltration into the lungs was confirmed in both strains of mice by injection of anti-IL-4 and anti-IL-5 monoclonal antibodies (mAbs). However, following OVA stimulation, in vitro IL-4 and IL-5 production in splenocyte cultures from C57BL/6 mice was lower than in splenocyte cultures from BALB/c mice. These results indicate that C57BL/6 mice induce Th2-type responses in the lungs, while BALB/c mice induce T helper 1 (Th1)-type responses in the lungs, despite considerable production of IL-4 and IL-5 from splenocytes. Therefore, local immune responses are more important in the induction of allergic inflammation in the lungs and are different from systemic immune responses, which are thought to depend on genetic background.     

CTLA—4Ig治疗C57BL／6小鼠自身免疫性肝炎的实验研究

目的 研究CTLA-4Ig在治疗C57BL/6小鼠自身免疫性肝炎上的作用。方法 通过用C57BL/6近交系小鼠的肝特异性抗原与弗氏完全佐剂混合物免疫攻击小鼠,随后用CTLA-4Ig治疗,观察小鼠的临床经过、血生化、肝脏组织学改变。结果 随着免疫次数的增多,治疗组临床经过、血生化、肝脏组织学改变逐步与正常对照相似,而病理模型组与前两组有明显差异;血生化可见天冬氨酸氨基转移酶、球蛋白显著升高;肝组织学检测可见炎细胞的浸润,肝细胞的肿胀、灶性坏死甚至广泛坏死;肝组织免疫荧光检测提示有大量免疫球蛋白沉着。结论 CTLA-4Ig能有效地治疗C57BL/6小鼠自身免疫性肝炎。     

The induction of immune complex deposits in mice by peroral and parenteral administration of mercuric chloride: strain dependent susceptibility.

Female Balb/c and SJL mice exposed to HgCl2 by subcutaneous injection or via drinking water for up to 6 months showed immunostimulation with increased concentrations of immunoglobulins (Ig) in the serum. The Ig isotype pattern was dependent both on the strain and the use of immunopotentiation by addition of Freund's complete adjuvant (FCA). Balb/c mice showed a T cell dependent pattern, whereas the SJL mice developed an increase also in T cell independent isotypes. This latter pattern shifted to show T cell dependency after an initial addition of FCA. FCA also converted the lack of stimulation in C57BL/6J mice to a low response with a T cell dependent isotype pattern. No correlation emerged between the body burden of mercury, as assessed by the concentration in the kidneys, and the degree of immunostimulation by mercury. Mice showing a stimulation of the immune system developed mesangial immune complex (IC) glomerulonephritis and, later on, IC deposits in renal, splenic, and hepatic vessel walls with an isotype pattern corresponding to that seen in the serum.     

Accelerated atherosclerosis in ApoE deficient lupus mouse models

The accelerated development of atherosclerosis with increased risk of cardiovascular disease in systemic lupus erythematosus (SLE) patients is not well understood. An appropriate mouse model would greatly help to understand the mechanisms of this association. We have therefore combined the ApoE(-/-) model of atherosclerosis with three different murine models of SLE. We found that induction of cGVH in B6.ApoE(-/-) mice, breeding a Fas null gene onto the B6.ApoE(-/-) mice, and breeding the ApoE(-/-) defect onto MRL/lpr mice all caused a modest increase of atherosclerosis at 24 weeks of age compared to B6.ApoE(-/-) controls. B cells in B6.ApoE(-/-) mice had certain phenotypic differences compared to congenic C57BL/6 mice, as indicated by high expression of MHC II, Fas, CD86, and by increased number of cells bearing marginal zone phenotype. Furthermore, B6ApoE(-/-) mice had significant titers of anti-oxLDL and anti-cardiolipin autoantibodies compared to their B6 counterparts. Our studies also indicate that, following induction of cGVH, marginal zone B cells in B6.ApoE(-/-) are depleted, and there is considerable increase in anti-oxLDL and anti-cardiolipin abs along with secretion of lupus-specific autoantibodies, such as anti-dsDNA and anti-chromatin abs. Histological sections showed that cGVH and/or Fas deficiency could exacerbate atherosclerosis. The production of anti-oxLDL and anti-cardiolipin in ApoE(-/-) mice was also increased. These observations define a connection between induction of lupus-like symptoms and development of severe atherosclerosis in ApoE deficient lupus mouse models.     

Alveolar macrophage priming by intravenous administration of chitin particles, polymers of N-acetyl-D-glucosamine, in mice.

