Immunolocalization of membrane-type 1 MMP in human rheumatoid synovium tissues

Membrane-type 1 matrix metalloproteinase (MT1-MMP, also known as MMP14), the best characterized membrane-anchored MMP, is an important matrix-degrading proteinase that could digest a broad spectrum of extracellular matrix proteins and accelerate angiogenesis. We have previously reported that some MMPs involved in the angiogenesis and the pannus formation within the joint, leading to the erosion of articular cartilage and bone in the pathological process of rheumatoid arthritis (RA). In the present study, we used immunohistochemistry assay and con-focal scanning technique to study the detailed immunolocalization of MT1-MMP in human RA synovium tissues as well as the infiltrating immune cell subsets. Our results showed that the positive MT1-MMP immunostaining could be found in synoviocytes, vascular endothelial cells, infiltrating macrophages and monocytes in RA synovium tissues, while weak or negative immunostaining could be found in infiltrating T cells, B cells and NK cells, respectively. Moreover, the Ki-67⁺ highly proliferating synoviocytes also showed higher MT1-MMP expression in RA synoviocytes. Thus, the aberrant expression of MT1-MMP in RA synoviocytes as well as infiltrating immune cells may contribute to the proliferation of the synoviocytes, and the angiogenesis and the pannus formation in RA pathological progression.     

Tissue factor expression in rheumatoid synovium: a potential role in pannus invasion of rheumatoid arthritis

Angiogenesis, as well as pannus formation within the joint, plays an important role in the erosion of articular cartilage and bone in the pathological process of rheumatoid arthritis (RA). Tissue factor (TF), an essential initiator of the extrinsic pathway of blood coagulation, is also involved in the angiogenesis and the pannus formation of RA progression. In the present study, we used immunofluorescence and confocal scanning methods to characterize TF immunolocalization in RA synovium. We showed that positive staining of TF could be immunolocalized in synoviocytes, CD19⁺ B cells and CD68⁺ macrophages, whereas weak or negative staining of tissue factor could be found in CD34⁺ endothelial cells of neo-vessels, CD3⁺ T cells and CD14⁺ monocytes in RA synovium tissues. Our study demonstrates a detailed local expression of TF in the rheumatoid synovium, and supports the notion that TF, expressed not only by the synoviocytes themselves, but also the infiltrating CD19⁺ B cells and CD68⁺ macrophages, is involved in the pannus invasion in the progression of rheumatoid arthritis.     

A preliminary study on the characterization of follicular helper T (Tfh) cells in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is a chronic and systematic autoimmune inflammatory disease. Recently, a novel T cell subset, follicular helper CD4 T cell (Tfh cells) was found in relation to the pathogenesis and progression of RA, and increased numbers of circulating Tfh cells were found in RA patients. However, there is little evidence regarding the localization of Tfh cells in synovium tissues from RA patients, owing to the lack of an available method to characterize their localization in tissue. The aim of our present study was to characterize the Tfh cells in rheumatoid synovium tissues from RA patients by using immunohistochemistry and triple-fluorescence immunostaining methods. Our results showed that specific staining of CD4, CXCR5 and ICOS could be found on infiltrating immune cells in rheumatoid synovium tissues. The use of triple-fluorescence immunostaining and confocal laser scanning showed immunolocalization of CD4⁺CXCR5⁺ICOS⁺T cells (Tfh cells) in the rheumatoid synovium tissues, whereas these signals were absent in osteoarthritis (OA) synovium and in normal synovium tissues. Thus the data from our present preliminary study support the notion that CD4⁺CXCR5⁺ICOS⁺Tfh cells could be found in rheumatoid synovium tissues from RA patients, indicating the possibility that this T cell subset in synovium tissues may have important roles in the pathogenesis and progression of RA.     

