"Lymphoid" chemokine messenger RNA expression by epithelial cells in the chronic inflammatory lesion of the salivary glands of Sjögren's syndrome patients: possible participation in lymphoid structure formation.

OBJECTIVE: Many studies have shown that the microanatomic organization of infiltrating leukocytes in the salivary gland lesions of patients with Sjögren's syndrome (SS) resembles the structure of lymphoid organs. A newly defined set of chemokines referred to as "lymphoid," which orchestrate leukocyte microenvironmental homing and contribute to the formation of lymphoid structures, provides directional clues. The aim of this study was to investigate the possible existence of "lymphoid" chemokines in the chronic inflammatory lesions of SS patients and thus validate their potential involvement in the disease process. METHODS: Twelve patients with primary SS, 3 patients with secondary SS, 4 patients with other autoimmune disorders, and 4 control individuals were the subjects of this study. Reverse transcriptase-polymerase chain reaction analysis was performed in order to examine the messenger RNA (mRNA) expression of "lymphoid" chemokines. Furthermore, in situ hybridization studies revealed chemokine mRNA localization. Immunohistochemistry was also applied in order to identify the cell types that expressed the chemokine mRNA. RESULTS: STCP-1/monocyte-derived chemokine and TARC mRNA were expressed in the majority of patients with primary and secondary SS, in 2 of 4 patients with other autoimmune disorders, and in 2 of 4 controls. BCA-1, ELC, and PARC mRNA were only detected in patients with primary and secondary SS. SLC mRNA was also detected in 1 non-SS patient. The main cellular sources of chemokine mRNA were ductal epithelial cells and infiltrating mononuclear leukocytes. CONCLUSION: The expression pattern of "lymphoid" chemokine mRNA points further to the role of epithelial cells in the pathogenesis of SS and offers new insight into the potential mechanisms that could be involved in leukocyte attraction and in the in situ formation of secondary lymphoid tissue structures.

Sjögren's syndrome. Autoimmune epithelitis.

Sjögren's syndrome is a chronic autoimmune disorder characterized by mononuclear cell infiltration proximally to epithelial cells of exocrine glands. In recent years, several studies have tried to address the function of the components of the immunopathologic lesion in Sjögren's syndrome. The majority of the mononuclear infiltrating cells are CD4 positive T lymphocytes (60-70%) whereas B cells constitute one fourth of the infiltrating cells. Macrophages and natural killer cells are poorly represented in the lesion. Epithelial cells of minor salivary glands of patients with Sjögren's syndrome express proinflammatory cytokines (IL-1 beta, IL-6), protooncogenes (c-myc) and costimulatory molecules (B71, B72). The destruction of epithelial cells of Sjögren's syndrome patients is probably due to activation of several apoptotic pathways since epithelial cells express different apoptosis related molecules such as Fas, FasL, Bax, while mononuclear cells express Bcl-2, Perforin and Granzymes. Finally epithelial cells seem to exert a regenerative effort since they express trefoil proteins (pS2). The above properties give epithelial cells a significant role in the pathophysiology of the syndrome but the exact events which drive the immune system towards an autoimmune reaction remain obscure.

Autoantibodies to La/SSB in patients with primary Sjögren's syndrome (pSS) are associated with upregulation of La/SSB mRNA in minor salivary gland biopsies (MSGs).

