Dependence of murine obstructive airway disease on CD40 ligand.

BACKGROUND: Human lung transplantation carries a poor prognosis because of chronic rejection in the form of obliterative bronchiolitis syndrome (OBS). Using the mouse model of heterotopic tracheal transplantation, we examined the role of costimulation in the allograft rejection that characterizes obstructive airway disease (OAD). METHODS: C57BL/6 or BALB/c tracheae were implanted into wild-type control, CD28-/-, muMT (B-cell deficient), or CD40L-/- recipient mice. Grafts were explanted from 7 to 42 days posttransplantation and evaluated. RESULTS: Thickening of the basement membrane and a decrease in patent luminal area were first noted at 2 weeks in wild-type allogeneic trachea recipients and to a slightly lesser degree in CD28-/- recipients. In contrast, CD40L-/- recipient mice showed no evidence of cellular infiltrates or fibrosis in transplanted tracheae. To determine whether CD40L interacted with host or donor CD40, CD40-deficient tracheae were transplanted into CD40L+/+, CD40+/+ wild-type mice. Wild-type mice rejected CD40-/- tracheae. Tracheae were transplanted into B-cell-deficient mice to determine the role of B-cell CD40 in chronic pulmonary allograft rejection. The OAD reaction was identical in wild-type and B-cell-deficient mice. CONCLUSIONS: Development of OAD in the mouse trachea transplant model is primarily dependent on CD40L and is relatively CD28 independent. The ability of mice to reject CD40-/- tracheae demonstrated that host, not donor, CD40 is required for rejection. Furthermore, the ability of B-cell-deficient mice to reject allogeneic tracheae demonstrated that B-cell CD40-mediated responses are not required for the development of OAD.

Elimination of lymphocytes, but not eosinophils, by Fas-mediated apoptosis in murine schistosomiasis.

Infection with the helminth parasite Schistosoma mansoni is associated with a pathogenic granulomatous response to parasite eggs. Multiple cell types constitute the granuloma with eosinophils achieving numerical dominance. We hypothesize that eosinophil dominance is achieved by selective apoptosis in lymphocytes. We report here that lymphocytes from both the spleens and granulomas of S. mansoni-infected mice undergo apoptosis. We also show that granuloma lymphocytes are more susceptible to Fas-FasL-mediated apoptosis than spleen lymphocytes and this apoptosis may be related to antigen concentration. Conversely, eosinophils from the granuloma and spleens of S. mansoni-infected mice are resistant to apoptosis in vivo and are protected in vitro from Fas-FasL-mediated apoptosis by the absence of FasL expression in the presence of Fas expression. Finally, the apoptotic regulatory molecules Bcl-2, Bcl-xL, and Bax, do not appear to play a significant role in the regulation of eosinophil apoptosis in the schistosome granuloma.

ICOS ligand costimulation is required for T-cell encephalitogenicity.

The interaction of ICOS with its ligand on APC provides a costimulatory signal to previously activated T-cells. In these studies, we blocked the ICOS:ICOS ligand interaction with ICOS-Ig during the in vitro activation of MBP-reactive transgenic CD4(+) T-cells. The presence of ICOS-Ig in these cultures inhibited the ability of the transgenic T-cells to transfer EAE, although they entered the brains of the recipient mice. ICOS-Ig increased apoptosis in the transgenic T-cells, especially in the memory population. This enhanced apoptosis was accompanied by an increase in the BAX/BCL-2 mRNA ratio. ICOS-Ig did not prevent IL2 production, demonstrating that IL-2 production is ICOS ligand independent. IFN-gamma and IL-10 production by the transgenic T-cells, however, was suppressed. Finally, ICOS-Ig injection into mice after the first signs of EAE ameliorated clinical disease. Therefore, ICOSL provides a signal distinct from CD28 costimulation that is required for the activation and viability of encephalitogenic T-cells.

DNA hydrolysis by monoclonal autoantibody BV 04-01.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.

Differential cytokine and chemokine production characterizes experimental autoimmune meningitis and experimental autoimmune encephalomyelitis.

After primary immunization with myelin/oligodendrocyte glycoprotein, CD28(-/-) mice developed experimental autoimmune meningitis (EAM) rather than experimental autoimmune encephalomyelitis (EAE). Cytokine and chemokine production in EAE and EAM were compared to understand the differences in disease phenotype. T cells from the central nervous system lesions of mice with either EAE or EAM expressed intracellular TNF-alpha. Splenic T cells from mice with EAM produced TNF-alpha and IL-6 but no IL-2. Conversely, EAE-derived splenic T cells produced TNF-alpha and IL-2 but no IL-6. Altered T cell differentiation in EAM was not due to a Th1 to Th2 shift, because equivalent amounts of T cell IFN-gamma mRNA were produced in both diseases. Neutrophils also produced inflammatory mediators such as TNF-alpha and IL-6 in EAM. Autocrine production of MIP-2 mRNA was observed in neutrophils from mice with EAM but not EAE. Therefore, distinct patterns of cytokines and chemokines distinguish EAE and EAM.

Experimental autoimmune meningitis: a novel neurological disease in CD28-deficient mice.

