Lacrimal gland inflammation is responsible for ocular pathology in TGF-beta 1 null mice

Mice homozygous for a nonfunctional transforming growth factor-beta 1 gene develop rampant inflammation in vital organs that contributes to a shortened life span. The presence of circulating anti-nuclear anti-bodies, immune deposits in tissues, leukocyte infiltration, and increased major histocompatibility complex antigen expression resembles an autoimmune-like syndrome. One of the overt symptoms that appears in these mice lacking transforming growth factor-beta 1 is the development of dry crusty eyes that close persistently as their health declines. Histologically, the eyes appear normal with little or no inflammation. However, inflammatory lesions, predominantly lymphocytic, develop in the lacrimal glands, disrupting their structure and function and severely limiting their ability to generate tears. This histopathology and aberrant function mimic that of Sj?gren's syndrome, a human autoimmune disease characterized by dry eyes and dry mouth. Impeding the leukocyte infiltration into the glands with synthetic fibronectin peptides, which block adhesion, not only prevents the inflammatory pathology but also prevents the persistent eye closure characteristic of these mice.     

Effect of cyclic AMP level reduction on human neutrophil responses to formylated peptides

In an attempt to elucidate the mechanism of development of organ-specific autoimmune lesions resembling human Sj?gren's syndrome of MRL/lpr mice, we have analyzed local cytokine gene expressions and organ-specific autoantibody production in vivo. We have demonstrated that a major proportion of T cells bearing CD4 and V(beta)8 molecules are essentially responsible for triggering the autoimmunity in the salivary glands of MRL/lpr mice. The local cytokine gene expressions including interferon(IFN)-gamma, IL-12(p40) mRNAs were observed during the course of murine Sjogren's syndrome in MRL/lpr autoimmune strain. In particular, a high level of local expressions of IL-12 mRNA was detected earlier in the proinflammatory stage of autoimmune lesions. A significant level of local expression of MHC class-II(I-Ak) mRNA was detected before the onset of inflammatory lesions in the salivary glands, and I-Ak-positive epithelial duct cells were frequently observed in the salivary glands of MRL/lpr mice. In addition, we found the salivary gland-specific autoantibody in sera from MRL/lpr mice with early phase of autoimmune lesions by immunoblot analysis. These results suggest that cytokine gene stimulation and autoantibody production are essentially involved in the development of organ-specific autoimmune lesions in Sj?gren's syndrome of MRL/lpr mice.     

Immunohistochemical study of inflammatory infiltrates in minor salivary glands in Sj?gren's syndrome and other autoimmune diseases

BACKGROUND: A study of the phenotype, activation and adhesive cells factors and cytokines in minor salivary glands in patients with primary Sj?gren's syndrome (pSS), secondary Sj?gren's syndrome (sSS) and autoimmune diseases (AD) without Sj?gren's syndrome. PATIENTS AND METHODS: We have studied the minor salivary glands in 30 patients with pSS, 30 patients with sSS, 19 patients with AD without SS and 18 controls, using immunohistochemical techniques to analyze the molecular expression of CD3, CD4, CD8, CD20, CD25, CD14, CD56, CD11a, CDw50 (ICAM-3), HLA-DR, IL-1, TNF-alpha and IFN-gamma in lymphocytic infiltration and epithelial cells. RESULTS: Phenotype features were similar in patients with pSS and sSS, except that CD20+ lymphocyte expression was significantly higher in the sSS group (p = 0.023). The patients affected by AD without SS had activated lymphocytes in minor salivary glands in a similar manner to patients affected by pSS and sSS. No significant differences were found in HLA-DR expression in epithelial cells. We found unusual CD25 expression in epithelial cells in patients with SS but not in patients with AD without SS. The differences between pSS and sSS are related to SS theoretical time development and to immunosuppressive treatments. CONCLUSIONS: The immunohistochemical pattern of minor salivary glands is similar in patients with pSS and sSS. Patients with AD are likely to develop immunological changes in minor salivary glands attributable to activated lymphocytes.     