The expression and anatomical distribution of BTLA and its ligand HVEM in rheumatoid synovium

Co-inhibitory signaling from B and T lymphocyte attenuator (BTLA) can suppress lymphocyte activation and maintain peripheral tolerance. However, the expression and anatomical distribution of BTLA and its ligand, herpesvirus entry mediator (HVEM), in rheumatoid arthritis (RA) synovium have not been reported. In this study, we analyzed the expression of HVEM and BTLA in RA synovium by immunohistochemistry, and our results showed that both factors were observed in all four cases of RA samples. At the cellular level, both HVEM and BTLA were found on the cell membrane and in the cytoplasm. Fluorescence dual staining demonstrated that HVEM was chiefly on CD3⁺ T cells, CD68⁺ macrophages, and to a lesser extent was found on CD31⁺ endothelial cells. Similarly, the expression of BTLA was observed on infiltrated CD3⁺ T cells and CD68⁺ macrophages. The co-expression of HVEM and BTLA with some members of the B7 family in these sections was also analyzed, and the results showed that HVEM antigen was also found on B7-H3⁺ capillaries, while it was absent on B7-H1⁺, B7-DC⁺, B7-H4⁺, and Z39Ig⁺ cells. Interestingly, BTLA was observed on B7-H1⁺, B7-H4⁺, and HVEM⁺ cells in the synovium. The characteristic expression and distribution of BTLA/HVEM in the synovium indicated that their signaling probably affects the pathogenesis of RA, and a clear understanding of their functional roles may further elucidate the pathogenesis of this disease.     

Induction of Fas-dependent apoptosis in synovial infiltrating cells in rheumatoid arthritis

Apoptosis is a feature of the synovium of rheumatoid arthritis(RA). We have recently shown that RA synoviocytes were susceptibleto anti-Fas mAb and undergo apoptosis in vitro. To investigatewhether infiltrating mononuclear cells also undergo Fas-dependentapoptosis, double-labeling techniques combined with immunohistochemicalexamination with anti-CD3 mAb and the TdT-mediated dUTP-biotinnick end labeling (TUNEL) method to detect apoptotic cells,or in situ RT assay to detect Fas mRNA, were performed usingfrozen tissue sections. We also examined the in vitro inductionof Fas-dependent apoptosis in freshly isolated synovium infiltratingmononuclear cells (SIM), synovial stromal cells (SSC) and peripheralblood lymphocytes (PBL) using tissues from nine patients withRA and three with osteoarthritis (OA). The results showed expressionof Fas antigen and apoptotic cells in a number of CD3-bearingcells in RA synovial tissues. In vitro treatment with anti-FasmAb produced a significant apoptosis of RA SIM and SSC, whilenone of PBL, and neither SIM nor SSC from OA exhibited apoptosis.Moreover, 50% of CD4⁺, CD3⁺ and CD45RO⁺ cells, and >90% ofFas-expressing cells of RA SIM underwent apoptosis in responseto anti-Fas mAb, as detected by flow cytometry. Our resultssuggest that RA synovial infiltrating lymphocytes acquire highsusceptibility to anti-Fas mAb and undergo apoptosis. Such aphenomenon of infiltrating T cells in RA synovium may play animportant pathophysiological role and suggest a possible therapeuticeffect for anti-Fas mAb in RA.     

Cyclophilin A may contribute to the inflammatory processes in rheumatoid arthritis through induction of matrix degrading enzymes and inflammatory cytokines from macrophages

Cyclophilin A (CypA) levels increase in the sera and synovial fluids of rheumatoid arthritis (RA) patients, but the cell types expressing CypA and the function of CypA in the pathogenesis of RA are not known yet. Immunohistochemistry analyses revealed high level CypA staining in the macrophages in the lining layers of human RA and osteoarthritis synovium. Low level CypA staining was also detected in endothelial cells, lymphocytes, and smooth muscle cells in RA synovium. Further investigation of the CypA function using monocyte/macrophage cell lines revealed that CypA induced expression of cytokine/chemokines such as TNF-alpha, IL-8, MCP-1, and IL-1beta and matrix metalloproteinase (MMP)-9 through a pathway that is dependent on NFkappaB activation. Furthermore, MMP-9 staining pattern overlapped with that of CypA in both RA and OA synovium. Our data suggest that CypA may stimulate macrophages to degrade joint cartilage via MMP-9 expression and promote inflammation via pro-inflammatory cytokine secretion.     