C57BL/6 mice develop T-cell-mediated experimental autoimmune encephalomyelitis (EAE) after immunization with the neuroantigen myelin oligodendrocyte glycoprotein. (MOG). We immunized CD28-deficient C57BL/6 mice to determine the role of T cell costimulation in the immune response to MOG. CD28-/- mice developed experimental autoimmune meningitis (EAM). EAM is a fatal, acute disease characterized by simultaneous weakness in all limbs, photophobia, irritability, and spatial disorientation. Histologically, EAM consisted of an infiltrate of myeloid, monocytic, and lymphocytic leukocytes within the leptomeninges. In contrast, the brain parenchyma was unaffected. EAM was mediated by CD4+ T cells since CD4 depletion prevented the disease. Upon rechallenge, mice in which EAM was prevented by CD4+ cell depletion developed EAE not EAM. Therefore, the presence or absence of CD28 determines the initial phenotype of the immune response to MOG. EAM, which develops in the absence of CD28, is a unique experimental model for immune-mediated aseptic meningitis.

Activated eosinophils are the major source of Th2-associated cytokines in the schistosome granuloma.

Eosinophils are a numerically dominant cell population within the schistosome granuloma. These granuloma eosinophils can produce a variety of cytokines, including IL-2, IL-4, IL-5, and IFN-gamma. Therefore, eosinophils may play a key role in the determination of the unique cytokine microenvironment within the granuloma milieu. These studies investigated the potential role of eosinophils in the regulation of granuloma immunopathology. We have characterized spleen- and granuloma-derived eosinophils based on cellular activation and cytokine production during the development of murine schistosomiasis. Based on the criteria of hypodensity and CD69 expression, granuloma eosinophils were highly activated and very homogeneous at 7 and 11 wk postinfection. Splenic eosinophils were also activated at 7 wk postinfection, but were much more heterogeneous than their granuloma counterparts. By 11 wk postinfection, few hypodense splenic eosinophils were observed. Eosinophils represented the majority of cytokine-producing cells in the granuloma and were a dominant source of IL-4. Eosinophils also produced IL-2, IL-5, and IFN-gamma, using the criteria of mRNA in situ hybridization and intracellular cytokine staining by FACS. Granuloma eosinophil activation and cytokine production were greatest at the time of maximum granuloma formation, i.e., 10-12 wk after initial cercarial exposure. Therefore, locally activated eosinophils, not Th2 lymphocytes, produce the majority of Th2 cytokines in the granuloma milieu and may be important determinators of immunopathology in schistosomiasis.

The schistosome granuloma: characterization of lymphocyte migration, activation, and cytokine production.

Granuloma formation and its regulation are dependent on lymphocytes. Therefore, we compared the characteristics of lymphocytes derived from the spleens and granulomas of Schistosoma mansoni-infected mice during the course of their disease. We examined lymphocyte cell cycle kinetics, migration, expression of activation Ags (CD69 and IL-2R), cytokine production (IL-2, IL-4, IFN-gamma), and apoptosis. Lymphocytes in the G2/M phase of the cell cycle and high levels of lymphocyte intracellular IL-2 were found in the spleen but not in the granuloma. Cell trafficking experiments showed Ag-specific recruitment of schistosomal egg Ag (SEA)-reactive lymphoblasts into granulomas in vivo, as well as recruitment to, residence within, and egress from granulomas in vitro. Granuloma-derived lymphocytes were more highly activated than splenic lymphocytes based on higher levels of CD69 and IL-2R expression. While the granuloma microenvironment was rich in Th2 cytokines, during peak granuloma formation, the lymphocytes per se from the spleen and granuloma did not exhibit a dominant Th1 or Th2 cytokine profile, producing low but similar levels of IL-4 and IFN-gamma. The discrepancy between high IL-2R expression and low levels of IL-2 protein production by granuloma lymphocytes was associated with increased apoptosis in the granuloma compared with the spleen. These findings support the hypothesis that granulomas may play a role in the regulation of systemic pathology in schistosomiasis by adversely affecting the survival of SEA-reactive, immunopathogenic T lymphocytes.

DNA hydrolysis by monoclonal anti-ssDNA autoantibody BV 04-01: origins of catalytic activity.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3'-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.

Lupus-derived autoantibodies with dual autoactivity: anti-DNA and anti-Fc. I. Comparison of IgG autoreactivities with single-chain Fv derivatives.

Investigations into the intrinsic affinity and reactivity of autoanti-DNA active sites were initiated through the use of purified monoclonal IgG and the synthesis of single-chain Fv derivatives of murine monoclonal anti-DNA autoantibodies BV 04-01 and BV 17-45. Results showed that relative to the respective IgG hybridomas, only the BV 04-01 SCA derivative showed demonstrable reactivity with DNA. The monovalent single-chain derivative of BV 17-45 showed no reactivity with DNA in solution or solid-phase assays, even though the parental IgG had been previously described as high affinity. However, 17-45 displayed reactivity as a bivalent single-chain derivative. In addition, upon concentration, BV 17-45 IgG formed a highly stable, papain-resistant precipitate. Investigations into the nature of the precipitate revealed that BV 17-45 possessed significant, DNA-inhibitable autobinding to its own IgG molecule. BV 04-01 also possessed similar anti-self reactivity. Thus, both monoclonal autoantibodies examined in this study possessed dual binding specificity; anti-DNa and anti-self.