1,25-dihydroxycholecalciferol enhances the expression of MHC class II antigens and intercellular adhesion molecule-1 by human renal tubular epithelial cells

PURPOSE: Beside its role in calcium and phosphorus metabolism, 1,25-dihydroxycholecalciferol (1,25-D3) exerts multiple effects on cytokine and major histocompatibility complex (MHC) class II expression in monocytes and lymphocytes. In different renal diseases tubular epithelial cells express MHC class II molecules and cell adhesion molecules,, such as intracellular adhesion molecule-1 (ICAM-1). Therefore, modulation of MHC class II and ICAM-1 expression in renal tubular epithelial cells by 1,25-D3 may be relevant to lymphocyte adhesion to tubular epithelial cells and immune mediated renal injury. However, the expression of MHC class II antigens and cellular adhesion molecules by renal tubular epithelial cells in response to 1,25-D3 has not been investigated. MATERIALS AND METHODS: We generated human renal tubular epithelial cells and SV40 transfected tubular epithelial cells to investigate immune modulation of 1,25 on renal epithelial cells. Major histocompatibility complex class II molecules and ICAM-1 were detected by a specific enzyme linked immunoassay. RESULTS: We found a dose-dependent increase of both constitutive and induced MHC class II and ICAM-1 expression in tubular epithelial cells stimulated with 1,25-D3. Dose-dependent stimulation of MHC class II and ICAM-1 expression was not restricted to primary human renal tubular epithelial cells but was also detected in SV40 transfected cells. CONCLUSIONS: Expression of MHC class II and ICAM-1 is crucial for antigen presentation by and lymphocyte adhesion to renal tubular epithelial cells. Modulatory effects of 1,25-D3 on immune accessory function of renal tubular epithelial cells may be of clinical significance in renal diseases.     

Vascular changes in major and lingual minor salivary glands in primary Sj?gren's syndrome

A histological investigation of the vascular changes of three major and lingual minor salivary glands in primary Sj?gren's syndrome was carried out on eight autopsied Japanese patients. This study compares vascular lesions in salivary glands between one group of four short-term corticosteroid-treated patients (Cases 1, 3, 4 and 7) and the other group of four long-term corticosteroid-treated patients (Cases 2, 5, 6 and 8). We proposed the following five stages for morphogenesis of arteritis; (1) endothelial swelling, (2) thrombosis, (3) fibrinoid degeneration, (4) necrotizing panarteritis and (5) endarteritis obliterans. Endothelial swelling was seen in small-to-large arteries of major salivary glands and the tongue, and this finding was considered as the initial change of vascular lesion. Thrombosis was observed in the small arteries of both organs. Fibrinoid degeneration and necrotizing panarteritis were predominantly localized in small and middle-sized arteries. Endarteritis obliterans was observed in small and large arteries of major and lingual minor salivary glands in primary Sj?gren's syndrome. Vascular lesion of this type was common in the four patients who received corticosteroid for more than 12 months. Corticosteroid therapy appears to accelerate the fibrotic change of the vascular wall. Therefore, we suggest that essential vascular lesions of major and lingual minor salivary glands in primary Sj?gren's syndrome may include four types (endothelial swelling, thrombosis, fibrinoid degeneration and necrotizing panarteritis), excluding endarteritis obliterans.     

Correction of defective expression in MHC class II deficiency (bare lymphocyte syndrome) cells by retroviral transduction of CIITA

Retrovirus-mediated gene transfer was used to restore expression to MHC class II-negative patient cells from complementation group A(II) of MHC class II immunodeficiency or bare lymphocyte syndrome (BLS). The cells of these patients do not transcribe MHC class II genes due to a defect in the trans-acting factor, CIITA. We constructed a vector, pGAG/Ii-CIITA, with the MHC class II-associated invariant chain promoter driving CIITA expression. Cocultivation with the virus producer line was consistently shown to be the optimal method for infection of all cell types. The induction of MHC class II expression after virus infection was rapid, and high levels of expression were achieved in cell lines within 1 wk of infection. In addition, expression was easily detectable even in peripheral blood cells of a BLS patient within a few days. Cell lines maintained in vitro for several months remained positive, and the proportion of cells with surface expression of DR was correlated with the number of integrated proviruses. Moreover, transduced B lymphoblastoid cell lines readily established tumors in CB17-scid/scid mice, and the MHC class II-positive cells demonstrated a clear competitive advantage in vivo. Ultimately, we hope to use this transduction system to restore normal immune function to a BLS patient for which no other therapeutic option currently exists.     

