CD45 tyrosine phosphatase controls common gamma-chain cytokine-mediated STAT and extracellular signal-related kinase phosphorylation in activated human lymphoblasts: inhibition of proliferation without induction of apoptosis.

The objective of this study was to test whether CD45 signals can influence signaling processes in activated human lymphoblasts. To this end, we generated lymphoblasts which proliferate in response to common gamma-chain cytokines, but readily undergo apoptosis after cytokine withdrawal. In experiments with the CD45R0 mAb UCHL-1, but not control CD45 mAbs, we found significant inhibition of proliferation. Interestingly, the pan-CD45 mAb GAP8.3, which is most effective in inhibition of OKT-3-mediated proliferation in quiescent lymphocytes, was ineffective in lymphoblasts. Addition of CD3 mAb OKT-3 had no influence on IL-2-mediated proliferation (with or without UCHL-1). In contrast, after addition of OKT-3 to IL-4- and IL-7-stimulated proliferation assays, UCHL-1 signals could not significantly alter cellular proliferation. We did not find induction of apoptosis following CD45R0 signaling. In Western blots using mAbs detecting phosphorylated STAT-3, STAT-5, STAT-6, or extracellular signal-related kinase 1/2, we found that CD45R0 signaling could effectively diminish phosphorylation of these intracellular signaling components. Using RT-PCR, we found that CD45R0 signaling inhibited IL-2 mRNA production without major influence on IL-13, IL-5, or IFN-gamma mRNA levels. Costimulation with OKT-3 and IL-2 optimally induced secretion of IFN-gamma, TNF-alpha, and IL-5, which was not decreased by CD45 signals. In conclusion, we illustrate that CD45R0 signals control early cytokine receptor-associated signaling processes and mRNA and DNA synthesis in activated human lymphoblasts. Furthermore, we show the existence of CD45 epitopes (GAP8.3), which are active and critical for signaling in quiescent lymphocytes, but are nonfunctional in activated human lymphoblasts.

Chlorpromazine induces apoptosis in activated human lymphoblasts: a mechanism supporting the induction of drug-induced lupus erythematosus?

OBJECTIVE: Drug-induced lupus erythematosus is a serious side effect of certain medications, such as procainamide, quinidine, hydralazine, chlorpromazine, and isoniazid, the underlying pathogenesis of which is unresolved. In this study, we examined the influence of these drugs on the regulation of apoptosis, or programmed cell death, in quiescent and activated human lymphocytes. We also discuss the dysregulation of apoptosis as a pathogenetic factor in systemic lupus erythematosus. METHODS: Peripheral blood mononuclear cells or activated lymphoblasts from normal donors were incubated with different concentrations of each of the above-mentioned drugs. RESULTS: We did not find induction of apoptosis in quiescent cells over a broad concentration range. In contrast, lymphoblasts readily underwent apoptosis when cultured with chlorpromazine, but not any of the other drugs, after stimulation with interleukin-2 (IL-2) in a dose-, time- and cell cycle-dependent manner. By several lines of evidence, toxicity was ruled out. Characteristic features of apoptosis-like incorporation of propidium iodide (PI), such as increased annexin V binding, changes in mitochondrial membrane potential, and induction of DNA breaks (as evidenced by TUNEL techniques), could be induced in lymphoblasts after chlorpromazine treatment. Chlorpromazine did not cause apoptosis by inhibition of cytokine binding or blockade of early intracellular signaling. The protease inhibitor Z-VAD and the ceramide inhibitor sphingosine 1-phosphate effectively blocked chlorpromazine-induced apoptosis (by PI staining and by externalization of phosphatidylserine), in contrast to the caspase 3/CPP32 inhibitor DEVD, which had only minor effects. Western blot analysis revealed IL-2-mediated phosphorylation of extracellular signal-regulated kinase, which was sensitive to chlorpromazine. Using lymphoblasts from a patient with Canale-Smith syndrome, we found that chlorpromazine-mediated apoptosis is Fas/ APO-1 independent. CONCLUSION: These data suggest that chlorpromazine mediates apoptosis in human lymphoblasts through specific activation of intracellular proapoptotic signaling cascades. This mechanism might lead to an unsynchronized inflow of apoptotic break-down products and thereby to the induction of (auto)immunity against nuclear components.

Etiopathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disease of unknown etiology. Research efforts of the last few years have mainly focused on basic molecular and cellular pathogenetic processes of the disease. Consequently, this paper reviews the etiopathogenetic hallmarks, such as impaired amount and presentation of nuclear antigens, production of antinuclear antibodies by T-cell-dependent B cell stimulation and organ damage by anti-dsDNA antibodies or immune complexes that are discussed at the present time. In summary, the hypothesis of a dysregulation of apoptotic cell clearance is strongly supported and broadly discussed.

[Hypereosinophilia with myocardial involvement due to toxocariasis. Diagnosis of regional myocardial perfusion abnormalities by pulsed tissue Doppler echocardiography]. / Myokardiale Beteiligung des Eosinophilie-Syndroms bei einer Fadenwurminfektion. Nachweis regionaler Myokardrestriktion mittels Pulswellen-Gewebe-Doppler-Echokardiographie.

CASE REPORT: The case of a 57-year-old woman is reported who was admitted for peripheral hypereosinophilia. DIAGNOSIS: Detailed investigations revealed first of all a hypereosinophilic syndrome with infiltration of bone marrow and lung. The patient suffered more and more from angina pectoris with signs of heart failure. Coronary angiography was therefore carried out which showed normal coronary arteries. With suspicion of myocardial involvement endomyocardial biopsies were performed which revealed the presence of Löfflers endocarditis parietalis fibroplastica. Finally, serological studies for parasites disclosed a positive ELISA test for Toxocara, confirmed later to be rising. CONCLUSION: Myocardial involvement of hypereosinophilia, caused by Toxocara is not described until now. Further diagnostic by means of pulsed wave tissue Doppler echocardiography provided regional differentiation of a restrictive filling pattern which documented the importance of this new diagnostic tool in myocardial illness.

[Gene therapy in rheumatoid arthritis--an already feasible therapeutic principle?]. / Gentherapie der rheumatoiden Arthritis--ein bereits anwendbares Therapieprinzip?

Based upon our increasing knowledge of mechanisms underlying tissue destruction in RA patients, new therapeutic principles have been developed, aimed at blocking proinflammatory cytokines or using antiinflammatory cytokines. Both principles, however, have proven to be very effective. In addition, the availability of the methodology to transduce cells with genes has initiated first experiments in animal models to test whether gene therapy for arthritis is suitable, followed by a first, very carefully formulated protocol for human RA. Gene therapy for RA has to still be considered as an experimental form of therapy in a very early stage, not allowing even now any serious treatment offer to RA patients. Several problems, such as the question of a suitable vector system have not yet been solved. With more experiments this therapeutic principle might become available and might prove effective even in a disease with a systemic character like RA in the coming years.

[Pathogenesis of rheumatoid arthritis]. / Pathogenese der rheumatoiden Arthritis.

BACKGROUND: Despite ongoing intensive research using sophisticated new molecular tools and methods, the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) is still not completely understood. HYPOTHESES: In this paper the two favorite hypotheses of the pathogenesis of rheumatoid arthritis currently discussed are introduced and compared. Hypothesis 1 is focussing on the central role of the T cells and T cell dependent phenomena in the pathogenetic scenario of RA. In contrast, hypothesis 2 stresses the role of altered synovial fibroblasts and their specific features critical for the destruction of inflamed joints. Both hypotheses are thoroughly discussed and suggestions for further research activities are made. CONCLUSION: Insights in the pathogenesis of RA provide options to develop new therapeutic strategies aimed at the inhibition of pathogenetic relevant processes.

Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice.

Rheumatoid arthritis (RA) has been thought to be largely a T-cell-mediated disease. To evaluate the role of T-cell-independent pathways in RA, we examined the interaction between isolated RA synovial fibroblasts and normal human cartilage engrafted into SCID mice in the absence of T cells and other human cells. The expression of cartilage-de grading enzymes and adhesion molecules was examined by immunohistochemistry and in situ hybridization techniques. The RA synovial fibroblasts invaded the cartilage and kept their transformed appearing cellular shape. They expressed VCAM-1 and produced the cathepsins L and B at the site of invasion. We conclude that RA synovial fibroblasts maintain their invasive and destructive behavior over longer periods of time in the absence of human T cells, indicating that T-cell-independent pathways play a significant role in rheumatoid joint destruction.

Comparative analysis of cathepsin L, cathepsin D, and collagenase messenger RNA expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis, by in situ hybridization.