Recent studies have shown that minor salivary glands (MSGs) of patients with primary Sjögren's syndrome (pSS) are sites of anti-La/SSB autoantibody production. The aim of this study was to investigate the expression of La/SSB mRNA in MSGs of patients with pSS. La/SSB mRNA expression was studied by in situ hybridization in six biopsies of pSS patients with anti-La/SSB antibodies, nine pSS patients without anti-La/SSB and 10 patients with non-specific sialadenitis. Oligonucleotide probes corresponding to c-DNA encoding four linear epitopes of La/SSB (bp 423-471, bp 861-909, bp 903-954 and bp 1048-1092) were utilized. cDNA encoding linear epitopes of Ro52 (bp 786-837), Ro60 (bp 654-702) and the housekeeping genes of Sm and GAPDH were used as controls. The results were expressed as percent of positive cells by image analysis. Serum levels of anti-La/SSB autoantibodies were correlated with the presence and the intensity of La/SSB mRNA labeling. All pSS patients with anti-La/SSB antibodies in their serum expressed mRNA transcripts of epitopes 301-318 aa and 349-364 aa (encoded by the cDNA probes bp 903-954 and bp 1048-1092 respectively), predominantly in acinar and mononuclear cells of MSGs. These epitopes are the major targets of anti-La/SSB antibodies. Serum levels of anti-La/SSB antibodies were correlated with the number of positively stained cells in MSGs. Two of the nine pSS patients without anti-La/SSB autoantibodies and 2/10 non-pSS patients expressed the mRNA of the La/SSB molecule. The probes of RO52 and Ro60 epitopes did not react, while mRNA encoding the housekeeping genes of Sm and GAPDH was positive in all samples. In conclusion, pSS patients with anti-La/SSB antibodies showed upregulation of La/SSB mRNA in acinar and mononuclear cells of MSGs. Thus, active synthesis of La/SSB in MSGs of pSS seems to play an important role in the autoimmune response of the affected tissues.

CD4 cytotoxic and dendritic cells in the immunopathologic lesion of Sjögren's syndrome.

The existence of CD4+ T lymphocytes with cytotoxic activity in minor salivary gland (MSG) biopsies from Sjögren's syndrome (SS) patients was investigated using in situ double immunohistochemistry technique. The presence of dendritic cells (DC) in SS lesions was examined by using single and double immunohistochemistry methods and a panel of different MoAbs to specific cell surface markers (i.e. CD3, CD11c, DRC). Furthermore, the ultrastructural morphology of DC was characterized by electron microscopy (EM). Immunogold labelling technique using the DRC surface marker was also applied. Finally, we investigated the existence of germinal centres (GC) in the salivary gland lesions of SS patients. Seven patients with primary SS and five patients with non-specific sialadenitis were the subjects of this study. Our results indicate the existence of a CD4+ cytotoxic cell population that utilizes perforin-mediated cell destructions as they expressed perforin mRNA. Quantitative analysis of these cells revealed that they comprised approximately 20% of the existing T lymphocytes. We also identified a population of CD4+ T cells that expressed the CD11c activation marker. Furthermore, we observed a distinct cell subtype which expressed the DRC cell surface marker. These cells had the characteristic ultrastructural morphology of DC and were DRC+ when examined by immunoelectron microscopy. Finally, the formation of GC structures in the histopathologic lesions of the salivary glands was observed. The above findings indicate that both CD4+ cytotoxic T lymphocytes (CTL) and DC may be involved in the initiation and perpetuation of SS pathogenesis. Moreover, the formation of GC in the lesions reveals a possible mechanism for in situ differentiation and proliferation of activated B lymphocytes.

Lymphoma development in Sjögren's syndrome: novel p53 mutations.

OBJECTIVE: Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by lymphocytic infiltrations of the exocrine glands. Disease progression may lead to uncontrolled clonal proliferation of B lymphocytes and development of lymphoma. This study was undertaken to examine the possible involvement of the cell cycle checkpoint genes p53 and p21 in the pathophysiology of the syndrome. METHODS: Protein expression of p53 and p21 was studied, by immunohistochemistry and Western blot analysis, in minor salivary gland (MSG) biopsy specimens from 7 patients with SS and 5 control subjects. In addition, sequence analysis of the p53 gene was performed on DNA samples obtained from MSG biopsy samples of the same 7 patients with SS and from 4 patients with SS and in situ non-Hodgkin's lymphoma (NHL). RESULTS: The study revealed increased protein expression of p53 and p21 in MSG biopsy specimens from patients as compared with controls, while sequence analysis showed that the p53 gene was of the wild type. Furthermore, sequence analysis of the p53 gene from patients with SS and in situ NHL revealed 2 novel mutations in exon 5 of the p53 gene. These mutations are single-base substitutions and appear to be functional since exon 5 is included in the coding region of the p53 gene. CONCLUSION: This is the first report on wild-type p53 gene activation in SS. Our findings indicate a probable role for the DNA damage response genes in the pathogenesis of this syndrome. The novel mutations of the p53 gene implicate dysregulation of this tumor suppressor gene as a possible mechanism for lymphoma development in SS.

