Increased susceptibility to airway responses in CD40-deficient mice

The interaction between CD40 and its ligand (CD154) is crucial for IL-12 production and effective humoral immunity such as IgE production. Although the interaction seems to play a crucial role in asthmatic inflammation, previous studies investigating the role of the CD40 and CD154 interaction in experimental animal models of asthma are complicated due to multistep reactions in developing asthma. Here, in order to investigate the role of CD40 in the effector phase in the development of airway responses, we used CD40-deficient mice backcrossed with mice transgenic for an ovalbumin (OVA)-specific TCR (TCRtg). Using intranasal OVA administration followed by aerosol inhalation of OVA, greater airway hyperreactivity and eosinophilia in bronchoalveolar lavage fluid (BALF) were observed in CD40-deficient mice backcrossed with TCRtg mice (CD40-/-/ TCRtg mice), compared with control littermates (CD40+/+/ TCRtg mice). CD4+ helper T cell subset analysis of lung draining lymph nodes revealed that the Th1 component was significantly decreased in CD40-/-/ TCRtg mice. Airway hyperreactivity and airway eosinophilia significantly correlated with the predomination of Th2 cells. Cytokine measurements in BALF also showed decreased IL-12 and the predominance of Th2 cells in CD40-/-/ TCRtg mice. These results suggest that CD40 may play a protective role in developing asthma in the phase after establishing specific memory T cells through the regulation of the balance between Th1 and Th2 cells presumably via induction of IL-12.     

CD28-deficient mice generate an impaired Th2 response to Schistosoma mansoni infection

Engagement of CD28 on T cells provides a co-stimulatory signal necessary for T cell activation and differentiation. Recent findings suggest that priming of T helper (Th)2 cells is more dependent on CD28 activation than Th1 cells. The present study examines whether mice that lack expression of CD28 as a result of gene targeting are capable of generating a Th2 response characteristic during infection with the intravascular trematode parasite Schistosoma mansoni. Mutant and control mice were either inoculated in the footpad with S. mansoni eggs (a potent inducer of a Th2 response) or infected percutaneously with the parasite. Draining lymph nodes (after footpad injection) or spleen cells (after natural infection) were harvested at 12 days and 8 weeks, respectively, and examined for cytokine responses to egg antigens. CD28-deficient mice (−/−) generated diminished egg antigen-driven interleukin (IL)-4 and IL-5 production (by 5- to 17-fold, respectively) compared to CD28-expressing (+/+) littermates. In contrast, lymphocyte proliferation and interferon (IFN)-γ production to egg antigens were equivalent for mutant and control mice. Infected CD28−/− mice also had reduced immunoglobulin secretion. Serum levels of parasite antigen-specific IgG1 and polyclonal IgE were significantly diminished in CD28−/− compared to CD28+/+ mice. Lack of CD28 expression had no effect on granuloma formation around eggs trapped in the liver, but increased susceptibility of mice to primary schistosomiasis infection. These studies indicate that CD28 activation contributes to T cell priming required for generation of a Th2 response to an intravascular dwelling helminth parasite.     

Impaired CD40-signalling in Langerhans' cells from murine neonatal draining lymph nodes: implications for neonatally induced cutaneous tolerance

Cutaneous tolerance to antigens may be induced in mice through application of antigen during the first few days following birth. The mechanism governing this neonatally induced tolerance remains uncertain. We employed a contact hypersensitivity model to analyse dendritic cell (DC) function and the expression of classical and non-classical lymphocyte populations within the neonate. Examination of draining lymph node DC after antigenic challenge of the skin revealed these DC to be significantly deficient in their ability to stimulate antigen-specific T cell proliferation. Co-stimulatory molecule (CD40, CD80 and CD86) expression of these cells was deficient in comparison to adult DC, and functional tests revealed these cells to possess a critical absence of CD40 signalling. A numerical analysis of classical and non-classical lymphocyte expression demonstrated that while the neonatal spleen is devoid of T cells, the lymph nodes have a normal repertoire of T, B, gammadelta and CD4+CD25+ lymphocytes but an increased expression of natural killer (NK) cells. This study indicates that functionally deficient DC are likely contributors to neonatally induced cutaneous tolerance.     