Immunolocalization of aromatase in human minor salivary glands of the lower lip with primary Sj?gren's syndrome

The enzyme aromatase is involved in the conversion of androgens to estrogens and in the modulation of various androgenic and estrogenic actions. Abnormalities of estrogen metabolism have been postulated to play roles in the development and/or pathophysiology of Sj?gren's syndrome. In the present study, aromatase was immunolocalized in 75 cases of inflammatory disorders of human minor salivary glands of the lower lip. These included cases of primary Sj?gren's syndrome (19 cases), of chronic sialadenitis (34 cases) and of mucous extravasation cysts (22 cases), in order to clarify the possible involvement of in situ estrogen production in primary Sj?gren's syndrome. Aromatase immunoreactivity was detected in myoepithelial cells of acini and in interstitial cells adjacent to acini and ducts in 13/19 (68%) cases of primary Sj?gren's syndrome. In contrast, aromatase expression was detected in only six of 34 (18%) cases of chronic sialadenitis and in seven of 22 (32%) cases of mucous extravasation cyst. These results suggest that increased aromatase expression in minor salivary glands with primary Sj?gren's syndrome in premenopausal women may be involved in the biological features of primary Sj?gren's syndrome through the production of estrogens in situ and possibly through the aggravation of the inflammatory reaction.     

Regulation of class II MHC expression

The class II genes of the major histocompatibility complex (MHC) encode the alpha/beta heterodimeric glycoproteins that play a critical role in the induction of immune responses through presentation of processed antigen to CD4+ T lymphocytes. The constitutive expression of class II MHC antigens is restricted primarily to B cells, dendritic cells, thymic epithelium, and macrophages, although a wide variety of other cell types can be induced to express class II antigens after exposure to cytokines. The appropriate constitutive and inducible te constitutive and inducible expression of class II MHC antigens is essential for normal immune function; thus it is not surprising that aberrant expression on cell types normally class II MHC negative has been correlated with various autoimmune disorders, and lack of expression results in a severe combined immunodeficiency disorder called bare lymphocyte syndrome (BLS). In this review, we discuss the agents that both induce and inhibit class II MHC expression, the function of class II MHC antigens with an emphasis on the ability of these proteins to act as signal transducing molecules, and the molecular regulation of class II MHC expression.     

Transfer of human serum IgG to nonobese diabetic Igmu null mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sj?gren's syndrome

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sj?gren's syndrome. NOD.Igmu null mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igmu null mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab')2 fragments from parental NOD mice or human primary Sj?gren's syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sj?gren's syndrome.     

Value of quantitative salivary gland scintigraphy in the early stage of Sj?gren's syndrome

The aim of this study was to test the impact of quantitative salivary gland scintigraphy in patients with suspected Sj?gren's syndrome. Thirteen patients with suspected Sj?gren's syndrome were investigated. During clinical work-up, three had severe and four had mild Sj?gren's syndrome, while six were normal. Quantitative salivary gland scintigraphy was performed using a standardized method. The normal data-base consisted of 172 patients without any evidence of salivary gland malfunction. Visual and quantitative comparisons of the patients' scintigrams were made. In the patients with severe Sj?gren's syndrome, uptake was 0.10 +/- 0.04% and 0.09 +/- 0.03% in the parotid and submandibular glands respectively, confirming the visual diagnosis. In the patients without Sj?gren's syndrome, concordance between the visual and quantitative evaluations could also be shown. In contrast, among the patients with mild Sj?gren's syndrome, uptake was diminished (P < 0.05), amounting to 0.21 +/- 0.05% and 0.16 +/- 0.02% in the parotid and submandibular glands respectively, while visual analysis indicated normal parenchymatous function. In conclusion, quantitative salivary gland scintigraphy is essential for the reliable detection of parenchymatous malfunction at an early stage of Sj?gren's syndrome, which may be missed by visual analysis alone.     