OBJECTIVE: To compare the expression of cathepsin L, cathepsin D, and collagenase messenger RNA (mRNA) in synovial specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: The expression of cathepsins L and D as well as collagenase mRNA in synovial tissues from 8 patients with RA, 6 patients with OA, and 2 patients with noninflamed joints was evaluated using in situ hybridization with digoxigenin-labeled RNA probes. RESULTS: Both RA and OA synovial tissue expressed cathepsins L and D as well as collagenase mRNA. The expression of the cathepsins was markedly higher in interstitial regions and, to some extent, in perivascular infiltrates of RA synovial tissue compared with OA specimens. CONCLUSION: Cathepsins L and D mRNA are expressed differently in RA and OA synovial tissues, supporting the concept that these enzymes may contribute to the influx of mononuclear cells into RA synovium. Moreover, the data reveal that the expression of collagenase and cathepsins in RA and OA synovial lining is otherwise largely similar, and suggest that the adhesion of synovial cells to cartilage mediates the invasive destructive process in RA.

Expression of E-selectin messenger RNA and protein in rheumatoid arthritis.

OBJECTIVE: To examine the de novo synthesis and cellular distribution of the E-selectin adhesion molecule in synovial tissues obtained from patients with rheumatoid arthritis (RA). METHODS: Immunohistochemistry techniques combined with in situ hybridization were used to examine RA synovium. RESULTS: There were numerous endothelial cells positive for E-selectin and E-selectin messenger RNA in the RA synovial membranes. Moreover, E-selectin expression appeared to correlate with inflammatory activity. CONCLUSION: The strong vascular expression of E-selectin indicates an activation of endothelial cells in the recruitment of cells associated with the chronic inflammation of RA.

Expression of vascular cell adhesion molecule-1 mRNA and protein in rheumatoid synovium demonstrated by in situ hybridization and immunohistochemistry.

BACKGROUND: Vascular cell adhesion molecule-1 (VCAM-1) is expressed in synovial tissue of patients with rheumatoid arthritis. VCAM-1-protein has been demonstrated in nonvascular cells beside a vascular expression of this molecule. There are conflicting results about the nonvascular cell types expressing VCAM-1. EXPERIMENTAL DESIGN: For the evaluation of VCAM-1 expression in rheumatoid synovium, this molecule has been demonstrated by alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. Furthermore, VCAM-1 mRNA has been demonstrated by in situ hybridization to evaluate de novo synthesis of this molecule in vivo. To elucidate the nature of the cell types expressing VCAM-1 mRNA, this molecule has been shown by combined in situ hybridization for VCAM-1 and immunohistochemistry in the same tissue section. Double labeling has been performed with anti-collagen type IV monoclonal antibodies to delineate endothelial cells and pericytes and with anti-CD68 antibodies to elucidate the expression of VCAM-1 mRNA in fibroblast-like (type B) or macrophage-like (type A) synoviocytes. RESULTS: Although it has been reported that VCAM-1 occurs on endothelial cells after cytokine stimulation, we show that vascular expression of VCAM-1 mRNA and protein was minimal and restricted to small vessels beneath the lining cell layer. Further expression of VCAM-1 mRNA could be demonstrated in pericytes outside the collagen type IV containing vascular basement membrane. With respect to the expression of VCAM-1 in the synovial lining layer, we could clearly demonstrate by combined in situ hybridization and immunohistochemistry that CD68 positive cells of the monocyte/macrophage lineage in the lining layer (type A cells) do not express VCAM-1 mRNA and that the expression of VCAM-1 mRNA in the lining layer was restricted to fibroblast-like synoviocytes (type B cells). Scattered stromal cells revealing VCAM-1 mRNA were also CD68 negative. CONCLUSIONS: The strong expression of VCAM-1 in the fibroblast-like cells of RA synovium and the lack of expression in the vascular endothelium suggest that the major role of VCAM-1 appears to be associated with the proliferating synovial cells prone to attach and subsequently invade articular cartilage.

Detection of insulin-like growth factor I and II in synovial tissue specimens of patients with rheumatoid arthritis and osteoarthritis by in situ hybridization.