Impaired IgE response in SWAP-70-deficient mice

Protein SWAP-70 was initially isolated from nuclei of activated B cells and was implicated in the immunoglobulin class switch process. After B cell activation the protein translocates from the cytoplasm to the nucleus, and may serve to signal nuclear processes. We have generated mice deficient in SWAP-70 and found three main differences when compared to wild-type mice: (i) their B lymphocytes are two- to threefold more sensitive to gamma-irradiation than B cells of wild type; (ii) SWAP-70-deficient mice developed autoantibodies at a much higher frequency; and (iii) the CD40 signaling pathway is compromised in the mutant mice. CD40-dependent switching to the IgE isotype is reduced five- to eightfold in vitro. In SWAP-70-deficient mice, IgE levels prior to immunization were six- to sevenfold lower than in wild-type mice, and after immunization three- to fourfold lower. CD40-induced proliferation was transiently increased in the mutant. LPS-induced switching to other isotypes, however, and LPS-induced proliferation were normal. We propose that SWAP-70 serves a specific role in the CD40 signaling pathway, in particular in the IgE response.     

The superantigen toxic shock syndrome toxin-1 induces CD40 ligand expression and modulates lgE isotype switching

Isotype switching to IgE requires two signals. The first signalis provided by the cytokines IL-4 or IL-13, and the second signalis delivered by the interaction between the B cell antigen CD40and its ligand (CD40L) which is expressed on activated T cells.Since superantigens have been shown to activate T cells, weexamined the effect of the superantigen toxic shock syndrometoxin-1 (TSST-1) on CD40L expression on T cells. TSST-1 inducedexpression of CD40L in both freshly isolated T cells and inT cell lines expanded by re-stimulation with TSST-1. CD40L waspreferentially expressed in the V_ß2 subset of T cellsexpanded by TSST-1. We next examined the effect of TSST-1 onIgE synthesis by human peripheral blood mononuclear cells (PBMC).Addition of TSST-1 to PBMC inhibited IL-4-induced IgE synthesisin a dose-dependent manner. This inhibition was reversed partlyby adding a neutralizing antibody to IFN-. In contrast, TSST-1induced high amounts of IgE synthesis in the presence of IL-4at low T: B cell ratios (0.5: 10 to 4: 10), a condition whichcircumvents the inhibitory effect of IFN-. TSST-1 inductionof IgE synthesis was inhibited by a mAb to CD40L. These resultsindicate that superantigens induce CD40L expression in T cellsand cause isotype switching in B cells which is mediated byCD40L-CD40 interaction.     

IgG-mediated anaphylaxis via Fcγ receptor in CD40-deficient mice

Anaphylaxis denotes an immediate hypersensitivity reaction to allergen, exclusively mediated by IgE antibodies. However, IgE antibodies do not explain all the syndromes that are encountered. We investigated potent IgG-mediated anaphylaxis in CD40-deficient mice that lack the immunoglobulin class switching for T cell-dependent antigens. Immunization with ovalbumin did not induce either humoral responses of IgG, IgA, and IgE, or systemic anaphylaxis in CD40-deficient mice. Although systemic anaphylaxis by active immunization was not observed in CD40-deficient mice, both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis assessed by mouse blood pressure monitoring with cervical artery catheterization did take place when antigen-specific IgG was transferred and then antigen challenge given. Further, to investigate the inflammatory pathway of IgG-mediated immediate hypersensitivity reactions, we focused on the Fcγ receptor (FcγR) function. Pretreatment of the mice with the anti-FcγRII/FcγRIII MoAb clearly blocked the response of PCA and passive systemic anaphylaxis, suggesting that they were initiated through FcγR. In conclusion, we directly demonstrate the IgG-mediated anaphylaxis and its triggering mechanism through FcγR in in vivo conditions. In addition to IgE-mediated anaphylaxis, IgG-mediated anaphylaxis should be considered and the blocking of FcγR would provide one of the therapeutic targets for the control of IgG-mediated hypersensitivity diseases.     

Impaired myocardial capillarogenesis and increased adaptive capillary growth in FGF2-deficient mice

Basic fibroblast growth factor (FGF2) plays a major role in angiogenesis and capillary growth. In contrast to vascular endothelial growth factor, which is required for proliferation and survival of endothelial cells, FGF2 does not seem to be essential since the Fgf2 knockout is not lethal. Therefore, the precise genetic and physiological roles of FGF2 for capillary development and adaptation remain to be determined. Here we show that myocardial capillary supply is normal at birth, but significantly reduced by approximately 25% in adult Fgf2+/- and Fgf2-/- mice as compared with wild-type littermates. In contrast, after induction of myocardial hypertrophy by continuous infusion of angiotensin II (ANG II) for 6 days marked capillary growth was seen in both Fgf2+/- and Fgf2-/- mice, but not in wild-type littermates. These data demonstrate that two intact Fgf2 genes are necessary for normal capillary development after birth, whereas FGF2 seems to be dispensable for adaptive myocardial capillary growth in the adult mouse.     