Altered apoptosis of inflammatory neutrophils in MMP-9-deficient mice is due to lower expression and activity of caspase-3

Matrix metalloproteinase 9 (MMP-9) is a Zn²⁺-dependent endopeptidase that degrades some of the components of basement membranes and extracellular matrix and thus participates in leukocyte infiltration during inflammation. In a model of zymosan peritonitis, neutrophil infiltration in MMP-deficient (MMP-9^−/−) mice was significantly weaker at the time of their maximal influx in wild-type mice (6 h). However, during the late stages of peritonitis (24 h) an extended accumulation of neutrophils was observed in MMP-9^−/− versus the wild-type mice. Recently, we reported that the ratio of apoptosis of inflammatory leukocytes is impaired in MMP-9^−/− mice during late peritonitis and the process depends on COX-1-driven PGE₂. Here we scrutinized the alterations in apoptotic mechanisms by comparisons between MMP-9^−/− and the wild-type mice. Altered apoptosis occurred only during late (24 h) peritonitis and concerned only neutrophils, and not macrophages, mast cells or lymphocytes. Furthermore, expression and activity of caspases was altered in MMP-9^−/− animals, delayed for caspase-8 and -9, and decreased in the case of caspase-3. Also the expression of Bax/Bcl-2 proteins was changed in MMP-9^−/− mice. These changes, and in particular the impaired neutrophil apoptosis and weaker caspase-3 activity, were restored by the selective COX-1 inhibition. We conclude that in mice lacking MMP-9 the enhanced COX-1-PGE₂ decreases caspase-3 expression and activity leading to impaired apoptosis of inflammatory neutrophils resulting in abnormal accumulation of the cells at the inflammatory focus. The data also reinforce the notion that MMP-9 is a key enzyme in neutrophil biology.     

宫颈癌COX-2、MMP-9和CD105表达与肿瘤血管生成及侵袭转移的关系

目的探讨环氧合酶-2(cyclooxygenase,COX-2)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)在宫颈癌局部肿瘤血管生成及肿瘤侵袭转移过程中的作用。方法采用免疫组化SP法检测32例正常宫颈上皮及68例宫颈癌中COX-2、MMP-9的表达。并用CD105标记新生血管内皮细胞,计算肿瘤内微血管密度(microvascular density,MVD)。结果COX-2、MMP-9在宫颈癌组织中的阳性表达率均显著高于正常宫颈上皮中的表达(P<0.01)。COX-2、MMP-9阳性组的MVD均显著高于COX-2、MMP-9阴性组(P<0.01),淋巴结转移组COX-2、MMP-9阳性率均高于无淋巴结转移组(P<0.05)。结论COX-2、MMP-9阳性表达可能在宫颈癌血管生成和侵袭转移中起重要作用,两者在宫颈癌血管生成中可能具有协同作用。检测宫颈癌中COX-2、MMP-9表达对进一步了解宫颈癌局部血管生成情况及判断预后有一定的应用价值。     

CD169 identifies an anti‐tumour macrophage subpopulation in human hepatocellular carcinoma

Macrophages are a major component of most solid tumours and can exert both anti‐ and pro‐tumourigenic functions. Although the immunosuppressive/pro‐tumour roles of macrophages have been widely examined, significantly less is known about macrophage subpopulations that have potential anti‐tumour properties in humans. In the present study, a population of CD169⁺ macrophages with relatively high expression levels of HLA‐DR and CD86 was identified in human hepatocellular carcinoma tissues. The frequency of CD169‐expressing macrophages within cancer nests was significantly lower than that found in paired non‐tumour areas. In vitro experiments revealed that in the presence of anti‐CD3 stimulation, CD169⁺ macrophages could significantly enhance the proliferation, cytotoxicity, and cytokine production capacity of CD8⁺ T cells in a CD169 molecule‐dependent manner. Autocrine TGF‐β produced by tumour‐stimulated macrophages was involved in the down‐regulation of CD169 expression on these cells. Moreover, the accumulation of CD169⁺ macrophages in tumour tissues was negatively associated with disease progression and predicted favourable survival in hepatocellular carcinoma patients, which was in contrast to the trend observed for total CD68⁺ macrophages. Therefore, CD169 might act as a co‐stimulatory molecule for cytotoxic T‐cell activation, and could define a population of tumour‐infiltrating macrophages with potential anti‐tumour properties in human hepatocellular carcinoma tissues. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Histopathological findings of pericarditis in a patient with multisystem inflammatory syndrome in children associated with COVID-19: A case report

