The production of CXCR3-agonistic chemokines by synovial fibroblasts from patients with rheumatoid arthritis

The inflamed synovial tissue of rheumatoid arthritis (RA) is characterized by an infiltration with Th1 cells that predominantly express the chemokine receptors CXCR3 and CCR5. In this study, we investigated the production of the CXCR3-agonistic chemokines CXCL9, CXCL10, and CXCL11 by synovial tissue cells and synovial fibroblast-cell lines (fourth or fifth passage) from RA patients. Concentrations of all CXCR3 ligands in synovial fluids were markedly higher in RA patients than in osteoarthritis (OA) patients. Synovial tissue cells from RA patients more strongly expressed mRNAs for CXCR3 ligands and spontaneously secreted larger amounts of these chemokine proteins than the cells from OA patients. The mRNA expression of all CXCR3 ligands was induced in synovial fibroblasts from RA patients after stimulation with interferon gamma (IFN-), tumor necrosis factor alpha (TNF-), or interleukin-1 beta (IL-1). However, synovial fibroblasts significantly secreted CXCL9 and CXCL10 proteins, but not CXCL11 protein, after IFN- stimulation and secreted only CXCL10 protein after TNF- or IL-1 stimulation. When stimulated with a combination of IFN- and TNF-, these cells were able to secrete large amounts of all three chemokines. These results indicate that synovial fibroblasts may be involved in perpetuating the Th1 immune response by producing the Th1-associated CXCR3 ligands, and the synergistic effect of IFN- and TNF- may be important for their chemokine production in RA joints.     

Serum zinc and copper in active rheumatoid arthritis: Correlation with interleukin 1β and tumour necrosis factor α

Serum zinc and copper levels and serum interleukin 1 (IL1) and tumour necrosis factor (TNF) levels were evaluated in 57 female patients with active rheumatoid arthritis (RA) to investigate a possible role of IL1 and TNF on zinc and copper homeostasis in RA. Serum zinc levels were significantly lower and serum copper levels significantly higher in RA patients when compared with osteoarthritis or asymmetrical psoriatic oligoarthritis patients and with normal controls. No differences were observed in serum IgM rheumatoid factor positive and serum IgM rheumatoid factor negative patients as regards serum zinc and copper concentration. In RA patients the erythrocyte sedimentation rate and acute-phase proteins correlated negatively with serum zinc and positively with serum copper. IL1 and TNF were found to correlate negatively with zinc and positively with copper in RA patients. Lower levels of zinc may be due to an accumulation of zinc-containing proteins in the liver and in the inflamed joints in RA. Elevated serum copper levels seem to be linked to the increased synthesis of ceruloplasmin by the liver.     

High levels of macrophage inflammatory protein-1α correlate with prolactin in female patients with active rheumatoid arthritis

The relationship between the endocrine immune modulator prolactin and the macrophage activation parameter MIP-1 (macrophage inflammatory protein-1) was investigated in 61 females with rheumatoid arthritis (RA). The chemokine MIP-1 was found to be twice as high in active than in inactive RA. Parallel to the inflammatory activity (acute phase response and joint count) and high levels of MIP-1 there were markable changes of serum prolactin. MIP-1 seems to have an influence on the pituitary hormone secretion. A significant correletion between prolactin and MIP-1 (r=0.67;p0.00001) point out the bidirectional influence of the immune and endocrine system in RA.     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Results of a multicenter placebo-controlled double-blind randomized phase III clinical study of treatment of rheumatoid arthritis with recombinant interferon-gamma

Summary In a multicenter placebo-controlled double-blind randomized clinical study, 91 patients with rheumatoid arthritis were given 28 days' treatment with recombinant interferon-gamma (50 g daily for 20 days, then 50 g each second day up to day 28, given by subcutaneous injection). The aim of the study was to provide a methodologically clear demonstration of the efficacy of treatment with interferon-gamma, using criteria that could be handled by statistical tests. Evaluatable documentation was available for 79 patients, of whom 40 were treated with the active compound. The principal criterion for the statistical evaluation of the therapeutic success was improvement of the Ritchie joint pain index or Lansbury joint pain index by at least 30% within 28 days. The chi-square test showed superiority of the interferon arm over the placebo arm with an error probability of a<1%. In addition, efficacy of interferon-gamma was demonstrated in respect of practically all parameters investigated. The frequency of side-effects, including febrile reactions, was the same for the active compound and the placebo. During interferon treatment the daily maximum body temperature was raised by 0.3°C on average, but was below 37.2°C at all times.     

