The Mr 95,000 gelatin-binding protein in differentiated human macrophages and granulocytes

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.     

Lysis of fibrillar collagen by neutrophils in synovial fluid. A role for surface-bound immunoglobulins

Synovial fluids (SF) contain inhibitors capable of neutralizing the activity of proteases secreted by inflammatory cells and fibroblasts. To further define a potential role for SF polymorphonuclear neutrophils (PMN) in mediating joint destruction, peripheral blood PMN were suspended in SF and incubated with reconstituted collagen fibrils. Incubation of PMN-SF mixtures with collagen fibrils precoated with monomeric IgG resulted in significant lysis of the underlying fibrils relative to that seen with uncoated fibrils. Augmented fibril lysis by PMN-SF mixtures in which the PMN were activated with fluid-phase ligands such as phorbol myristate acetate or heat-aggregated IgG was not seen. Lysis of IgG-coated fibrils by PMN-SF was inhibited in the presence of EDTA or sodium azide. PMN-mediated resorption of fibrillar collagen occurred despite the presence of protease inhibitors in the SF at a concentration capable of neutralizing human neutrophil collagenase. These results suggest that the focal release and activation of human neutrophil collagenase during PMN stimulation by tissue-bound immunoglobulins may mediate the resorption of joint tissue collagens in rheumatoid arthritis, even in the presence of protease inhibitors.     

Transdifferentiation of polymorphonuclear neutrophils to dendritic-like cells at the site of inflammation in rheumatoid arthritis: evidence for activation by T cells

OBJECTIVES: To investigate infiltrated cells in the synovial fluid (SF) of inflamed joints of patients with rheumatoid arthritis (RA), with special reference to polymorphonuclear neutrophils (PMN) and their interaction with T cells. METHODS: Expression on PMN of activation associated receptors CD14, CD64, CD83, and major histocompatibility complex (MHC) class II was examined in the SF of 15 patients with RA, as were the infiltrated T cells. SF cytokines were determined by enzyme linked immunosorbent assay (ELISA). To mimic the in vivo situation, co-culture experiments were carried out using PMN and T cells of healthy donors. RESULTS: The SF contained activated T lymphocytes and abundant PMN. SF PMN expression of CD14 and CD64 was enhanced compared with peripheral blood. Of special interest was the observation that only the SF PMN expressed MHC class II antigens and CD83. Exposure to SF, which contained considerable amounts of cytokines (for example, interferon gamma (IFNgamma), tumour necrosis factor alpha, and interleukin 2), induced a similar receptor pattern on blood derived PMN of healthy donors. Furthermore, PMN acquired MHC class II and CD83 within 24 to 48 hours, when co-cultured with autologous T cells or T cell lines. This effect was also achieved by T cell supernatants, was dependent on protein synthesis, and could be inhibited by antibodies against IFNgamma. CONCLUSIONS: SF PMN from patients with RA undergo major alterations, including transdifferentiation to cells with dendritic-like characteristics, probably induced by T cell derived cytokines. Because MHC class II positive PMN are known to activate T cells, the mutual activation of PMN and T cells might contribute to the perpetuation of the local inflammatory process, and eventually to the destructive process in RA.     

Fibronectin fragments and their role in inflammatory arthritis

OBJECTIVES: To identify potential immunopathogenic links between fibronectin (Fn) fragmentation and the inflammatory response in chronic joint disease. METHODS: Scientific papers involving studies of Fn fragments and inflammatory processes important in the pathogenesis of arthritis, including chondrolysis, synoviocyte growth and adhesion, polymorphonuclear leukocyte (PMN) and monocyte function, proteolysis, and immune complex activation were reviewed. In addition, reports identifying Fn fragments in synovial fluid (SF) were assessed. RESULTS: A series of Fn fragments have been identified in arthritic SF by several investigators. Fn and fragments ranging from 30 to 200 kd are present in elevated concentrations in inflammatory SF. SF Fn fragments display reduced affinity for fibrin and collagen. The 29- and 50-kd amino terminal fragments mediate release of proteoglycan from articular cartilage by RGD-independent mechanisms. Fn fragments can induce fibroblast gene expression of metalloproteinases or can act as proteinases themselves. A 90-kd plasmin generated fragment possesses homology with streptokinase. Fragments mediate PMN chemotaxis and enhance proliferation of CD4+ lymphocytes as well as binding to the C1q component of complement and influencing the behavior of immune complexes. CONCLUSIONS: Fn fragments can be functionally and biochemically characterized in diseased SF. Modification of fragment formation and inhibition of fragment function may have potential therapeutic value in the interruption of chronic synovial inflammation.     