OBJECTIVE: To study the expression of insulin-like growth factor I and II (IGF I and II) in synovial tissue specimen of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Synovial tissue sections were examined for the expression of IGF I and II by in situ hybridization using digoxigenin labeled antisense and sense RNA probes. RESULTS: The antisense probe of IGF I reacted with all specimens. IGF II mRNA was expressed in 7/7 RA and 4/5 OA tissues. Cells of the synovial lining and subsynovial layer bound both antisense probes, whereas inflammatory infiltrates of RA tissues were labeled rarely. CONCLUSION: The significant number of cells in the synovium that express IGF I and II mRNA suggests a role of IGF in repair mechanisms of articular cartilage in response to injury and effects on fibroblast growth within the synovium.

A new double labeling technique for combined in situ hybridization and immunohistochemical analysis.

BACKGROUND: A reliable, sensitive, and specific double labeling technique is required that allows the simultaneous visualization of in situ hybridization products and antigens. Currently used double labeling techniques are limited by various problems including the numerous disadvantages associated with radioactive labels, the time-dependent loss of fluorescence signals, and the high background that is associated with various peroxidase techniques. Therefore, the aim of this study was to develop an improved double labeling technique. EXPERIMENTAL DESIGN: Riboprobes were used for detection of mRNA of cathepsin D, vascular cell adhesion molecule, and endothelial leukocyte adhesion molecule in in situ hybridization and monoclonal antibodies specific for macrophages and basement membranes (collagen type IV) for immunohistochemical analysis. The in situ hybridization and immunohistochemical analysis were used to characterize cathepsin D messenger ribonucleic acid (mRNA) in macrophages expressing cells and the expression of adhesion molecule mRNA in endothelial cells delineated by the vascular basement membrane expressing collagen type IV. RESULTS: The application of in situ hybridization detection systems before immunohistochemical analysis was shown to give reliable results. In situ hybridization with digoxigenin labeled riboprobes using alkaline phosphatase linked Fab fragments visualized by 4-nitro blue tetrazolim chloride/5-bromo-4-chloro-3-indolphosphate combined with immunohistochemical detection of the antigen by the alkaline phosphatase anti-alkaline phosphatase-technique with new fuchsin as substrate is a reliable double labeling technique. Using this protocol, we could show that the reaction product is stable, there is virtually no background, and both reaction products can be easily distinguished. Vascular cell adhesion molecule-1 mRNA is expressed only in endothelial cells and certain fibroblast-like cells that do not label with antibodies against macrophages, whereas cathepsin D mRNA is coexpressed with macrophages. We also demonstrated that endothelial leukocyte adhesion molecule-1 mRNA is strongly expressed in endothelial cells that can be localized within the boundaries of the vascular basement membrane. CONCLUSIONS: A new and reliable double labeling technique for the simultaneous evaluation of in situ hybridization and immunohistochemical analysis is described that is suitable for various applications.

A new model for rheumatoid arthritis generated by engraftment of rheumatoid synovial tissue and normal human cartilage into SCID mice.

OBJECTIVE: A new animal model was used to study the interaction between rheumatoid synovial cells and cartilage and to explore the cellular basis of rheumatoid joint destruction. METHODS: Fresh synovial tissue derived from patients with rheumatoid arthritis was implanted with normal human cartilage into SCID mice, either subcutaneously or under the renal capsule, for up to 304 days. The implants were analyzed by light and electron microscopy, as well as by immunohistochemistry and in situ hybridization. RESULTS: Human synovial tissue and cartilage implanted in SCID mice are maintained by the animals for up to 304 days. After 35 days, focal erosions occur at the site of attachment of synovial lining cells to the cartilage. After 105 days, a pannus-like formation, consisting of proliferating synovial fibroblast-like cells invading the cartilage, is observed. The fibroblast nature of these cells was supported by observation of only focal expression of the macrophage markers CD14 and CD68. Cells at the immediate site of cartilage destruction express messenger RNA for cathepsin L, whereas cathepsin D messenger RNA was detected in subsynovial regions away from the site of destruction. The human origin of the tissue involved in cartilage destruction was demonstrated using monoclonal antibodies to HLA-ABC and human type IV collagen. CONCLUSION: The present approach introduces a novel in vivo model of rheumatoid arthritis for the study of the molecular and cellular mechanisms of rheumatoid joint destruction at sites of synovial attachment to cartilage. In this model, the SCID mouse acts as a useful host for studying the properties of rheumatoid synovium in the absence of circulating human blood components.