Distribution of estrogen receptor subtypes,expression of their variant forms,and clinicopathological characteristics of human colorectal cancer

We examined the expression of estrogen receptor (ER) messenger RNAs (ER-, ER-, and ER- isoforms) in colorectal tumor samples and corresponding normal mucosa, paying particular attention exons 3 and 5 of both ER mRNA subtypes that likely suffer deletions, and may encode proteins that have lost either DNA- or ligand-binding moieties. Then we correlated these findings with the clinicopathological properties of the tumors. Our results demonstrated that in all patients the two ER subtype mRNAs were coexpressed in wild-type form. In 10% of the patients the ER- mRNA was also present as an exon-5-deleted form that encoded any aberrant protein. Immunohistochemical analysis revealed that the ER- protein was present in tumor stroma, but not in infiltrating lymphocytes. ER-1 and ER-2, isoforms of ER-, were up-regulated in malignant tissues, whereas the ER-5 isoform, was found to be equally expressed, at very low levels, in the two tissue compartments. No correlations between ER levels and clinicopathological parameters were found. This suggests that the ER- mRNA levels are independent of the tumor characteristics.     

Distribution of β-Adrenoceptor Subtypes in Gastrointestinal Tract of Nondiabetic and Diabetic BB Rats A Longitudinal Study

The effects of aging and diabetes on thedistribution of -adrenoceptor subtypes in the gutwere investigated in the BB rat.[¹²⁵I]Cyanopindolol binding to 10-msections was evaluated using film autoradiography. Cyanopindolol binding to -,₁-, and₂-adrenoceptors was displaced by 1M propranolol, 50 nM ICI-89-406, and 100 nMICI-118-551, respectively. -Adrenoceptor bindingwas highest in the circular muscle of proximal colon and lowest in thepylorus of 4- to 5-month-old rats. Aging (8- to10-month-old vs. 4- to 5-month-old rats) was associatedwith increased -adrenoceptor binding in thepylorus and reduced binding in the proximal colon.Diabetes had a time-dependent effect on the level of-adrenoceptor binding. It was increased in theantral and pyloric stomach but longer periods ofdiabetes caused a reduction in -adrenoceptorbinding in the pylorus. Those in the intestine werereduced time-dependently and involved₁- or ₂-adrenoceptorsor both.     

Changes in expression levels of genes involved in fatty acid metabolism: upregulation of all three members of the PPAR family (α, γ, δ) and the newly described adiponectin receptor 2, but not adiponectin receptor 1 during neonatal cardiac development of the rat

During neonatal cardiac development, the heart changes its substrate preference from glucose to fatty acids. The aim of this study was to investigate the changes in mRNA expression levels of genes involved in the control of cardiac fatty acid metabolism in the transition from neonatal to adult life. Methods mRNA expression levels for peroxisome proliferator activated receptor (PPAR) , and , PPAR co–factor 1 and (PGC–1 and ), 9–cis retinoc–acid–activated receptor , and (RXR , , ), 5–AMP activated protein kinase (AMPK) 1 and 2, adiponectin receptor 1 and 2 (AR 1 and AR 2) were measured in heart tissue of neonatal 0–day, 7–day and 21– day old rats. Results mRNA expression of all three members of the PPAR family were upregulated significantly from day 0 to day 21 ( +117%, +133%, +203%). In addition, m–RNA expression of all RXR isoforms increased from day 0 to day 7 ( +125%, +69%; +41%). AR 2 exhibited a small but significant increase in mRNA expression (+ 46%). Conclusions We were able to demonstrate for the first time that in addition to PPAR, also PPAR and , as well as all RXR isoforms and AR 2 are upregulated in the heart during neonatal development.Drs. Steinmetz and Quentin contributed equally to this publication     