Multisystem inflammatory syndrome in children (MIS-C), which is associated with the novel coronavirus disease 2019 (COVID-19), has been described as an inflammatory complication of exposure to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It carries a risk of serious and lethal complications, including cardiogenic shock. Here, we report the pathological findings of the pericardium in a 10-year-old child with MIS-C, who developed pericarditis-induced cardiac tamponade. In the patient's pericardium, the numbers of infiltrating CD68⁺ macrophages; CD3⁺, CD4⁺, and CD8⁺ T cells; and myeloperoxidase⁺ granulocytes were increased, although the number of CD20⁺ B cells was not. These findings provide a clue to understanding the pathophysiology of MIS-C.     

中性粒细胞增强类风湿关节炎滑膜细胞产生基质金属蛋白酶及其细胞侵袭力

目的：研究中性粒细胞表面CD147分子对类风湿关节炎（RA）成纤维样滑膜细胞（FLS）产生基质金属蛋白酶（MMP）及细胞侵袭力的作用。方法：取自关节镜或骨科手术治疗的RA、骨关节炎（OA）患者的滑膜组织，体外分离培养FLS，通过流式细胞术（FCM）检测细胞表面分子及形态学方法鉴定。采用全反式维甲酸（ATRA）诱导HL-60细胞（人早幼粒白血病细胞株）诱导中性粒细胞分化，并用硝基蓝四氮唑（NBT）还原反应测定其分化程度。进而以FCM测定分化前后的HL-60细胞及FLS表面CD147的表达，并通过明胶酶谱和重组基质侵袭实验检测分化前后HL-60细胞对FLS MMP表达和细胞侵袭能力的作用以及CD147拮抗肽（AP-9）对这种MMP表达和细胞侵袭能力的抑制作用。结果：成功建立了FLS分离培养和HL-60细胞分化的实验体系。培养的FLS具有典型成纤维细胞的形态，均一的高度表达CD90（〉98％）而不表达CD14。分化后的HL-60细胞CD147表达上调，RAFLS细胞CD147表达水平低于分化前及分化后HL-60细胞（P〈0．05）。明胶酶谱试验显示：①与OAFLS相比，单培养的RAFLS表达较高水平的酶原及活化形式的MMP-2（P〈0．05）；②分化前／后的HL-60细胞分别与FLS共培养，酶原及活化形式的MMP-2和MMP-9的表达水平较这些细胞单独培养均显著升高（P〈0．05），且分化后的HL-60细胞与FLS共培养酶原及活化形式MMP-2表达水平较分化前HL-60细胞与FLS共培养明显升高（P〈0．05）；③AP-9显著抑制两共培养组中MMP-2和MMP-9的上调作用（P〈0．05）。侵袭实验显示：RAFLS侵袭能力高于OA FLS（P〈0．05）；分化前后的HL-60均可增加RAFLS的侵袭能力（P〈0．05），且分化后强于分化前（P〈0．05）；AP-9可抑制这一促进作用，降低FLS的侵袭能力（P〈0．05）。结论：中性粒细胞可能通过CD147促进RA患者滑膜FLS MMP-2、MMP-9的表达和活化，增强FLS的侵袭能力，这种促进作用可被CD147拈抗肽所抑制，将为RA关节软骨和骨损伤机制和治疗的进一步研究提供了重要的线索。     

Expression and location of IL-17A,E, F and their receptors in colorectal adenocarcinoma: Comparison with benign intestinal disease