Levels of synovial fluid interleukin-1 receptor antagonist in rheumatoid arthritis and other arthropathies. potential contribution from synovial fluid neutrophils

Objective. To measure synovial fluid (SF) levels of interleukin-1 receptor antagonist (IL-1ra) and to determine the capacity of SF neutrophils (PMN) to synthesize and release IL-1ra. Methods. A sensitive and specific enzyme-linked immunosorbent assay was used to measure SF IL-1ra protein concentrations and IL-1ra production by isolated SF PMN. Results. SF IL-1ra levels were elevated in 13 of 16 samples from patients with rheumatoid arthritis (RA) (mean 17.1 ng/ml), in 6 of 18 samples from patients with infectious or inflammatory, non-RA arthropathies (mean 10.6 ng/ml), and in none of 11 noninflammatory SF samples. SF IL-1ra levels correlated with SF PMN concentrations (r = 0.680, P < 0.00001). Isolated SF PMN contained preexisting IL-1ra protein in the absence of messenger RNA (mRNA). In addition, both lipopolysaccharide and granulocyte-macrophage colonystimulating factor induced modest increases in IL-1ra mRNA by cultured SF PMN. Conclusion. IL-1ra levels are increased in >80% of RA SF samples. SF PMN produce IL-1ra, possibly contributing to the levels of IL-1ra present within the SF.     

SYNOVIAL FLUID POLYMORPHONUCLEAR LEUCOCYTES FROM PATIENTS WITH RHEUMATOID ARTHRITIS HAVE REDUCED MPO AND NADPH-OXIDASE ACTIVITY

Synovial fluid (SF) polymorphonuclear leucocytes (PMN) frompatients with rheumatoid arthritis (RA) were compared to RAand normal circulating blood PMN. RA SF PMN were as viable asblood PMN and remained viable for up to 24 h in culture. Measurementof myeloperoxidase indicated that RA SF PMN had degranulatedand secreted their myeloperoxidase prior to isolation, 26.5±11.7%being found extracellularly compared to less than 2.9% in RAand normal blood PMN. RA SF PMN alone showed a decrease in basalNADPH-oxidase activity as well as an increase in responsivenessto stimulation by chemotactic peptide during culture. Stimulationof PMN with phorbol-12-myrisitate-13-acetate evoked equivalentresponses in each population before and after culture. Theseresults demonstrate a major difference in resting and receptor-mediatedactivation of superoxide release by RA SF PMN. Together, theseresults have important implications in identifying the roleof the activated PMN in RA. KEY WORDS: Arthritis, White cells, Synovial fluid, Myeloperoxidase, Superoxide anion, nFMLP, Pholasin, chemiluminescence     

Phospholipase A2 activity associated with synovial fluid cells

High activity of phospholipase A2 (PLA2) has been detected in synovial fluids (SF) in inflammatory arthritides. Since the source(s) of SF PLA2 has not been identified, we tested PLA2 content in SF cells obtained from 11 SF. Cell sonicates were prepared at 5 X 10(6) cells/ml. In the supernatants of the sonicated SF cells (n = 11), PLA2 activity ranged from 38-755 U/ml, mean 368 +/- 243 (SD) U/ml, compared to 5-64 U/ml, mean 31 +/- 15 (SD) U/ml in sonicates of normal peripheral blood PMN (n = 5) (p less than 0.0001). Spontaneous release of PLA2 from unstimulated SF cells ranged from 26-365 U/ml, mean 131 +/- 144 (SD) U/ml (n = 5), whereas spontaneous release from peripheral blood PMN was negligible. Neither 10(-8) M FMLP nor 5 X 10(-6) M dexamethasone altered extracellular PLA2 release. To assess whether PLA2 adsorbs passively to cell membranes through hydrophobic interaction, normal peripheral PMN were incubated in crude SF (n = 7) with PLA2 ranging from 4,000-24,300 U/ml, or with purified human SF PLA2 or Naja naja PLA2. We found that soluble PLA2 adsorbed to PMN membranes in a concentration dependent fashion. PLA2 activity in sonicates of PMN incubated in crude SF ranged from 185-358 U/ml compared to controls of 21-64 U/ml. Sonicates of PMN incubated with purified human SF PLA2 (5,000-30,000 U/ml) showed progressive concentration dependent increase in PLA2 from 7 +/- 4 to 212 +/- 11 (SD) U/ml (p less than 0.001). PMN incubated with Naja naja PLA2 also showed marked increase in the content of PLA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phagocytosis and intracellular killing of Staphylococcus aureus by polymorphonuclear cells from synovial fluid of patients with rheumatoid arthritis