Effects of Cytokines on the Binding of Leukocytes to Cultured Rat Hepatocytes and on the Expression of ICAM-1 by Hepatocytes

Adhesions of leukocytes to hepatocytes andsinusoidal endothelial cells mediates the induction andprogression of hepatic injury. However, in contrast toendothelial cells, information regarding the regulation of interactions between leukocytes andhepatocytes is limited. In the present study, weinvestigated the effect of inflammatory mediatorsincluding lipopolysaccharide (LPS), staphylococcalenterotoxin B (SEB), interferon- (IFN-), tumornecrosis factor- (TNF-), andinterleukin-1 (IL-1) on the adhesion ofpolymorphonuclear leukocytes or lymphocytes to primarycultured rat hepatocytes, and on the expression of intercellular adhesionmolecule-1 (ICAM-1) gene in hepatocytes. Bothpolymorphonuclear leukocyte and lymphocyte adhesion tohepatocytes were enhanced after exposure of hepatocytes to IFN- and TNF-, but not afterexposure to LPS, SEB or IL-1. The adhesion inducedby either IFN- or TNF- was inhibited bymonoclonal antibodies against ICAM-1 or lymphocytefunction-associated antigen-1 (LFA-1). Nonstimulated hepatocytesexpressed faintly ICAM-1 mRNA, which increased slightlyduring the culture period. ICAM-1 mRNA expression wasup-regulated to a greater extent by incubating hepatocytes with IFN- or TNF-,and peaked after 12 hr of incubation with TNF-and after 24 hr with IFN-. These results indicatethat IFN- and TNF- induce the expressionof ICAM-1 on parenchymal hepatocytes and that theLFA-1-ICAM-1 pathway plays an important role in theinteraction between hepatocytes and neutrophils orlymphocytes.     

Cytokines affecting megakaryocytopoiesis in rheumatoid arthritis with thrombocytosis

Particular interleukins, namely interleukin (IL)-6, IL-4 and IL-1, with pro-inflammatory mediator activities have also been shown to be involved in the regulation of megakaryocytopoiesis. As rheumatoid arthritis (RA) is not uncommonly associated with reactive thrombocytosis, we investigated serum IL-la, IL-1, IL-4 and IL-6 concentrations in RA patients with marked thrombocytosis, and compared them to the levels in RA patients with normal platelet counts and healthy volunteers. IL-1, IL-4 and IL-6 concentrations were found to be correlated with the disease activity in both groups of RA patients, with higher serum levels of each cytokine in the thrombocythaemic group. Significant positive correlations of IL-1 and IL-4 with the platelet counts were documented only in patients with thrombocytosis. According to our results, IL-6 and IL-1 were found to be good indicators of disease activity in RA, while IL-1 and IL-4 seemed to be related more with the process of reactive thrombocytosis secondary to rheumatoid inflammation.     

Degradation in vivo of articular cartilage in rheumatoid arthritis and juvenile chronic arthritis by cathepsin G and elastase from polymorphonuclear leukocytes