Lupus-derived autoantibodies with dual autoactivity: anti-DNA and anti-Fc. II. Fine specificity of anti-self autoreactivity.

The anti-immunoglobulin reactivity of two monoclonal, dual specific, autoantibodies, BV 17-45 and BV 04-01 was examined. The current study further defined the anti-immunoglobulin autoreactivity of these MoAbs to be Fc-specific. Both BV 17-45 and BV 04-01 bound their own Fc domains in addition to Fc regions of other MoAbs of similar isotype with varying levels of activity. The different anti-Fc reactivity patterns of BV 17-45 and BV 04-01 suggested that these MoAbs recognized distinct epitopes. Neither BV 17-45 nor BV 04-01 bound Fab fragments or single-chain antibody derivatives, which confirmed that the anti-immunoglobulin reactivity of these autoantibodies was Fc-specific. In addition, abrogation of anti-Fc reactivity was observed when affinity-labelled MoAbs were used as coating antigens in solid-phase ELISAs. These results implied that active-site ligand binding induced conformational changes which altered the Fc epitope(s) recognized by BV 17-45 and BV 04-01.

Relative conformational stabilities of single-chain pocket and groove-shaped antibody active sites including HCDR transplant intermediates.

Stability measurements of SCA 04-01/212 (anti-ssDNA) which possesses a groove-shaped active site were performed by Gdn-HCl-induced unfolding, analyzed assuming a simple two-state equilibrium, and expressed as the free energy of unfolding, delta Gn-u. A delta Gn-u of 1.44 +/- 0.13 kcal/mol was determined experimentally for SCA 04-01/212. In addition, the conformational stabilities of HCDR transplants, hybrid antibody molecules resulting from the transplantation of HCDRs from SCA 4-4-20 (anti-fluorescein) into the corresponding regions of 04-01 in all combinations, were determined using the identical protocol applied to SCA 04-01. On the basis of the results of these stability experiments, the HCDR transplants were categorized into three groups, representing low, intermediate, and high stability. Data were discussed in terms of the relationships between structure-function and conformational stability pertaining to the groove-shaped antibody active site of SCA 04-01/212 and the pocket-shaped active site of SCA 4-4-20/212.

Elucidation of anti-ssDNA autoantibody BV 04-01 binding interactions with homooligonucleotides.

Binding interactions of various synthetic oligohomonucleotides with anti-ssDNA autoantibody BV 04-01 (IgG2b, kappa) and the corresponding single-chain antibody (SCA) 04-01/212 were studied. Oligonucleotide binding to IgG or SCA resulted in quenching of the protein's tryptophan fluorescence permitting direct assessment of ligand binding under equilibrium conditions. The effect of oligothymidylate length, (dT)n, on tryptophan quenching was evaluated. The equilibrium dissociation constants (Kd) for the binding of (dT)6 and (dT)8 were the same [(1.3 +/- 0.02) x 10(-7) M], while decreasing the length of the oligothymidylate to (dT)3 increased the Kd an order of magnitude. To assess base specificity, the comparative binding of other hexahomonucleotides was examined. Neither (dA)6 nor (dC)6 showed measurable binding, while the dissociation constant for (dG)6 was (7.1 +/- 0.3) x 10(-7) M. Fluorescence lifetime quenching data correlated with the steady-state binding results and indicated that the quenching process contains both dynamic and static components. The ability of BV 04-01 to bind (dT)6 and (dG)6 nucleotides was further supported by fluorescence anisotrophy studies with fluorescein-labeled hexadeoxynucleotides. Various levels of tryptophan fluorescence quenching upon titration with oligothymidylates of different length, as well as the similar affinities for (dT)6 and (dG)6, supported the concept that the groove-type binding pocket in BV 04-01 consists of binding subsites that cooperatively adapt for efficient binding of oligonucleotides.

Construction, characterization, and selected site-specific mutagenesis of an anti-single-stranded DNA single-chain autoantibody.

Single-chain antibodies are comprised of immunoglobulin light and heavy chain variable domains joined through a polypeptide linker. A single-chain autoantibody, containing the 14-amino acid 212-polypeptide linker (GSTSGSGKSSEGKG), was constructed based on the light and heavy chain variable region gene sequences of anti-single-stranded DNA autoantibody BV04-01 (IgG2b, kappa). Following protein expression in Escherichia coli, denaturation, refolding, and affinity purification, single-chain autoantibody 04-01 binding with single-stranded DNA and poly(dT) was characterized in solid-phase and solution-phase assays. Homopolymer ligand binding results demonstrated that single-chain autoantibody 04-01 possessed anti-DNA binding properties similar to BV04-01 IgG and Fab fragments. Based on x-ray crystallographic analyses of BV04-01, site-specific mutagenesis studies were conducted on 2 residues (L32Tyr and H100aTrp) involved in aromatic stacking interactions with the middle thymidine of a (dT)3 ligand.