Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

Immunohistochemical colocalization of growth hormone (GH) and α subunit in human GH secreting pituitary adenomas

Summary This immunohistochemical study disclosed that 9 of 15 GH secreting pituitary adenomas contained subunit positive cells. These cases also contained PRL positive adenoma cells, but LH was negative. Of these 9 cases, 4 cases showed occasional FSH containing cells, 2 of these also contained a few TSH positive cells. By mirror section technique, variable numbers of adenoma cells were found to contain both GH and subunit. Immunoelectron microscopically, both GH and subunit were localized in secretory granules which suggested their co-release from the tumour cells. The presence of GH and subunit in rough endoplasmic reticulum indicated their active production in the tumour. In the normal adult anterior pituitary gland, about 10% of GH cells contain FSH , and LH subunits and had appearances suggesting the co-production of GH and FSH as well as LH. The colocalization of GH and FSH is considered to be associated with the neoplastic transformation GH cells which possess the intrinsic potentiality of differentiation toward subunit. However, the mechanism for the lack or deficiency of subunits in the neoplastic condition remains to be further investigated.     

Neutrophil elastase α1-proteinase inhibitor complexes in pleural effusions

Summary Polymorphonuclear (PMN) granulocyte derived neutrophil elastase (NE) is rapidly antagonized by ₁-proteinase inhibitor (₁ PI) in vivo. To determine the clinical value of elastase ₁-proteinase inhibitor complexes (E-₁ PI) in pleural effusions, fluid samples of 99 patients were examined. Fifty-six had malignant effusions, 30 had nonmalignant exudates (pleural protein above 3 g/dl) mainly of inflammatory origin, and 13 patients had low protein transudates (below 3 g/dl) due to congestive heart failure. Nonmalignant exudates showed significantly higher (P<0.001) concentrations of E-₁ PI compared with malignant effusions or low protein transudates (P<0.001). Malignant exudates secondary to lung cancer were characterized by higher (P<0.001) median pleural E-₁ PI concentrations compared to malignant exudates due to primarily extrathoracic malignancies. Total pleural leukocyte counts and pleural neutrophil counts were performed in 68 effusions. By this means no clear-cut differentiation between malignant and nonmalignant exudates seems possible except for marked empyema. In conclusion, E-₁ PI complexes in pleural fluid may better reflect the stage of inflammation of pleural effusions rather than mere pleural leukocyte counts. Low levels of E-₁ PI complexes (<75 ng/ml) in pleural exudates with protein values above 3 g/dl are characteristic of malignant exudates. Determination of E-₁ PI in pleural exudates may serve as a sensitive marker of inflammation and useful adjunct to pleural cytology in aspects of differential diagnosis of pleural effusions.Abbreviations E-₁ PI elastase ₁ proteinase inhibitor complexes - NE neutrophil elastase - PMN polymorphonuclear     

Dysregulated Cytokine Production in Human Cystic Fibrosis Bronchial Epithelial Cells

Although pulmonary inflammation is an important pathologic event in cystic fibrosis (CF), the relationship between expression of the CF gene and the inflammatory response is unclear. We studied tumor necrosis factor (TNF) and IL-1 stimulated production of IL-6 and IL-8 by CF, corrected CF, and normal human bronchial epithelial cells in culture. During the first 24 hours of TNF stimulation, CF cells produced significantly more IL-8 than normal or corrected CF cells. In the second 24 hours of TNF stimulation, IL-6 and IL-8 generation ceased in normal and corrected CF cells but accelerated in CF cells, resulting in marked IL-6 and IL-8 accumulation in CF cells. Similar results were found when cells were stimulated with IL-1. Finally, when CF cells were grown at 27°C (a culture condition which results in transport of CF transmembrane conductance regulator, CFTR, to the cell membrane and normalization of chloride conductance) TNF-stimulated production of IL-6 and IL-8 reverted to normal. We conclude that dysregulation of cytokine generation by CF bronchial epithelial cells is directly related to expression of mutant CFTR and these observations provide a potential mechanism for persistence of airway inflammation in CF.     

Chemically modified ribozyme targeting TNF-alpha mRNA regulates TNF-alpha and IL-6 synthesis in synovial fibroblasts of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is chronic polyarthritis in which a variety of inflammatory cytokines play a role. Since tumor necrosis factor- (TNF-) is one of the most important cytokines in the pathogenesis of RA, we evaluated the feasibility of ribozymes as a therapeutic agent to control the inflammatory process of RA synovium. A hammerhead ribozyme against TNF- was chemically modified to increase nuclease resistance and added to RA fibroblastlike cell cultures without using a delivery system. The cellular uptake of fluorescent-labeled ribozyme into synovial cells was found to last at least 48 hr by confocal laser scanning microscopy. The ribozyme targeting TNF- gene inhibited both the expression of TNF- mRNA and the secretion of TNF- and IL-6. The cytotoxic effect by the ribozyme on synovial cells was negligible when determined by an alamar blue assay. Chemically modified ribozymes designed to suppress the TNF- gene may be potential as a therapeutic agent for rheumatoid arthritis.     