Summary Peroxidase-anti-peroxidase (PAP) staining and specific antibodies against cathepsin G and elastase from polymorphonuclear leukocytes (PMN) were applied to pannus-free and microscopically intact superficial articular cartilage. Restricted local deposits containing cathepsin G and elastase were found in three of ten patients with seropositive rheumatoid arthritis (RA), in one of three patients with seronegative RA and in one patient with juvenile chronic arthritis (JCA). Similarly, localized deposits of IgG and C3 were found in the patients with seropositive RA and JCA, but not in the patient with seronegative RA. Adjacent sections exhibited esterase activity in and around the PMN. In proteinase-positive areas from patients with seropositive RA the inhibitors ₁-proteinase inhibitor (₁-PI) and ₂-macroglobulin (₂-MG) were present in two of three and one of three patients, respectively. In JCA only ₁-proteinase inhibitor was present, and in seronegative RA no inhibitors were found. No staining of articular cartilage was observed in a patient with psoriatic arthritis. One of three cases with osteoarthritis exhibited patchy superficial staining for IgG only. In articular cartilage covered by pannus, in three patients with seropositive RA, in one with seronegative RA and in the patient with JCA a few regions with variably dense PMN infiltrates were observed. Cathepsin G, elastase and esterase activity were found in and around the PMN. In one of the three patients with seropositive RA the adjacent cartilage-pannus junction exhibited distinct staining for cathepsin G and elastase, but not for IgG/C3 and proteinase inhibitors. The findings suggest that cathepsin G and elastase from PMN are involved in the breakdown of rheumatoid articular cartilage in the presence, as well as in the absence, of entrapped immune-complex-like material, and that proteinase inhibitors are often lacking in such regions.This paper is dedicated to Prof. Dr. A. Böni and Prof. Dr. H. Mathies for their 75th and 70th birthdays     

Oligosaccharides accumulated in the bovineβ-mannosidosis kidney

Summary The phenotype of bovine-mannosidosis (-mannosidase deficiency), recently identified in Salers cattle, is similar to the caprine form of the disease (Abbittet al., 1991). This investigation was designed to characterize accumulated kidney oligosaccharides in bovine-mannosidosis. Oligosaccharides were extracted from the kidney of an affected Salers calf and purified by chromatographic techniques. The amount of accumulating oligosaccharides in 1 g of wet tissue was about 21µmol. Structures of derivatized oligosaccharides were characterized by high-performance liquid chromatography, mass spectrometry, methylation analysis and sequential exoglycosidase digestions. The major accumulating oligosaccharides were Man1-4GlcNAc and Man1-4GlcNAc1-4GlcNAc. Oligosaccharides accumulating in minor amounts were Man1-4GlcNAc1-4Man1-4GlcNAc, Man1-6Man1-4GlcNAc1-4GlcNAc and Man1-4GlcNAc1-4Man1-4GlcNAc1-4GlcNAc. As in caprine-mannosidosis, oligosaccharides with terminal-mannose residues and cleaved as well as uncleaved chitobiose linkages were identified in bovine-mannosidosis kidney. The accumulating oligosaccharides in tissue were thus identical in bovine and caprine-mannosidosis; however, the source of the novel oligosaccharides remains to be determined.     

Increased Expression of Transforming Growth Factor-β1 During Gastric Ulcer Healing in Rats

This study was done to investigate theexpression and localization of transforming growthfactor-₁ (TGF-₁) inthe gastric ulcerated tissues produced by acetic-acidduring the healing process, by northern blot analysis and immunohistochemicaltechnique. Ulcerated TGF-₁ mRNA levelswere significantly increased from days 3 to 18, in asimilar manner to extracellular matrix proteins, andreturned to control levels at the scarred phase.Immunoreactive TGF-₁ was localized inepithelial cells beneath proliferative zone in intacttissues. 1 In ulcerated tissues, TGF-₁was localized in macrophages in the ulcer bed and in fibroblasts ormyofibroblasts in the granulation tissues. Treatmentwith prostaglandin E₁ (PGE₁)further stimulated ulcerated TGF-₁expression, being associated with the acceleration of gastric ulcer healing, while treatment withindomethacin reduced TGF-₁ expression,being accompanied by the delayed ulcer healing. Thecombination of PGE₁ and indomethacin reversedthe indomethacin-induced decrease in ulcerated TGF-₁.Thus, TGF-₁ may be implicated in theacceleration of gastric ulcer healing.     