Intravenous (i.v.) administration of phagocytosable chitin particles (1 to 10 microm) in C57BL/6 mice and SCID mice primed alveolar macrophages (Mphi) within 3 days to yield up to a 50-fold increase in their oxidative burst when elicited in vitro with phorbol myristate acetate (PMA). C57BL/6 mice pretreated with monoclonal antibodies (MAbs) against mouse gamma interferon (IFN-gamma) or NK1.1 showed a markedly decreased level of alveolar Mphi priming following injection of chitin particles. To confirm IFN-gamma production in vitro, spleen cells isolated from normal C57BL/6 mice and SCID mice were cultured with chitin particles. Significant IFN-gamma production was observed following stimulation with chitin but not with chitosan or latex beads. When spleen cells were treated with anti-NK1.1 MAb, IFN-gamma production was significantly inhibited. Another set of experiments showed that when C57BL/6 mice were pretreated i.v. with a small dose IFN-gamma, a higher level of priming was induced with not only phagocytosable chitin particles but also phagocytosable chitosan and even latex beads. Likewise, the spleen cell cultures preconditioned with IFN-gamma provided an up-regulation of IFN-gamma production by these phagocytosable particles. Taken together, the in vivo and in vitro results suggest that (i) the alveolar Mphi priming mechanism is due, at least in part, to direct activation of Mphi by IFN-gamma, which is produced by NK1.1+ CD4- cells; (ii) IFN-gamma would have an autocrine-like effect on Mphi and make them more responsive to particle priming; and (iii) phagocytosis of particulates, probably by a postmembrane event such as interiorization, appears to be important for the up-regulation of alveolar Mphi priming and IFN-gamma production.     

"Viable motheaten," a new allele at the motheaten locus. I. Pathology

A new spontaneous autosomal recessive mutation has recently occurred at the motheaten (me) locus on Chromosome 6 in strain C57BL/6J mice. Homozygotes for the new allele, designated "viable motheaten" (mev), have a mean life span of 61 +/- 2.4 days, compared with only 22 +/- 1.3 days for C57BL/6J-me/me mice. Like the original motheaten mutation, the immediate cause of death in mev/mev mice appears to be severe pneumonitis associated with accumulations of macrophages, granulocytes, and lymphocytes in the lungs. However, because of its longer life span, progression of the disease in mev/mev mice is more amenable to investigation. Eosinophilic crystalline material in alveolar macrophages from mev/mev mice is associated with extravasation of erythrocytes into alveoli. These crystals are morphologically indistinguishable from hematoidin, which results from hemoglobin breakdown following uptake of erythrocytes by macrophages. Severe macrocytic hypochromic anemia with abnormalities in size and shape of erythrocytes develops by 7 weeks. A two-fold increase in peripheral leukocyte count and a five-fold increase in the percentage of neutrophils is seen by 10 weeks. Viable motheaten mice develop focal granulocytic skin lesions by 4 days of age, show depletion of cells from the thymus cortex by 4 weeks, and lack lymphoid follicles in the lymph nodes, spleen, and Peyer's patches. Excessive erythropoiesis and myelopoiesis in the spleen result in marked splenomegaly. Lymph nodes and spleens from mev/mev mice contain increased numbers of plasma cells by 3 weeks; and by 6 weeks, large numbers of atypical plasma cells with Russell bodies are evident. Development of glomerulonephritis by 10 weeks is characterized by granular depositis of immunoglobulin and complement within glomeruli. A twofold increase of blood urea nitrogen levels is present by 15 weeks. Sterility of male mev/mev mice is associated with Leydig cell depletion in the testes, lowered testosterone levels, and impaired spermatogenesis.     

Serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA in C57BL/6 mice and their congenics at the lpr (lymphoproliferation) locus

The serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA were determined in mice of C57BL/6 background, from weaning to one year of age, by quantitative isotype-specific, indirect double sandwich enzyme-linked immunosorbent assays (ELISAs). Only limited data could be obtained for the IgG2a isotype in the present study. The mean serum Ig levels found for 6-month-old B6 mice were 0.22 mg/ml for IgM, 0.28 mg/ml for IgG1, 1.22 mg/ml for IgG2b, 0.18 mg/ml for IgG3, 0.075 mg/ml for IgA and about 0.7 mg/ml for IgG2a. In comparison with mice of the wild strain, C57BL/6 mice homozygous at the lpr (lymphoproliferation) locus showed very high increases in serum Ig levels when older than 20 weeks. With 6-month-old B6 lpr mice, increases in concentration were found for all tested heavy chain isotypes: 6 to 6.5-fold for IgA (0.45 mg/ml) and IgG1 (1.82 mg/ml), 9-fold for IgG3 (1.6 mg/ml), 11 to 11.5-fold for IgM (2.44 mg/ml) and IgG2b (13.8 mg/ml) and about 8-fold for IgG2a (5.5 mg/ml). Therefore homozygosity at the lpr locus provides the conditions for generalized, poly-isotypic rather than isotype-specific restricted Ig enhancement. This observation may be more compatible with hyperinducibility of all B-cell subclasses than with excessive production of T-cell-derived factors whose activity would be expected to be restricted to some T-dependent subclasses, and at least to affect IgM-committed B cells to a lesser extent than other B-cell classes.     

Lack of endothelial nitric oxide synthase aggravates murine accelerated anti-glomerular basement membrane glomerulonephritis

Nitric oxide (NO) radicals generated by endothelial nitric oxide synthase (eNOS) are involved in the regulation of vascular tone. In addition, NO radicals derived from eNOS inhibit platelet aggregation and leukocyte adhesion to the endothelium and, thus, may have anti-inflammatory effects. To study the role of eNOS in renal inflammation, the development of accelerated anti-glomerular basement membrane (GBM) glomerulonephritis was examined in mice lacking a functional gene for eNOS and compared with wild-type (WT) C57BL/B6j mice. WT C57BL/6j mice (n = 12) and eNOS knockout (-/-) mice (n = 12) were immunized intraperitoneally with sheep IgG (0.2 mg in complete Freund's adjuvant). At day 6.5 after immunization, mice received a single i.v. injection of sheep anti-mouse GBM (1 mg in 200 microl PBS). Mice were sacrificed at day 1 and 10 after induction of the disease. All WT mice survived until day 10, whereas 1 eNOS-/- mouse died and 2 more became moribund, requiring sacrifice. At day 10, eNOS-/- mice had higher levels of blood urea nitrogen than WT mice (P < 0.02), although proteinuria was comparable. Immunofluorescence microscopy documented similar IgG deposition in both WT and eNOS-/- mice, but eNOS-/- mice had more extensive glomerular staining for fibrin at day 10 (P < 0.007). At day 10, light microscopy demonstrated that eNOS-/- mice had more severe glomerular thrombosis (P < 0.003) and influx of neutrophils (P < 0. 006), but similar degrees of overall glomerular endocapillary hypercellularity and crescent formation. In conclusion, accelerated anti-GBM glomerulonephritis is severely aggravated in eNOS-/- mice, especially with respect to glomerular capillary thrombosis and neutrophil infiltration. These results indicate that NO radicals generated by eNOS play a protective role during renal inflammation.     

Effect of complement component C3 deficiency on experimental Lyme borreliosis in mice

Mice deficient in complement component C3 (C3(-/-)) and syngeneic C57BL/6 control mice were challenged with Borrelia burgdorferi to determine the role of complement in immune clearance and joint histopathology during experimental Lyme borreliosis. Tibiotarsal joint, ear, and heart tissues were monitored for spirochete numbers at 2, 4, 8, and 12 weeks postinoculation with 10(5) B. burgdorferi B31 clone 5A4 by using quantitative real-time PCR. The spirochete load in joint and ear tissue remained higher in the C3(-/-) mice than in the wild-type counterparts throughout the 12-week study, whereas the numbers in heart tissue of both groups of mice decreased substantially at 8 to 12 weeks postinfection. Histopathology scores for joint tissue were generally higher in the C3(-/-) mice compared to C57BL/6 controls at 2 and 4 weeks postinfection, which may reflect the presence of higher numbers of bacteria in the joints at these early time points. Levels of anti-B. burgdorferi immunoglobulin G tended to be reduced in the C3(-/-) mice compared to control mice. Furthermore, a 5.5-fold-lower number of the complement-sensitive Borrelia garinii was needed to infect C3(-/-) mice compared to C57BL/6 mice, indicating that its sensitivity to complement is one barrier to infection of the mouse model by B. garinii. These results indicate that the complement system may be important in controlling the early dissemination and progression of B. burgdorferi infection.     

Btk regulates localization, in vivo activation, and class switching of anti-DNA B cells

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk-/- mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk-/- mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk-/- mice. A transgene expressing a low level of Btk in B cells (Btk(lo)) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btk(lo) B cells. anti-DNA IgG was not observed in 56R.Btk(lo) mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.