Suppression of MHC class II expression by human class II trans-activator constructs lacking the N-terminal domain

The class II trans-activator (CIITA) is a bi- or multi-functional domain protein which plays a critical role in the expression of MHC class II genes. We report that removal of the N-terminal 151 amino acids, encompassing all of the acidic domain but leaving intact the proline/serine/threonine-rich domain, results in a mutant protein with potent suppressive properties for MHC class II expression. HeLa cells stably or transiently transfected with mutant CIITA constructs showed up to 99% suppression of MHC class II antigen induction by IFN-gamma and marked suppression of HLA-DRA mRNA expression. Transient transfection of a B lymphoma line resulted in up to 89% reduction of constitutive MHC class II expression within 5 days and suppression of HLA-DRA mRNA synthesis.     

Mutation of RFXAP, a regulator of MHC class II genes, in primary MHC class II deficiency

BACKGROUND: Major-histocompatibility-complex (MHC) class II deficiency is an autosomal recessive primary immunodeficiency disease in which MHC class II molecules are absent. It is a genetically heterogeneous disease of gene regulation resulting from defects in several transactivating genes that regulate the expression of MHC class II genes. The mutations responsible for MHC class II deficiency are classified according to complementation group (a group in which the phenotype remains uncorrected in pairwise fusions of cells). There are three known complementation groups (A, B, and C). METHODS: To elucidate the genetic defect in patients with MHC class II deficiency that was not classified genetically, we performed direct complementation assays with the three genes known to regulate the expression of MHC class II genes, CIITA, RFX5, and RFXAP, and the relevant mutations were identified in each patient. RESULTS: Mutations in the RFXAP gene were found in three patients from unrelated families, and the resulting defect was classified as belonging to a novel complementation group (D). Transfection with the wild-type RFXAP gene restored the expression of MHC class II molecules in the patients' cells. CONCLUSIONS: Mutations in a novel MHC class II transactivating factor, RFXAP, can cause MHC class II deficiency. These mutations abolish the expression of MHC class II genes and lead to the same clinical picture of immunodeficiency as in patients with mutations in the other two MHC class II regulatory genes.     

Human T cell leukemia virus type I expression in salivary glands of infected patients

Human T cell leukemia virus type I (HTLV-I) sequences were sought in labial salivary glands of patients with HTLV-I-associated myelopathy or tropical spastic paraparesis and of seropositive neurologically healthy carriers. HTLV-I proviral DNA was found by polymerase chain reaction amplification in DNA extracted from lip biopsies of every patient. Viral RNA was found by in situ hybridization in the acini epithelium, as well as in lymphocytic infiltrates. This observation suggests that HTLV-I expression in labial salivary glands could participate in the inflammatory lesions observed in these patients. Some seronegative patients with Sj?gren's syndrome or dryness syndrome were also positive for viral transactivator tax DNA (41% in Martinique and 16% in non-HTLV-I-endemic region). Despite histologic signs of lymphocytic infiltration, no viral expression was found in the labial salivary glands of these patients.     