Presence of Two Signaling TGF-β Receptors in Human Pancreatic Cancer Correlates with Advanced Tumor Stage

Transforming growth factor- (TGF-)signal transduction is mediated via specific cellsurface signaling TGF- receptors, most notably thetype I ALK5 (TR-I_ALK5)and the type II(TR-II). We evaluated TR-I_ALK5 andTR-II expression in 41 human pancreatic cancertissue samples and correlated these findings withclinical data of the patients. Northern blot analysisindicated that, in comparison with the normal pancreas,pancreatic adenocarcinomas exhibited 8.0-fold and4.5-fold increases (P < 0.01), respectively, in mRNAlevels encoding TR-I_ALK5 andTR-II. In situ hybridization showed that both TR-I_ALK5 mRNAwere highly expressed in the majority of pancreaticcancer cells. Immunohistochemical analysis ofTR-I_ALK5 and TR-II revealedpositive immunostaining in 73% and 56% of the tumors, respectively. Both receptorswere concomitantly present in 54% of the pancreaticcancer samples. The presence ofTR-I_ALK5 or TR-II and theconcomitant presence of TR-I_ALK5 and TR-II in the cancer cells was associatedwith advanced tumor stage (P < 0.01). These findingsshow that in many human pancreatic cancers, increasedlevels of the two signaling TRs are present. The presence of the signaling TRs inadvanced tumor stages indicates a role in diseaseprogression.     

Hypertrophic osteoarthropathy: Endothelium and platelet function

Summary Hypertrophic osteoarthropathy (HOA) is characterized by finger clubbing, periostosis and arthritis. The pathogenesis of hypertrophic osteoarthropathy is still uncertain. Earlier studies have been focused on the potential role of platelet and endothelium in the pathogenesis of HOA.The aim of this study was to evaluate the circulating levels of endothelin-1 (ET-1), -thromboglobulin (-TG) and platelet-derived growth factor (PDGF) in 21 HOA patients.The circulating levels of ET-1, -TG were significantly higher in HOA patients vs healthy controls, but not vs controls with lung diseases. On the contrary, PDGF was significantly higher in HOA patients vs healthy controls and vs subjects with lung diseases.These findings suggest that endothelium/platelet unit may play a role in the pathogenesis of HOA, and PDGF could induce the changes observed in HOA.     

Enhanced Expression of Transforming Growth Factor (TGF) -α Precursor and TGF-β1 During Paneth Cell Regeneration

An intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induces selective killing and rapid regeneration of Paneth cells, which have a large amount of zinc in their cytoplasmic granules. We examined the expression pattern of transforming growth factor (TGF) - and TGF-₁ in this regenerative process. Messenger RNA expression of TGF- and TGF-₁ reached their peaks at 12 and 24 hr after dithizone injection, respectively. Protein expression of TGF- precursor and TGF-₁ increased to a maximum at 24 and 72 hr, respectively. Their immunoreactivities were localized in the epithelial cells in the vicinity of Paneth cells, whereas they were prominent in the upper half of the crypts in control rats. In conclusion, destruction of Paneth cells induced TGF- precursor expression, followed by an increase of TGF-₁ especially in the crypt bases. This unique expression pattern of two growth factors may be involved in rapid regeneration of Paneth cells.     

Double blind controlled phase III multicenter clinical trial with interferon gamma in rheumatoid arthritis

Summary The controlled clinical trial reported here is part of a multicenter clinical and basic research project, sponsored by the German Federal Minister of Science and Technology, directed by a standing commission of the president of the Max-Planck-Gesellschaft, and coordinated by the Max-Planck-Institut für Biochemie, München. Overall, 249 patients with rheumatoid arthritis (RA) were enrolled by 16 participating hospitals. In addition to NSAID treatment, patients were randomly given either interferon gamma (IFN-) or placebo. In the IFN- group, 107 patients were evaluated and in the control group, 116 patients were evaluated. The response rate after 3 months of treatment, according to joint pain indexes, was significantly higher in the IFN- group with an error probability of 1%. IFN- was able to reduce the quantity of corticosteroids administered. Compared with the control group, the IFN- group benefited considering all parameters measured. Most important side effects were transient fever and transient influenza-like symptoms; all other adverse events were comparable in both groups.