Impaired T cell activation and increased Th2 lineage commitment in Annexin-1-deficient T cells

Annexin-1 is a well-known endogenous anti-inflammatory protein that modulates the activation of cells of the innate immune system such as neutrophils and macrophages. We have recently reported a positive role for the exogenous protein on T cell differentiation, however, whether such a role holds true for the endogenous protein has yet to be determined. This aspect has been investigated here finding that Annexin-1-deficient T cells display an impaired activation and proliferation in response to anti-CD3 plus anti-CD28 stimulation. Furthermore, differentiation of T cells from Annexin-1-deficient mice in Th0/Th1/Th2 or Th17 skewing conditions demonstrated an increased Th2 phenotype compared to cells from control littermates. Similar results were obtained when we analyzed the Th1/Th2 profile of lymph node cells obtained from mice immunized with keyhole limpet hemocyanin or the inflammatory infiltrate in mouse model of allergic inflammation. These results demonstrate a novel modulatory role of endogenous Annexin-1 in TCR signaling and T cell differentiation and suggest this protein might play a dual and complementary role in the innate and adaptive immune response.     

Inhibition of B-cell death does not restore T-cell-dependent immune responses in CD40-deficient mice

Signalling through CD40 is essential for the development of immunoglobulin G (IgG) antibody responses, germinal centres and B-cell memory against T-dependent antigens. In addition, engagement of CD40 in B cells promotes cell survival by inducing the expression of anti-apoptotic members of the bcl-2 family of cell-death regulators. In the present study we analysed whether T-dependent immune responses can be developed in mice deficient in CD40 if the anti-apoptotic activity mediated by the engagement of CD40 in B cells is compensated by the constitutive over-expression of anti-apoptotic genes of the bcl-2 family. We showed that the over-expression of either hbcl-2 or hbcl-xL transgenes in B cells is not sufficient to restore IgG antibody responses and germinal centre formation in CD40-deficient mice. These results indicate that CD40 functions, other than those mediated through survival, are required for the establishment of T-dependent B-cell responses.     

The 129 genetic background affects susceptibility to glomerulosclerosis in tensin2-deficient mice

The ICGN mouse strain is a glomerulosclerosis (GS) model that shows significant proteinuria, podocyte morphological abnormalities and increased extracellular matrix accumulation in the glomeruli, which represent the final common pathology associated with a variety of kidney diseases leading to end-stage renal failure. Previously, we demonstrated that GS in ICGN mice can be attributed to the deletion mutation of the tensin2 (Tns2) gene (Tns2(nep)). Further, the C57BL/6J (B6) mouse is resistant to GS caused by this mutation. 129/Sv is also a popular strain; however, its susceptibility to GS has not been defined. Thus, to determine whether 129/Sv is resistant or susceptible to GS, we produced a congenic strain carrying Tns2(nep) on the 129(+Ter)/Sv (129T) background. 129T congenic mice (129T-Tns2(nep)) did not exhibit albuminuria, renal anemia, increases in BUN, or any severe pathological changes until at least 16 weeks of age. These results indicate that 129T is resistant to GS. Although their usage in biomedical studies is already widespread, 129/Sv mice may afford a late-onset and unique strain applicable to kidney disease research.     

Impaired contact hypersensitivity to trinitrochlorobenzene in interleukin-4-deficient mice

We have examined the role of endogenously produced interleukin-4 (IL-4) in the contact hypersensitivity (CH) reaction to the haptene trinitrochlorobenzene (TNCB). The CH reaction was abolished in IL-4 genetically deficient mice (IL-4 KO), when compared to wild-type (wt) mice. The CH reaction was restored by treatment with IL-4 and further analysis revealed that IL-4 exerted its action both at the induction and effector stages of the CH reaction. Despite failure to develop a CH reaction, IL-4 KO mice developed a T helper type 1 (Th1) response to TNCB, in terms of lymphokine production in vitro. Furthermore, the number of Vgamma3+ cells accumulating in the lymph nodes of TNCB-immune IL-4 KO mice was normal. The recruitment of mononuclear cells and vascular leakage at the challenge site were consistently reduced in IL-4 KO mice and were restored by injection of IL-4. This suggests that IL-4 acts as a proinflammatory mediator in CH, perhaps favouring the accumulation of mononuclear cells at the site of inflammation. Among Th2-type cytokines, IL-13, but not IL-10, was shown to restore the CH reaction to TNCB in IL-4 KO mice. However, IL-4 KO mice developed a normal CH response to oxazolone, indicating that IL-4 was required for the CH reaction to TNCB, but not for that to oxazolone.     