Presence of two isoforms of Na,K-ATPase with different pharmacological and immunological properties in the rat kidney

Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the ₁ isoform of the catalytic subunit, whereas the collecting duct expresses an ₃-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC₅₀ 5.10^–6 M) which is recognized by an anti- ₃ antibody and another moiety of lower affinity for ouabain (IC₅₀ 5.10^–4 M) which is recognized by an anti- ₁ antibody. Whether these two subpopulations correspond to different isoforms of the subunit of Na,K-ATPase ( ₁ and ₃-like) remains to be determined.     

Low In Vitro Production of Interferon-γ and Tumor Necrosis Factor-α in HIV-Seronegative Patients with Pulmonary Disease Caused by Nontuberculous Mycobacteria

We studied 32 HIV-seronegative patients with pulmonary disease caused by nontuberculous mycobacteria (NTM). Immunologic studies included lymphocyte subset analysis by flow cytometry, measurement of interferon- (IFN-) and tumor necrosis factor- (TNF-) production followingin vitro stimulation of diluted whole blood (DWB) and peripheral blood mononuclear cells (PBMC) by phytohemagglutinin (PHA), anti-CD3 as well as purified protein derivative of tuberculin (PPD), and in four cases with different amounts of the very mycobacterium, which caused disease in these patients. Data were compared to those of 30 HIV-seronegative patients with disease byMycobacterium tuberculosis (MTb). Following -CD3-stimulation of PBMC, NTM patients showed lower IFN-(P < 0.00005) and lower TNF-(P < 0.02). For a subgroup of tuberculin skin test-positive NTM patients we found significantly lower PPD-induced IFN- releases in cultured DWB(P < 0.0002) and PBMC(P < 0.0004) compared to MTb patients. Data for PPD-induced TNF- release for this subgroup were also significant(P < 0.001 andP < 0.05, respectively). The four NTM patients with poor PPD-induced IFN- response hardly showed increased cytokine production on stimulation with their specific mycobacterium. The lower production capacity of IFN- and TNF- of NTM patients compared to the MTb patients points to an immunologic imbalance forming the basis for their increased susceptibility to pulmonary infections by nontuberculous mycobacteria.     

alpha1-acid glycoprotein possesses in vitro pro- and antiinflammatory activities

We studied the effects of ₁-acid glycoprotein on tumor necrosis factor- (TNF-) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to ₁-acid glycoprotein: first, stimulation of TNF- and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-/IL-10 ratio remained unchanged after addition of native ₁-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of ₁-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.     

Intracerebral Production of Tumor Necrosis Factor-α, a Local Neuroprotective Agent, in Alzheimer Disease and Vascular Dementia

The local pattern of proinflammatory cytokine release was studied in Alzheimer disease (AD) and vascular dementia (VAD), by measuring intrathecal levels of IL-1, IL-6, TNF-, and its naturally occurring antagonists, soluble TNF receptors I and II. The cytokine levels were related to neuronal damage, as measured by the intrathecal tau concentration, to cerebral apoptosis assessed by levels of Fas/APO-1 and bcl-2, and to clinical variables. In vitro analysis was performed to study the effect of TNF- on the production of bcl-2, an antiapoptotic factor, by human neuronal cells. Patients with both AD and VAD displayed significantly higher intrathecal levels of TNF- compared to controls. In addition, patients with AD showed significantly negative correlations between the intrathecal levels of TNF- and the levels of Fas/APO-1 as well as of tau protein. The level of bcl-2 in supernatants of TNF--exposed cultures of human neuronal cells was up to three times higher than in control supernatants. Our study demonstrates intrathecal production of TNF- in patients with dementias, suggesting that this cytokine may have a neuroprotective role in these neurodegenerative conditions as evidenced by negative correlations between this cytokine and (i) levels of intrathecal Fas/APO-1 and (ii) levels of tau protein, both parameters closely related to brain damage. Our in vitro data suggest that TNF- exerts its neuroprotective effect by stimulating neuronal cells to express bcl-2, a molecule which downregulates apoptosis.     

Immunohistochemical localization of αl-antitrypsin and αl-antichymotrypsin in salivary pleomorphic adenomas

Summary Immunohistochemical identification of l-antitrypsin (l-AT) and l-antichymotrypsin (l-ACh) in pleomorphic adenomas of salivary glands is reported in order to compare their distribution profiles with those of lysozyme and lactoferrin, already described elsewhere.Normal salivary glands indicated positive l-AT staining in ductal segments and had no l-ACh in any glandular cell. Pleomorphic adenomas displayed moderate positivity to l-AT staining in duct-like, tubular and glandular epithelia which was particularly intense in luminal cells. The limited number of tumour cells which showed duct-like structures with a single cellular layer arrangement, displayed the highest staining to l-ACh. Strongly l-AT positive tumour cells located on the inner side of luminal cavities were also markedly positive to l-ACh. Spindle shaped tumour cells existed outside tubular and ductal structures and were negative to l-AT and l-ACh.Distribution of l-AT in salivary glands was similar to that of lysozyme as is usual in ductal segments or their transformed cells, and occurrence of l-ACh localization rather resembled that of lactoferrin, with occurrence in acinar compartments and changed epithelia within acini.The biological role of a specific immunohistochemical distribution of l-AT and l-ACh in pleomorphic adenomas may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases.     