The research aimed to investigate secretion, expression and location of IL-17 relative ligands, IL-17 relative receptors, infiltrating inflammatory cells and parenchymal structural cells in colorectal cancer (CRC) compared with ulcerative colitis (UC) and benign hyperplastic polyp. 29 human intestinal tissues with CRC, 17 with UC and 7 with polyp were stained using immunohistochemistry to evaluate immunoreactivity for IL-17 family relative ligands including IL-17A, E, F and their respective relative receptors such as IL-17RA, IL-17RB and IL-17RC. At the same time the infiltration of inflammatory cells including lymphocytes, phagocytes, mast cells and neutrophils and parenchymal structural cell changes involving vascular endothelial cells and CD90⁺ fibroblast cells were also evaluated using the same methods The immunoreactivity or positive inflammatory cells of all the sections were analyzed using professional image analysis software to determine statistical significance. The immunoreactivity for IL-17A, IL-17RA, IL-17E, IL-17RB and IL-17F showed significant decrease in CRC tissue when compared to UC (p?=?0.00001. respectively). The reduction of above IL-17 relative ligands and receptors was accompanied by an obvious decrease in the number of infiltrating neutrophils and mast cells in CRC (p?=?0.00001 and p?=?0.007, respectively) but accompanied by a marked increase of CD31⁺ blood vessels (p?=?0.001). The immunoreactivity of IL-17A, IL-17RA, IL-17E, IL-17RB and IL-17F and the numbers of infiltrating neutrophils and mast cells showed significant decrease in CRC tissues when compared to those in polyp (p?<?0.05). In contrast, the immunoreactivity of IL-17RC and the numbers of CD3⁺ 1ymphocytes were elevated in CRC when compared with those in polyp (p?=?0.0001, p?=?0.007, respectively). In CRC tissues, positive correlations between IL-17A, IL-17RA with CD68⁺ macrophages were observed respectively (r?=?0.621, p?=?0.0001; r?=?0.75, p?=?0.0001). IL-17 cytokine family including ligands and their corresponding receptors were secreted and expressed by infiltrating inflammatory cells. Not only infiltrating lymphocytes but also increased blood endothelial cells were relative significantly to genesis and progression of CRC.     

Rheumatoid arthritis fibroblast‐like synoviocytes co‐cultured with PBMC increased peripheral CD4+CXCR5+ICOS+ T cell numbers

‘Circulating’ T follicular helper cells (Tfh), characterized by their surface phenotypes CD4⁺chemokine receptor 5 (CXCR5)⁺ inducible co‐stimulatory molecule (ICOS)⁺, have been identified as the CD4⁺ T cell subset specialized in supporting the activation, expansion and differentiation of B cells. Fibroblast‐like synoviocytes (FLS) are critical in promoting inflammation and cartilage destruction in rheumatoid arthritis (RA), and the interaction between FLS and T cells is considered to facilitate FLS activation and T cell recruitment. However, it remains unknown whether RA‐FLS co‐cultured with activated peripheral blood mononuclear cells (PBMC) has immunoregulatory effects on peripheral Tfh. In the present study, we co‐cultured RA‐FLS with or without anti‐CD3/CD28‐stimulated PBMC. The results showed that RA‐FLS co‐cultured with stimulated PBMC could increase the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells of RA PBMC possibly via the production of interleukin (IL)‐6, a critical cytokine involved in the differentiation of Tfh cells. We also observed increased reactive oxygen species (ROS) levels in the co‐culture system of RA‐FLS and PBMC. The percentage of CD4⁺CXCR5⁺ICOS⁺ T cells was decreased when ROS production was inhibited by N‐acetyl‐L‐cysteine (NAC), a specific inhibitor which can decrease ROS production. In addition, we showed that the higher levels of tumour necrosis factor (TNF)‐α and IL‐1β in the co‐culture system and the blocking of TNF receptor 2 (TNF‐R2) and IL‐1β receptor (IL‐1βR) both decreased the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells. Our study reveals a novel mechanistic insight into how the interaction of RA‐FLS and PBMC participates in the RA pathogenesis, and also provides support for the biologicals application for RA.     

MMP-2 activation and stepwise progression of pulmonary adenocarcinoma: analysis of MMP-2 and MMP-9 with gelatin zymography