Expression of B7 costimulatory molecules by salivary gland epithelial cells in patients with Sjögren's syndrome.

OBJECTIVE: To investigate the expression of B7 costimulatory molecules in the lymphoepithelial lesions of salivary gland (SG) biopsy tissues and in SG epithelial cell lines derived from patients with Sjögren's syndrome (SS). METHODS: B7.1 and B7.2 protein expression was studied by immunohistochemistry in minor SGs obtained from 11 patients with SS and 10 disease control patients with nonspecific sialadenitis and in cultured SG epithelial cell lines obtained from minor SGs from 15 SS patients and 15 control patients. B7.1 and B7.2 messenger RNA (mRNA) expression by SG epithelial cell lines was examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In biopsy tissues from SS patients, but not control patients, ductal and acinar epithelial cells showed increased expression of both B7.1 and B7.2. Intense spontaneous B7.1 protein expression (as well as HLA-ABC, but not B7.2 or HLA-DR) was also found in 73% of SG epithelial cell lines from SS patients versus 13% of those from control patients (P < 0.01). Interferon-y treatment induced, or up-regulated, B7.1, B7.2, and HLA-DR expression in all SG epithelial cell lines tested. B7.1 and B7.2 expression by SG epithelial cell lines was also verified at the mRNA level by RT-PCR. CONCLUSION: Human SG epithelia are intrinsically capable of expressing B7 proteins upon activation. In SS patients, the expression of B7 molecules by SG epithelial tissues and by SG epithelial cell lines indicates the activated status of SG epithelial cells in this disorder and, possibly, their capacity for presenting antigens to T cells.

Progesterone induces calcitonin gene expression in human endometrium within the putative window of implantation.

The human endometrium acquires the ability to implant the developing embryo within a specific time window that is thought to open between days 19-24 of the secretory phase of the menstrual cycle. During this period the endometrium undergoes pronounced structural and functional changes induced by the ovarian steroids, estrogen and progesterone, that prepare it to be receptive to invasion by the embryo. The identification of reliable biochemical markers to assess this critical receptive phase in the context of the natural cycle remains one of the major challenges in the study of human reproduction. Our previous studies in a rat model system demonstrated that the expression of calcitonin, a peptide hormone involved in calcium homeostasis, is transiently induced by progesterone in the glandular epithelium at the onset of implantation. Attenuation of calcitonin synthesis in the uterus during the preimplantation phase by administration of calcitonin antisense oligodeoxynucleotides severely impairs implantation of rat embryos, suggesting that this peptide hormone plays a critical role in uterine receptivity. To investigate whether calcitonin is also expressed in the human endometrium during implantation, we monitored the spatio-temporal expression of calcitonin on various days of the menstrual cycle. Our studies employing RT-PCR showed that calcitonin messenger ribonucleic acid is expressed in human endometrium during the postovulatory midsecretory phase (days 17-25) of the menstrual cycle, with maximal expression occurring between days 19-21. Very little calcitonin expression was detected in the endometrium in either the preovulatory proliferative (days 5-14) or the late secretory (days 26-28) phase. In situ hybridization and immunocytochemical analyses localized the calcitonin expression predominantly in the glandular epithelial cells of the endometrium. Our studies further showed that calcitonin expression in the human endometrium is under progesterone regulation. Treatment of women with an antiprogestin, mifepristone (RU-486), drastically reduced calcitonin expression in the endometrium. Collectively, these findings reveal that progesterone-induced expression of calcitonin in the secretory endometrium temporally coincides with the putative window of implantation in the human.

Modes of epithelial cell death and repair in Sjögren's syndrome (SS).

