CD8⁺ cells regulate the T helper-17 response in an experimental murine model of Sjögren syndrome.

This study investigated the regulatory function of CD8⁺ cells in T helper-17 (Th17) cell-mediated corneal epithelial barrier disruption that develops in a murine desiccating stress (DS) model that resembles Sjögren syndrome. CD8⁺ cell depletion promoted generation of interleukin-17A (IL-17A)-producing CD4⁺ T cells via activation of dendritic cells in both the ocular surface and draining cervical lymph nodes in C57BL/6 mice subjected to DS. T-cell-deficient nude recipient mice receiving adoptively transferred CD4⁺ T cells from CD8⁺ cell-depleted donors exposed to DS displayed increased CD4⁺ T-cell infiltration and elevated IL-17A and CC-chemokine attractant ligand 20 levels in the ocular surface, which was associated with greater corneal barrier disruption. Enhanced DS-specific corneal barrier disruption in CD8-depleted donor mice correlated with a Th17-mediated expression of matrix metalloproteinases (MMP-3 and MMP-9) in the recipient corneal epithelium. Co-transfer of CD8⁺CD103⁺ regulatory T cells did not affect the ability of DS-specific pathogenic CD4⁺ T cells to infiltrate and cause ocular surface disease in the nude recipients, showing that CD8⁺ cells regulate the efferent arm of DS-induced immune response. In summary, CD8⁺ regulatory cells suppress generation of a pathogenic Th17 response that has a pivotal role in DS-induced disruption of corneal barrier function.

IL-17 disrupts corneal barrier following desiccating stress.

T helper (Th)-17 is a recently identified subtype of Th response that has been implicated in host defense and autoimmunity. We investigated whether there is evidence for a Th-17 response in human and experimental murine dry eye (DE). Gene expression in the human DE conjunctiva showed increased levels of the Th-17 inducers, interleukin (IL)-23, IL-17A, and interferon-gamma (IFN-gamma). In the murine model, we found that desiccating stress increased matrix metalloproteinase-9, Th-17-associated genes (IL-6, IL-23, transforming growth factor-beta1 and -2, IL-23R, IL-17R, IL-17A, retinoid-related orphan receptor-gammat, and CC chemokine attractant ligand-20) and IFN-gamma in cornea and conjunctiva. Furthermore, we found a significantly increased concentration of IL-17 in tears and number of IL-17-producing cells on the ocular surface. Antibody neutralization of IL-17 ameliorated experimental DE-induced corneal epithelial barrier dysfunction and decreased the expression of matrix metalloproteinases 3 and 9. Taken together, these findings suggest that IL-17 has a role in corneal epithelial barrier disruption in DE.

Presentation of ovalbumin internalized via the immunoglobulin-A Fc receptor is enhanced through Fc receptor gamma-chain signaling.

The mechanism of enhanced presentation of ovalbumin (OVA) internalized as immunoglobulin A (IgA)-OVA via the IgA Fc receptor (FcalphaR) was analyzed by focusing on the role of the FcalphaR-associated gamma chain. Comparison of B-cell transfectants expressing FcalphaR plus wild-type (WT) gamma chain or gamma chain in which the immunoreceptor tyrosine-based activation motif (ITAM) was altered by tyrosine mutation or substitution with the ITAM of FcgammaRIIA showed that signaling-competent ITAM was not required for endocytosis of IgA-OVA. However, antigen presentation was impaired by ITAM changes. Signaling-competent gamma-chain ITAM appeared necessary for transport of ligated FcalphaR to a lamp-1(+) late endocytic compartment for remodeling and/or activation of that compartment and also for efficient degradation of IgA complexes. Moreover, FcalphaR ligation also activated efficient processing of nonreceptor-targeted antigen. The results suggest that gamma-chain signaling activates the antigen processing compartment.

The control and facilitation of MHC class II antigen processing by the BCR.

Antigens capable of cross-linking the BCR are preferentially captured, processed and presented to MHC-class-II-restricted T cells. Cross-linking antigens initiate tyrosine-kinase-dependent pathways that accelerate the delivery of antigen-receptor complexes to specialized late-endocytic processing compartments. Accelerated trafficking is mediated by the recruitment of signaling molecules required for transience through specific checkpoints along the endocytic pathway.

Ig alpha and Ig beta are required for efficient trafficking to late endosomes and to enhance antigen presentation.

The B cell Ag receptor (BCR) is a multimeric complex, containing Ig alpha and Ig beta, capable of internalizing and delivering specific Ags to specialized late endosomes, where they are processed into peptides for loading onto MHC class II molecules. By this mechanism, the presentation of receptor-selected epitopes to T cells is enhanced by several orders of magnitude. Previously, it has been reported that, under some circumstances, either Ig alpha or Ig beta can facilitate the presentation of Ags. However, we now demonstrate that if these Ags are at low concentrations and temporally restricted, both Ig alpha and Ig beta are required. When compared with the BCR, chimeric complexes containing either chain alone were internalized but failed to access the MHC class II-enriched compartment (MIIC) or induce the aggregation and fusion of its constituent vesicles. Furthermore, Ig alpha/Ig beta complexes in which the immunoreceptor tyrosine-based activation motif tyrosines of Ig alpha were mutated were also incapable of accessing the MIIC or of facilitating the presentation of Ag. These data indicate that both Ig alpha and Ig beta contribute signaling, and possibly other functions, to the BCR that are necessary and sufficient to reconstitute the trafficking and Ag-processing enhancing capacities of the intact receptor complex.

Cutting edge: signals from the B lymphocyte antigen receptor regulate MHC class II containing late endosomes.