Defective Th1 and Th2 cytokine synthesis in the T-T cell presentation model for lack of CD40/CD40 ligand interaction

In this study, T or NK cell clones used as antigen-presenting cells (T- or NK-APC) were shown to be significantly less efficient than professional APC in inducing Th1 and Th2 cytokines by antigen-specific T cell clones. This phenomenon was not related to a limited engagement of TCR by T-APC, since comparable thresholds of TCR down-regulation were shown when antigen was presented by either T-APC or professional APC. Rather, the stimulatory T-APC weakness was due to their inability, because they are CD40⁻, to provide the appropriate co-stimuli to responder T cells both indirectly via IL-12, and partially via direct CD40L triggering on T cells. Indeed, the simultaneous addition of IL-12 and reagents directly engaging CD40L on responder T cells restored T cell cytokine synthesis when antigen was presented by T-APC. In addition, either IL-12 production or blocking of T cell cytokine synthesis by anti-IL-12 p75 antibodies was evident only when professional APC were used in our antigen-specific system. The down-regulation of cytokine synthesis in the system of T-T cell presentation could represent a novel mechanism of immune regulation, which may intervene to switch off detrimental Th1- or Th2-mediated responses induced by antigen presentation among activated T cells infiltrating inflamed tissues.     

Altered airway responsiveness in CD38-deficient mice

Cyclic ADP-ribose (cADPR) mobilizes calcium from intracellular stores and contributes to agonist-induced intracellular calcium elevation in airway smooth muscle (ASM). In this study we determined the functional role of CD38/cADPR signaling in the regulation of airway tone using CD38 deficient (cd38(-/-)) mice. The responsiveness to different doses of methacholine, as determined by changes in lung resistance and dynamic compliance, was significantly (P < or = 0.05) lower in cd38(-/-) mice compared with wild-type controls. To determine the mechanism responsible for the reduced responsiveness, we measured the intracellular calcium responses to contractile agonists in ASM cells. In ASM cells isolated from cd38(-/-) mice, the intracellular calcium responses to acetylcholine and endothelin-1 were significantly lower than in controls. Pretreatment of ASM cells with a cADPR antagonist resulted in attenuated intracellular calcium responses to endothelin-1 in cells isolated from wild-type mice, but not in those isolated from the cd38(-/-) mice. Very low cADPR levels and no detectable ADP-ribosyl cyclase activity were observed in lung tissue from cd38(-/-) mice, suggesting that CD38 is a critical source for cADPR synthesis. The results of the present study demonstrate that CD38/cADPR contributes to airway smooth muscle tone and responsiveness through its effects on agonist-induced elevation of intracellular calcium in ASM cells.     

Impaired control of Brucella melitensis infection in Rag1-deficient mice

After intranasal inoculation, Brucella melitensis chronically infects the mononuclear phagocyte system in BALB/c mice, but it causes no apparent illness. Adaptive immunity, which can be transferred by either T cells or antibody from immune to naive animals, confers resistance to challenge infection. The role of innate, non-B-, non-T-cell-mediated immunity in control of murine brucellosis, however, is unknown. In the present study, we documented that BALB/c and C57BL/6 mice had a similar course of infection after intranasal administration of 16M, validating the usefulness of the model in the latter mouse strain. We then compared the course of infection in Rag1 knockout mice (C57BL/6 background) (referred to here as RAG-1 mice) which have no B or T cells as a consequence of deletion of Rag1 (recombination-activating gene 1), with infection in normal C57BL/6 animals after intranasal administration of B. melitensis 16M. C57BL/6 mice cleared brucellae from their lungs by 8 to 12 weeks and controlled infection in the liver and spleen at a low level. In contrast, RAG-1 mice failed to reduce the number of bacteria in any of these organs. From 1 to 4 weeks after inoculation, the number of splenic bacteria increased from 2 to 4.5 logs and remained at that level. In contrast to the consistently high numbers of brucellae observed in the spleens, the number of bacteria rose in the livers sampled for up to 20 weeks. Immunohistologic examination at 8 weeks after infection disclosed foci of persistent pneumonia and large amounts of Brucella antigen in macrophages in lung, liver, and spleen in RAG-1, but not C57BL/6, mice. These studies indicate that T- and B-cell-independent immunity can control Brucella infection at a high level in the murine spleen, but not in the liver. Immunity mediated by T and/or B cells is required for clearance of bacteria from spleen and lung and for control of bacterial replication in the liver.     