We evaluated possible modes of epithelial cell destruction and restoration in minor salivary gland biopsies from patients with SS. Minor salivary gland biopsies from 10 primary Sjögren's syndrome (pSS) patients and eight control individuals were evaluated by immunohistochemical staining for the expression of apoptosis-related molecules, substances released by activated cytotoxic T cells, as well as proteins involved in epithelial cell repair. The results were analysed by computer screen analysis and they were expressed as average percentages. Apoptosis-promoting molecules, Fas antigen and Fas ligand were observed in ductal and acinar epithelial cells as well as in infiltrating mononuclear cells of minor salivary glands from SS patients in comparison with control biopsies. Bax protein, which acts as a death-promoter message, was expressed in the ductal and acinar epithelial cells and in mononuclear infiltrating cells of SS patients compared with control individuals, while Bcl-2, an inhibitor of apoptosis, was primarily found in the lymphocytic infiltrates. In situ DNA fragmentation assay (TUNEL) revealed that epithelial cells were apoptotic in patients with SS compared with control subjects. Immunohistochemical staining for perforin and granzyme B, released from granules of activated cytotoxic lymphocytes, revealed their presence in lymphocytic infiltrates of patients with SS compared with control biopsies. pS2, a member of the trefoil protein family which functions as promoter of epithelial cell repair and cell proliferation, was expressed in epithelial cells in biopsies from SS patients. These studies suggest that the functional epithelium of minor salivary glands in patients with SS appears to be influenced by both intrinsic and extrinsic mechanisms of destruction, while a defensive mechanism of epithelial restoration seems to be active.

Calcitonin is a progesterone-regulated marker that forecasts the receptive state of endometrium during implantation.

Previous studies established that in the rat, the uterus can accept a developing blastocyst for implantation only during a limited period of time on day 5 of gestation, termed the receptive phase. Our previous studies showed that the expression of calcitonin, a peptide hormone that regulates calcium homeostasis, is induced in rat uterus between days 3-5 of gestation and is switched off once the implantation process has progressed to day 6. In the present study, we analyze in detail how the expression of calcitonin messenger RNA (mRNA) in the uterus is regulated by the steroid hormones progesterone and estrogen and explore the possibility that calcitonin may serve as a potential marker of uterine receptivity. We demonstrate by in situ hybridization that calcitonin mRNA is synthesized specifically in the glandular epithelial cells between days 3-5 of pregnancy. Interestingly, calcitonin synthesis is also induced in these cells during pseudopregnancy, indicating that this peptide hormone is produced in the endometrium in response to maternal, rather than embryonic, signals. We also demonstrate that calcitonin mRNA expression during pseudopregnancy, like that in normal pregnancy, is under progesterone regulation. We further examined the steroid hormone regulation of uterine calcitonin expression in a delayed implantation model. In pregnant rats in which implantation is blocked upon removal of both ovaries on day 4 of gestation, continued administration of progesterone sustains calcitonin expression in the uterus for several days in the absence of estrogen. Administration of estrogen, which allows delayed implantation, also rapidly reduces calcitonin expression, indicating a role for this steroid hormone in turning off calcitonin gene expression. In gene transfection studies, expression of the progesterone receptor B isoform in cultured endometrial cells induces RNA synthesis from a reporter gene containing a 1.3-kb calcitonin promoter fragment in a hormone-dependent manner. As expected, mifepristone-complexed progesterone receptor B isoform fails to activate the calcitonin promoter. Progesterone acting through its nuclear receptor therefore regulates the expression of the calcitonin gene at the level of transcription. Finally, using RIA we investigated whether calcitonin is secreted from its glandular site of synthesis at the time of implantation by analyzing uterine flushings obtained from pregnant rats. We report the detection of a significant amount of calcitonin in the luminal secretions collected on day 4 and a lower amount on day 5 of gestation, whereas similar samples collected from animals on either day 3 or 6 of gestation did not contain detectable amounts of this peptide hormone. A transient burst of calcitonin secretion into the uterine lumen therefore occurs immediately preceding implantation. Based on these results, we propose that calcitonin is a measurable marker that forecasts the receptive state of rat endometrium during blastocyst implantation.