The B lymphocyte response to protein Ag is dependent upon the successful presentation to T cells of Ag-derived, MHC class II-restricted peptides. The B cell Ag receptor (BCR) facilitates this process by internalizing ligand and delivering it to specialized compartment(s) (MHC class II peptide-loading compartments (MIIC)) where it is processed into peptides and loaded onto MHC class II. In addition to efficiently targeting Ag, the BCR can provide tyrosine kinase-dependent signals that augment the presentation of Ag, possibly by enhancing the generation of immunogenic peptides. However, the mechanism by which this occurs is unclear. Herein, we report that the BCR signals a reorganization, fusion, and acidification of an MHC-like compartment into an invariant chain- and MHC class II-rich complex of large vesicles. This complex becomes the primary target for endocytosed receptors. These data suggest that signals generated by the BCR regulate the site of Ag processing.

Inhibition of JAK3 and STAT6 tyrosine phosphorylation by the immunosuppressive drug leflunomide leads to a block in IgG1 production.

Leflunomide is an immunosuppressive drug capable of inhibiting T and B cell responses in vivo. A number of studies demonstrate that leflunomide functions both as a pyrimidine synthesis inhibitor and as a tyrosine kinase inhibitor. We previously reported that leflunomide inhibits LPS-stimulated B cell proliferation, cell cycle progression, and IgM secretion. This inhibition can be reversed by the addition of exogenous uridine, suggesting that leflunomide functions as a pyrimidine synthesis inhibitor in B cells. We report here that while the addition of uridine restored proliferation and IgM secretion to leflunomide-treated LPS-stimulated B cells, as determined by metabolic labeling and immunoprecipitation, it did not completely restore secretion of IgG Ab. We hypothesized that leflunomide inhibits LPS-induced IgG secretion by inhibiting tyrosine kinase activity required for isotype switch. We tested this hypothesis in a well-defined model of isotype switch, LPS plus IL-4 induction of IgG1. Leflunomide inhibited IgG1 secretion in this model in a dose-dependent manner. The signal transduction pathway utilized by IL-4 to induce IgG1 involves tyrosine phosphorylation of the IL-4 receptor, JAK1, JAK3, and STAT6 proteins induced by IL-4 binding to the IL-4R. Leflunomide diminished the tyrosine phosphorylation of JAK3 and STAT6 in the absence or presence of uridine. In gel mobility shift studies, STAT6 binding to the STAT6 DNA binding site in the IgG1 promoter decreased in the presence of leflunomide or leflunomide plus uridine. Taken together, these data suggest that leflunomide acts as a tyrosine kinase inhibitor to block IgG1 production.

Regulation of B cell function by the immunosuppressive agent leflunomide.

Leflunomide is an immunosuppressive drug capable of inhibiting cellular and humoral mediated responses in vivo. The mechanism responsible for suppression of B cell antibody responses in vivo has not been identified. In this study we demonstrate that leflunomide functions to inhibit murine B cell antibody production by directly acting on the B cell. Experiments performed in vivo showed that both T cell-dependent as well as T cell-independent antigen responses were suppressed by leflunomide. Initial in vitro experiments demonstrated that leflunomide inhibited B cell antibody production by decreasing B cell proliferation. The suppression of B cell proliferation induced by a variety of stimuli that use different signal cascade components suggested that leflunomide was acting on a common component required for B cell proliferation. Kinetic studies with LPS activated B cells revealed that leflunomide retained its inhibitory activity when added as late as 24 hr after stimulation in an 88-hr assay. By analyzing the cell cycle of LPS-stimulated B cells we observed that leflunomide targets two different stages in cell cycle transition: (1) from G1 to S phase and (2) from S phase to G2/M phase. Analysis of one of the cyclin-dependent kinases, Cdk2 protein, by Western blot revealed that Cdk2 levels were decreased, in the presence of leflunomide, 48 hr after stimulation. These data further confirmed that leflunomide inhibited B cell progression through the S phase. We also present evidence that the addition of exogenous uridine reversed the antiproliferative activity of leflunomide. This indicated that leflunomide acted as a pyrimidine synthesis inhibitor, thereby inhibiting B cell proliferation and cell cycle progression.

Involvement of LFA-1 and ICAM-1 in the herpetic disease resulting from HSV-1 corneal infection.

Herpes simplex virus type 1 (HSV-1) corneal infection in immunologically normal mice results in a transient epithelial lesion followed in about 2 weeks by a potentially blinding inflammatory response in the corneal stroma, and a mild blepharitis. Similarly infected T cell-deficient mice do not develop corneal stromal inflammation, but exhibit severe periocular skin disease and succumb to viral encephalitis. The role of certain adhesion molecules in both T cell activation, and in the extravasation of inflammatory cells from the blood into inflammatory sites is now being established. These studies investigated the involvement of the adhesion pair LFA-1/ICAM-1 in the disease that results from HSV-1 corneal infection in mice. Treatment of mice with mAb to LFA-1 beginning 1 day before HSV-1 corneal infection resulted in a delay in the onset of stromal inflammation, but ultimately stromal inflammation developed to a normal extent. This treatment also caused a significant exacerbation of periocular skin disease, but did not render mice susceptible to encephalitis. Treatment with mAb to ICAM-1 beginning 1 day before HSV-1 corneal infection caused an acceleration of both stromal inflammation and periocular skin disease, and rendered mice uniformly susceptible to lethal encephalitis. Treatment with either mAb beginning 6 days after HSV-1 corneal infection did not significantly affect the clinical course of herpetic disease. Our findings suggest that LFA-1 may play a role in the early phase of corneal stromal inflammation following HSV-1 corneal infection. Both LFA-1 and ICAM-1 appear to be important for protection of the skin from HSV-1 infection.(ABSTRACT TRUNCATED AT 250 WORDS)