The phagocytosis and intracellular killing by synovial fluid (SF) polymorphonuclear cells (PMN) of 10 patients with rheumatoid arthritis was studied. PMN phagocytosis was assessed by morphologic and microbiologic methods and intracellular killing was measured independently of continuous phagocytosis of Staphylococcus aureus. Phagocytosis of S aureus by SF PMN or peripheral blood (PB) PMN was as effective in the presence of synovial fluid as in the presence of serum. On average, SF PMN ingested S aureus opsonized with synovial fluid and serum more efficiently than patient or donor PB PMN did. Enhanced ingestion of S aureus was associated with increased expression of C3 receptors on the membrane of SF PMN. In the presence of heat-inactivated synovial fluid, the capacity of SF PMN to ingest S aureus was greater than that of patient or donor PB PMN. Under these conditions, phagocytic activity was correlated with Fc receptor expression. SF PMN was found to be as active in killing S aureus as PB PMN from healthy donors.     

The transsynovial lymphocytic ratio. Characterization of blood and synovial fluid lymphocytes from patients with arthritic diseases

Monoclonal antibodies and flow cytometry techniques were used to analyze and compare the distribution of lymphocyte subpopulations of peripheral blood and synovial fluid (SF) from 70 patients, 43 with rheumatoid arthritis (RA), 10 with ankylosing spondylitis (AS) or reactive synovitis, 10 with psoriatic arthritis and 7 with other inflammatory arthritic diseases. Patients with RA had significantly reduced number of CD8+ T cells and greater CD4/CD8 ratios in peripheral blood, a greater number of CD4+ T cells and lower CD4/CD8 ratio in SF. No significant difference was found between the groups with AS, reactive synovitis and psoriatic arthritis. The simultaneous analysis of peripheral blood and SF lymphocyte subpopulations allowed us to establish a transsynovial lymphocytic ratio which reflects CD4/CD8 variations on both peripheral blood and SF, 2 easily accessible compartments for physicians. This new ratio may distinguish RA from other inflammatory arthritic diseases.     

Chemotaxis and chemiluminescence responses of synovial fluid polymorphonuclear leucocytes during acute reactive arthritis.

The chemotaxis and chemiluminescence responses of polymorphonuclear leucocytes (PMN) of synovial fluid and peripheral blood from patients with acute reactive arthritis were studied. Rates of chemotactic and chemokinetic migration of synovial fluid PMN were significantly decreased. In addition, chemiluminescence responses tended to be depressed, suggesting that the cells were deactivated for both chemotaxis and production of oxygen derived free radicals. Such deactivation has been described previously as a characteristic of synovial fluid PMN in rheumatoid arthritis. Compared with those with a mild disease, patients with severe acute reactive arthritis had higher chemiluminescence responses of synovial fluid PMN to phorbol myristate acetate during acute disease and developed increased migration of peripheral blood PMN towards zymosan treated serum after recovery from the disease. This supports the view that hyperreactive PMN contribute to the development of severe inflammatory symptoms in acute reactive arthritis.     

SINGLE CELL IMAGING REVEALS ABNORMAL INTRACELLULAR CALCIUM SIGNALS WITHIN RHEUMATOID SYNOVIAL NEUTROPHILS

Intracellular calcium (Ca²⁺) signalling in synovial fluid (SF)polymorphonuclear leucocytes(PMN) from patients with rheumatoidarthritis (RA) was compared to RA and normal circulating bloodPMN using single cell imaging. RA SF PMN stimulated by the peptidef-Met-Leu-Phe (FMLP) showed a striking difference in the releaseof Ca²⁺ from the intracellular store compared to RA and normalcirculating blood PMN. Stimulation caused the release of a verydispersed, nonrestricted ‘cloud’ of Ca²⁺ in 60%of RA SF PMN compared to the highly localized and restricted‘cloud’ observed in only 30% of normal circulatingPMN. In the presence of extracellular Ca²⁺, both RA SF and normalblood PMN showed heterogeneity in both the timing and magnitudeof their cytosolic free Ca²⁺ signalling. These observationsimply that the Ca²⁺ signalling mechanism in RA SF and RA bloodPMN has been primed in a way which could exacerbate the releaseof inflammatory mediators. This may have serious implicationsfor explaining the aberrant behaviour of SF PMN in RA. KEY WORDS: Rheumatoid arthritis, Polymorphonuclear leucocytes, Synovial fluid, FMLP, Cytosolic free Ca²⁺, Intracellular Ca²⁺ ‘cloud’     

Septic versus inflammatory arthritis: discriminating the ability of serum inflammatory markers