Heparin-binding epidermal growth factor-like growth factor in experimental models of membranous and minimal change nephropathy.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently described member of the epidermal growth factor (EGF) family. It binds to heparan sulfate proteoglycans via a cationic domain and is a potent mitogen for epithelial cells, fibroblasts and vascular smooth muscle cells. In the present study we have attempted to identify changes in quantity and distribution of HB-EGF in two models of acute glomerular epithelial cell injury, using Western blotting, immunohistochemistry and in situ hybridization. Prior to disease induction, Western blots showed some expression of HB-EGF protein within glomeruli. Within the first three days in the acute puromycin aminonucleoside (PAN) and passive Heymann nephritis (PHN) models, immunohistochemistry and in situ hybridization demonstrated an up-regulation of HB-EGF mRNA and protein in glomerular epithelial cells (GEC). In both cases, increased protein and mRNA was found prior to the onset of proteinuria and continued until day 21 post-induction, the last time point studied. Early in the course of the models, HB-EGF was localized to the cytoplasm of glomerular epithelial cells. At day 21, however, HB-EGF protein was distributed in a nodular pattern within GEC and along the glomerular basement membrane (GBM) in both models, suggesting that the secreted form might bind to the membrane. The increase in HB-EGF protein within glomeruli was confirmed by Western blots of glomerular membrane protein which, however, demonstrated a single 29 kDa species, consistent with the transmembrane form. These data are not consistent with binding of the secreted form of HB-EGF to the GBM. The transmembrane form of HB-EGF is able to signal in a juxtracrine fashion, so increased expression of HB-EGF mRNA and protein by GEC might contribute to the genesis of proteinuria through the initiation of abortive GEC mitogenesis.

Immunopathology of Sjögren's syndrome.

Sjögren's syndrome is a chronic autoimmune disorder characterized by mononuclear cell infiltration around epithelial cells of exocrine glands. In recent years, several studies have tried to elucidate the components of the immunopathologic interaction in Sjögren's syndrome as well as the function of these components. The majority of the mononuclear infiltrating cells are CD4 positive T lymphocytes (60-70%) whereas B cells constitute one fourth of the infiltrating cells. Macrophages and natural killer cells are poorly represented in the lesion. Epithelial cells of minor salivary glands of patients with Sjögren's syndrome express several cytokines (IL-1 beta, IL-6, NO), protooncogenes (c-myc), autoantigens (Ro, La, Fodrin) and costimulatory molecules (B71, B72). The characteristic destruction of epithelial cells of Sjögren's syndrome patients is probably due to activation of several apoptotic pathways since epithelial cells express different apoptosis related molecules such as Fas, FasL, Bax, while mononuclear cells express Perforin and Granzymes. Finally epithelial cells seem to exert a regenerative effort since they express trefoil proteins (pS2). The above mentioned properties give epithelial cells the leading role in the pathophysiology of the syndrome but the exact causative agent which drives the immune system towards an autoimmune reaction still remains obscure.