Small pulmonary adenocarcinomas can be classified on the basis of their histological characteristics and prognosis, and when classified as such, the prognosis of replacing-type adenocarcinoma with active fibroblast proliferation is significantly worse than adenocarcinoma without fibroblast proliferation. In order to clarify the biological mechanisms of the key to the morphological changes associated with active fibroblast proliferation, we examined the activities of matrix metalloproteinase (MMP)-2 and MMP-9, which are important enzymes in the stromal invasion by cancers. The active MMP-2 and MMP-9 content of 40 pulmonary adenocarcinomas that were less than 20 mm in diameter was measured by the gelatin zymography method. The quantity of active MMP-2 in the pulmonary adenocarcinomas with active fibroblast proliferation was higher than in the pulmonary adenocarcinomas without proliferation (P < 0.001), but there were no correlations between the histological features and the activation of MMP-9. The presence of active fibroblast proliferation in small pulmonary adenocarcinomas suggests that the cancer cells have acquired the ability to invade through the action of active MMP-2, and this is thought to be one of the reasons for the worse prognosis of pulmonary adenocarcinoma with active fibroblast proliferation.     

CD206‐positive myeloid cells bind galectin‐9 and promote a tumor‐supportive microenvironment

In patients with metastatic melanoma, high blood levels of galectin‐9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin‐9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin‐9 with macrophages. We aimed to determine whether galectin‐9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor‐supportive. It was determined that incubation of CD68⁺ macrophages with galectin‐9 or anti‐CD206 blocked target binding and that both CD206 and galectin‐9 were detected by immunoprecipitation of cell lysates. CD206 and galectin‐9 had a binding affinity of 2.8 × 10^?7 m . Galectin‐9 causes CD206⁺ macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP‐1), but less macrophage‐derived chemokine (MDC). Galectin‐9 had no effect on classical monocyte subsets, but caused expansion of the non‐classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin‐9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin‐9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti‐tumor effects by macrophages. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Influenza A virus induction of oxidative stress and MMP-9 is associated with severe lung pathology in a mouse model

Infection by different strains of influenza virus presents different pictures. Whether the pathogenicity of influenza virus is defined by the ability of the virus to induce differential immunopathlogical responses in the lungs still remains unclear. We compared the immunopathological response induced by influenza virus A/WSN/33 (H1N1) and that by A/Panama-like (H3N2) virus in C57BL/6 mice. WSN virus, in contrast to Panama-like virus, induced high mortality and severe lung pathology accompanied by massive Gr-1⁺ and CD11b⁺ cell infiltration and high levels of CXCL6/GCP-2, CCL2/MCP-1 and TIMP-1 production. Infection by WSN virus but not by Panama-like virus induced up-regulation of the active and latent forms of MMP-9 in the lungs and MMP-2/9 inhibitor partially reduced WSN virus-induced lung pathology. Both Gr-1⁺ and CD11b⁺ cells in WSN virus-infected lungs produced reactive oxygen and nitrogen species (ROS/RNS). While wild type mice infected by WSN virus had severe lung pathology and the presence of oxidized phospholipids and numerous MMP-9⁺ cells in the lungs, ncf1 deficiency ablated their expression and manifested less lung pathology. Employing a pulmonary mouse model we demonstrated in this study that infection by virulent influenza virus is characterized by a heavy cellular infiltration, severe lung pathology which is accompanied by oxidative stress and MMP-9 production.     

Induction of fibroblast-like cells from CD34(+) progenitor cells of the bone marrow in rheumatoid arthritis

To assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA), we examined the capacity of CD34(+) cells from bone marrow to generate fibroblast-like type B synoviocytes. CD34(+) cells from the bone marrow of 22 RA patients differentiated into cells with fibroblast-like morphology, which expressed prolyl 4-hydroxylase, in the presence of stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha), much more effectively than CD34(+) cells from bone marrow of 15 control subjects (10 patients with osteoarthritis and 5 healthy individuals). The generation of fibroblast-like cells was not at all observed in cultures with SCF, GM-CSF, and interleukin 4 (IL-4) with or without TNF-alpha. Generation of fibroblast-like cells was correlated with matrix metalloproteinase (MMP)-1 levels in culture supernatants. Thus, MMP-1 levels were significantly higher in TNF-alpha-stimulated cultures of bone marrow CD34(+) cells from patients with RA than in those from the control group. These results indicate that bone marrow CD34(+) cells from patients with RA have abnormal capacities to respond to TNF-alpha and to differentiate into fibroblast-like cells producing MMP-1, suggesting that bone marrow CD34(+) progenitor cells might generate type B synoviocytes and thus could play an important role in the pathogenesis of RA.