Interferon-gamma deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts

We have hypothesized that T cell cytokines participate in the pathogenesis of graft arterial disease (GAD). This study tested the consequences of IFN-gamma deficiency on arterial and parenchymal pathology in murine cardiac allografts. Hearts from C-H-2(bm12)KhEg (bm12, H-2(bm12)) were transplanted into C57/B6 (B6, H-2(b)), wild-type, or B6 IFN-gamma-deficient (GKO) recipients after immunosuppression by treatment with anti-CD4 and anti-CD8 mAbs. In wild-type recipients, myocardial rejection peaked at 4 wk, (grade 2. 1+/-0.3 out of 4, mean+/-SEM, n = 9), and by 8-12 wk evolved coronary arteriopathy. At 12 wk, the GAD score was 1.4+/-0.3, and the parenchymal rejection grade was 1.2+/-0.3 (n = 8). In GKO recipients of bm12 allografts, myocardial rejection persisted at 12 wk (grade 2.5+/-0.3, n = 6), but no GAD developed (score: 0.0+/-0.0, n = 6, P < 0.01 vs. wild-type). Mice treated with anti-IFN-gamma mAbs showed similar results. Isografts generally showed no arterial changes. In wild-type recipients, arterial and parenchymal cells showed increased MHC class II molecules, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 compared to normal or isografted hearts. The allografts in GKO recipients showed attenuated expression of these molecules (n = 6). Thus, development of GAD, but not parenchymal rejection, requires IFN-gamma. Reduced expression of MHC antigens and leukocyte adhesion molecules may contribute to the lack of coronary arteriopathy in hearts allografted into GKO mice.     

Vascular adhesion molecules and immunogenicity in blood vessels used as coronary artery bypass grafts

OBJECTIVE: The performance of coronary bypass grafts can be affected by a variety of circulating cell types. The initial event in any biological effect of such cells is adherence to the vascular endothelium prior to migration into the perivascular space. We aimed to investigate the expression of molecules that regulate cell adhesion in blood vessels employed as bypass conduits. METHODS: Segments of human saphenous vein, internal mammary artery, gastroepiploic artery and inferior epigastric artery were stained using specific monoclonal antibodies against the endothelial workers EN-4, Pal-E, von Willebrand factor small (vWF), and the cell adhesion molecules platelet-endothelium cell adhesion molecule (PECAM), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, the leucocyte marker (CD45) and major histocompatibility complex (MHC) class I and II antigens, with visualisation by ABC immunoperoxidase method. RESULTS: All vessels had a strong expression of the endothelial specific antigens EN4, vWF, and PECAM as well as MHC class I. However, there was less expression of Pal-E, ICAM-1, E-Selectin and of the DR determinant of MHC class II. VCAM-1, DP and DQ determinants of MHC class II were expressed to a weaker extent. There were no marked differences in the expression of all the molecules examined between the four vessel types. CONCLUSION: Thus vessels used as bypass grafts are immunogenic and possess the potential to attract and interact with blood elements. Definition of the molecules responsible could offer opportunities for modulating the response to such interactions.     

The protein tyrosine kinase p56lck regulates cell adhesion mediated by CD4 and major histocompatibility complex class II proteins

The CD4 protein is expressed on a subset of human T lymphocytes that recognize antigen in the context of major histocompatibility complex (MHC) class II molecules. Using Chinese hamster ovary (CHO) cells expressing human CD4, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In T lymphocytes, CD4 can also function as a signaling molecule, presumably through its intracellular association with p56lck, a member of the src family of protein tyrosine kinases. In the present report, we show that p56lck can affect cell adhesion mediated by CD4 and MHC class II molecules. The expression of wild-type p56lck in CHO-CD4 cells augments the binding of MHC class II+ B cells, whereas the expression of a mutant p56lck protein with elevated tyrosine kinase activity results in decreased binding of MHC class II+ B cells. Using site-specific mutants of p56lck, we demonstrate that the both the enzymatic activity of p56lck and its association with CD4 are required for this effect on CD4/MHC class II adhesion. Further, the binding of MHC class II+ B cells induces CD4 at the cell surface to become organized into structures resembling adhesions-type junctions. Both wild-type and mutant forms of p56lck influence CD4-mediated adhesion by regulating the formation of these structures. The wild-type lck protein enhances CD4/MHC class II adhesion by augmenting the formation of CD4-associated adherens junctions whereas the elevated tyrosine kinase activity of the mutant p56lck decreases CD4-mediated cell adhesion by preventing the formation of these structures.