The differential effects of IL-1 and TNF-α on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells

Objective and design:Interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF- on gene expression in the human chondrosarcoma cell line, SW1353. Materials and methods:SW1353 cells were stimulated singularly with IL-1, TNF-, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA. Results:Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-. The expression pattern of proIL-1, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1 is produced in the proform in IL-1-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation. Conclusions:IL-1 and TNF- each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1 appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.Received 15 September 2003; returned for revision 16 October 2003; accepted by J. S. Skotnicki 11 March 2004     

IFN-α Acts on T-Cell Receptor-Triggered Human Peripheral Leukocytes to Up-Regulate CCR5 Expression on CD4+ and CD8+ T Cells

Interleukin-12 (IL-12) as well as IL-2 was recently shown to up-regulate CCR5 expression on T-cell receptor (TCR)-triggered human T cells. Because of the functional similarity between interferon-alpha (IFN-) and IL-12, the present study investigated whether IFN- also up-regulates T cell CCR5 expression. CCR5 was marginally detected on T cells from unstimulated human peripheral blood leukocytes (PBLs) and only slightly induced on PBL T cells following stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies (mAbs). When anti-CD3/anti-CD28-triggered PBLs were exposed to IFN-, T cells expressed high levels of CCR5. The levels of CCR5 expression were comparable to those induced by IL-12. However, when purified T cells instead of unfractionated PBL were stimulated with anti-CD3/CD28 and then exposed to IL-12 or IFN-, CCR5 expression was induced by IL-12 but not by IFN-. IFN- was found to act on anti-CD3/anti-CD28-stimulated PBL to promote their IL-12 production. Moreover, addition of anti-IL-12 mAb to IFN--stimulated cultures of anti-CD3/CD28-pretreated PBL resulted in considerable inhibition of CCR5 expression. Together, these results indicate that IFN- as well as IL-12 up-regulates CCR5 expression on TCR-triggered T cells and that IFN- functions not by acting directly on T cells but via enhancing IL-12 production by PBL.     

In vitro response of mitochondrial succinate oxidase system to epinephrine in human blood lymphocytes from health individuals and patients with neurocirculatory dystonia

Activities of succinate dehydrogenase (SDH) and -glycerophosphate dehydrogenase (hyaloplasmic and mitochondrial: -GPDH_H and -GPDH_M) in peripheral blood lymphocytes, and the response of SDH activity to exogenous epinephrine in vitro (the epinephrine test) were studied in 20 healthy subjects and 46 patients with hypertensive neurocirculatory dystonia. Activities of SDH, -GPDH_H, and -GPDHM in blood lymphocytes and SDH adrenoreactivity in epinephrine test were higher in patients than in healthy controls. Treatment with hypotensive agents (isradipin and pyrroxan) moderated adrenoreactivity. Phytotherapy normalized the baseline activities of succinate oxidase system and -glycerophosphate pathway in blood lymphocytes.     

Expression of estrogen receptors-α and -β in primary breast neoplasms and tumors exposed to neoadjuvant hormonal therapy

We studied the content and expression of mRNA for estrogen receptors receptors- and - in breast tumors before and after 3-month neoadjuvant hormone therapy with antiestrogen tamoxifen and/or aromatase inhibitors. Expression of estrogen receptors- and - was most often detected in ER⁺PR⁺ tumors and most significantly decreased in these neoplasms after exemestane therapy. Immunocytochemical and radioligand assays showed that tamoxifen and anastrozole have little effect on the number of estrogen receptors- The number of progesterone receptors in tumors decreased by the end of anastrozole therapy. Estrogen receptors- were immunocytochemically revealed in 50% primary breast tumors. Anastrozole slightly decreased, while tamoxifen increased the incidence of these receptors. Interruption of signaling through estrogen receptors and suppression of estrogen biosynthesis had different effects on the receptor status of neoplasms and distribution of estrogen receptors- and -.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 559–562, November, 2004     

Antitumor effects of liposomal IL1α and TNFα against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1) and tumor necrosis factor alpha (TNF) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37°C. The biological activities mediated by liposomal IL1 and TNF were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 and TNF significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 and TNF displayed significant in vivo antitumor activity against the IL1- and TNF-resistant B16F10 metastatic murine melanoma.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.