Heparin-binding epidermal growth factor-like growth factor, an immediate-early gene for mesangial cells, is up-regulated in the Thy-1.1 model.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is one of five well-described growth factors which bind to and activate the EGF receptor. Since cultured mesangial cells synthesize HB-EGF and this cytokine is a potent mitogen for smooth muscle cells (SMC), a cell type similar to mesangial cells, we attempted to determine (1) whether HB-EGF mRNA is present in the proliferative phase of experimental mesangial proliferative glomerulonephritis, and (2) some of the factors which regulate its synthesis by mesangial cells. In this study we demonstrate that cultured rat mesangial cells (RMC) express HB-EGF mRNA and that transcript levels are markedly increased by serum with maximal induction occurring within 2 h. Stimulation with individual cytokines (EGF, TGF-alpha, PDGF, TGF-beta and TNF-alpha), by contrast, had only a minor effect. The increase in HB-EGF mRNA levels following addition of serum was rapid, transient and independent of protein synthesis, features characteristic of immediate-early genes. In normal rat kidneys, there was no detectable glomerular expression of HB-EGF mRNA as determined by in situ hybridization, although occasional tubular cross sections were positive. Within 30 min after induction of the Thy-1.1 model, however, cells within the glomerulus and an increased number of tubules were positive. The number of positive glomerular and tubular cells increased progressively at days 1 and 4 post-induction, but declined by day 9 and had returned to background at day 15. Within the glomerulus, HB-EGF was expressed by cells of Bowman's capsule and cells within the glomerular tuft. Using (a) combined in situ hybridization and immunohistochemical staining of the same section and (b) staining of sequential sections by immunohistochemistry or in situ hybridization, it was found that intrinsic glomerular cells which did not stain with the macrophage marker ED-1 expressed HB-EGF mRNA. Although some were probably glomerular epithelial cells because of their peripheral location, it was uncertain whether mesangial cells were also positive. Future studies will be directed towards firmer identification of the glomerular cells expressing HB-EGF mRNA and to define the functional role of HB-EGF in the Thy-1.1 model.

Elevation of human cerebrospinal fluid clusterin concentration is associated with acute neuropathology.

Clusterin is a serum glycoprotein which is an inhibitor of complement and is expressed in many tissues in cell injury and death. It has been identified normal and pathological brain tissue and is a component of normal human cerebrospinal fluid (CSF). We have measured the clusterin concentration of 115 abnormal and normal human CSF samples and related these data to the patient's clinical diagnoses. CSF clusterin levels in patients with neurodegenerative and meningeal disease were within the normal range. Twelve of 15 patients with demyelination, however, had significant elevation of CSF clusterin concentration. This was not a specific finding for multiple sclerosis as elevated clusterin levels were also seen in patients with other acute neuropathology. Determination of CSF clusterin concentration may be of clinical value in neurological diagnosis.

Essential fatty acid deficiency increases interstitial IA-positive cells in rat kidney and accelerates renal allograft rejection.

Essential fatty acid deficiency has been reported to result in depletion of interstitial macrophages from rat kidneys and to permit transplantation of these kidneys across a fully allogeneic barrier without need for immunosuppression. In view of the potential for application of this phenomenon to xenografts, this study attempted to confirm the observation. Kidneys from rats fed normal or essential fatty-acid-deficient diets were transplanted to DA recipients. The donors' livers and contralateral kidneys were analyzed for their fatty acid profile in liver phospholipids, and the kidneys were examined by immunohistology for interstitial Ia(+) cells. EFAD resulted in an increase in renal interstitial Ia(+) cells detected by MRC-OX6 (anti-RT1Bpublic) from 13.5 +/- 2.9 (control diet fed rats) to 22.8 +/- 3.6 in rats on a stringent EFAD diet. Graft survival of kidneys from these EFAD rats was significantly shorter than that of kidneys from control diet fed rats. In direct contrast to the original report, this study found that EFAD caused a marked increase in renal interstitial Ia(+) cells and a reduction in allograft survival of EFAD donor kidneys.

Immunohistological detection of C5b-9 complement complexes in normal and pathological human livers.

The immunohistological localization of components and neoantigens of the C5b-9 human terminal complement complex was studied in 30 human liver biopsies. C5b-9, apparently in the soluble SC5b-9 form, was invariably detected in normal liver capsule and normal portal tract connective tissue. In livers with fibrosis and or cirrhosis, the pathological connective tissue contained variable amounts of SC5b-9 which was distributed in a similar way to that seen in normal livers. There was no significant C5b-9 deposition outside of the portal tract and capsule in any of the liver biopsies. In particular, in pathological livers, there was no deposition in relation to cellular infiltrates or areas of hepatic necrosis. These data support the concept that C5b-9 is a common component of connective tissue but do not indicate that C5b-9 mediated pathways are involved in the pathogenesis of hepatic injury.