CD19-deficient mice exhibit poor responsiveness to oral immunization despite evidence of unaltered total IgA levels, germinal centers and IgA-isotype switching in Peyer's patches

CD19 exhibits a critical role as a response regulator in B cells, influencing activation, differentiation and survival. Accordingly, CD19-deficient mice largely lack B-1 cells, and their conventional B-2 cells are poor responders to thymus-dependent antigen. Since both B-1 and B-2 cells may contribute to the total intestinal IgA production, we investigated whether lack of CD19 negatively affected mucosal immunity. We found that CD19(-/-) mice have near normal total IgA levels in serum and gut mucosa and, contrary to systemic lymphoid tissues, Peyer's patches (PP) had germinal centers to which also IgA+ B cells localized. However, the mice demonstrated severely impaired responses to oral immunization with keyhole limpet hemocyanin plus cholera toxin adjuvant. Mucosal responses to oral immunization were significantly more impaired than systemic responses. Despite normal specific IL-4 production, a selective defect in Th2-regulated B cell isotypes was observed, with poor or no mucosal IgA, low serum IgG1 and no IgE, but intact serum IgA and IgG2a production. Ex vivo experiments revealed strongly inhibited CD40-stimulated proliferation and IgA differentiation in CD19-deficient PP B cells. Taken together, an impaired CD40 responsiveness selectively affected Th2, but not Th1, coordinated B cell responses. The CD19(-/-) mice provide compelling evidence for the differential regulation of serum and mucosal IgA immunity.     

Exposure to a farming environment has allergen-specific protective effects on TH2-dependent isotype switching in response to common inhalants

BACKGROUND: IgE synthesis by human B cells results from allergen-dependent, T(H)2-mediated isotype switching. Exposure to a farming environment protects against IgE responses. OBJECTIVE: We reconstructed allergen-dependent switching patterns in vivo to identify the level or levels at which farm exposure acts to protect against atopy. METHODS: Serum IgG1 to IgG4 and IgE to grass (rPhl p 1 and rPhl p 5), cat (rFel d 1), and mite (rDer p 2) were assessed by means of ELISA in the Allergy and Endotoxin study population (812 children). Farm exposure was defined as currently living on a farm, exposure to stables/farm milk in the first year of life, or both. RESULTS: Farm exposure did not affect allergen-specific IgG2 and IgG3 levels but had complex allergen-specific effects on IgG1, IgG4, and IgE levels. Exposure protected against grass-specific responses at every step along the IgG1/IgG4/IgE switching pathway but had no significant effect on mite responses. Protection from cat responses was concentrated at the IgG1 level. For all allergens, failure to express IgG1 was associated with low prevalence of IgG4 or IgE responses. Notably, coexpression of IgG1, IgG4, and IgE to grass was associated with increased risk of allergic disease and higher IgE levels compared with production of IgG1 and IgE without IgG4, suggesting IgG4 coexpression marks stronger activation of T(H)2-dependent events. CONCLUSION: The protective effects of farm exposure were confined to T(H)2-dependent IgG1, IgG4, and IgE expression and were allergen and switch stage specific, suggesting that distinct mechanisms regulate individual steps within allergen-induced class switching in vivo. CLINICAL IMPLICATIONS: Environmental interventions to prevent IgE expression might need to be tailored to specific allergens.     

CD40 ligation triggers COX-2 expression in endothelial cells: evidence that CD40-mediated IL-6 synthesis is COX-2-dependent

OBJECTIVE: To investigate whether CD40-CD154 interactions on HUVEC can trigger COX-2 synthesis as well as PGE2 and PGI2 secretion in vitro and explore whether the CD40-triggered prostanoids provide costimulatory signals for IL-6 secretion in this cell type. MATERIALS AND METHODS: COX-2 protein expression was examined in HUVEC using Western blot analysis. ELISAs were employed to assess PGE2, PGI2 and IL-6 synthesis. RESULTS: We found that COX-2 expression is upregulated when HUVEC are cultured with CD154+ D1.1 cells but not CD154- B2.7 cells. This effect was specifically inhibited by anti-CD154 mAb, and was amplified by the presence of IFNgamma. Analysis of cell supernatants showed a concomitant rise in PGE2 and PGI2 secretion triggered by CD154+ D1.1 cells, or rsCD154. Use of selective (NS-398) and non-selective (ibuprofen) COX-2 inhibitors effectively inhibited prostanoid synthesis triggered by CD40 ligation. Reduction in prostanoid levels by NS-398 was accompanied by a reduction in IL-6 secretion levels triggered by CD40 ligation. Furthermore, exogenously added PGE2 triggered a dose-dependent IL-6 secretion, which was unaffected by NS-398. CONCLUSIONS: These studies demonstrate that CD40 ligation upregulates HUVEC COX-2 expression and function. Moreover, the data strongly suggest that CD154-induced IL-6 secretion in HUVEC is dependent on COX-2 activity.