Early diagnosis of septic arthritis is very important. Few studies showed diagnostic accuracy of serum inflammatory markers in septic arthritis. The aim of our study was to compare the serum and synovial fluid markers [procalcitonin, serum IL-6, TNF-α, C-reactive protein, erythrocyte sedimentation rate, synovial fluid white blood cell counts and PMN percentage] in septic and inflammatory arthritis. Seventy-five patients, including 25 and 50 septic and non-septic arthritis, were enrolled in the study. The serum and synovial fluid markers [procalcitonin, serum IL-6, TNF-α, C-reactive protein, erythrocyte sedimentation rate, synovial fluid white blood cell counts, and PMN percentage] were compared in septic and inflammatory arthritis. Patients with septic arthritis had significantly elevated levels of procalcitonin, serum TNF-α, C-reactive protein, erythrocyte sedimentation rate, synovial fluid white blood cell counts, and PMN percentage in comparison with the inflammatory arthritis group (P < 0.00). Serum IL-6 level does not differ among the two groups. In a receiver operating characteristic curve analysis, synovial fluid WBC counts, PMN percentage, TNF-α, ESR, and serum PCT preformed best in distinguishing between septic and non-septic arthritis. Our study suggests that PCT can be used to diagnose the septic arthritis, but more studies warranted in order to determine the specificity and sensitivity of the test.     

Characterization of heparan sulfate-proteoglycan of glomerular basement membranes.

Native and de novo synthesized heparan sulfate-proteoglycan (HS-PG) of basement membranes from isolated whole glomeruli were characterized. Sepharose CL-6B chromatograms of [35S]sulfate-labeled de novo synthesized HS-PG extracted from whole glomeruli indicated identical molecular weight characteristics to that isolated from purified basement membranes (Mr of intact HS-PG approximately equal to 130,000; Mr of chains approximately equal to 25,000). Electron microscopic autoradiography showed that almost all radioactive grains were localized to the basement membranes proper. The estimated Mr of core protein approximately equal to 18,000. The sedimentation coefficient of native intact HS-PG was 5.56 S, corresponding to a Mr between 150,000-250,000, a value in accord with gel filtration data on newly synthesized HS-PG. Physicochemical characteristics of HS-PG of native functional basement membranes differed remarkably from that isolated from the basement membrane-producing tumor, Engelbreth-Holm-Swarm sarcoma.     

EXPRESSION OF COMPLEMENT REGULATORY MOLECULES AND OTHER SURFACE MARKERS ON NEUTROPHILS FROM SYNOVIAL FLUID AND BLOOD OF PATIENTS WITH RHEUMATOID ARTHRITIS

In an effort to elucidate the activation status of neutrophils(PMN) in inflammatory joint disease the expression of relevantcell surface proteins was examined using immunofluorescenceand flow cytometry. Paired samples of SF and peripheral bloodwere obtained from 18 patients with RA and PMN purified usingmethods designed to minimize activation in vitro. We then usedflow cytometry to measure expression of the four membrane complementregulatory molecules, decay accelerating factor (DAF; CD55),complement receptor 1 (CR1; CD35), membrane cofactor protein(MCP; CD46) and CD59; two adhesion molecules of the integrinfamily LFA1 (     

Activated leucocytes express and secrete macrophage inflammatory protein-1alpha upon interaction with synovial fibroblasts of rheumatoid arthritis via a beta2-integrin/ICAM-1 mechanism

OBJECTIVE: To examine the expression and regulation of chemotactic factor, macrophage inflammatory protein-1alpha (MIP-1alpha) by fibroblast-like synoviocytes (FLS), monocytes and polymorphonuclear neutrophils (PMN) isolated from the synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS: Monocytes or PMN obtained from RA SF were co-cultured with unstimulated semiconfluent RA FLS. Culture supernatants were assayed for MIP-1alpha by enzyme-linked immunosorbent assay. The expression of MIP-1alpha mRNA and protein was also determined by Northern blot analyss and immunohistochemistry respectively. RESULTS: Interaction of activated leucocytes with FLS synergistically increased MIP-1alpha expression and secretion via a mechanism mediated by beta2-integrin/ intercellular adhesion molecule 1. CONCLUSION: MIP-1alpha expression within inflamed joints appears to be regulated not only by inflammatory cytokines but also by the physical interaction of activated leucocytes and FLS, and plays a crucial role in the progression and maintenance of RA synovitis.     

Increased basal phosphorylation of mitogen-activated protein kinases and reduced responsiveness to inflammatory cytokines in neutrophils from patients with rheumatoid arthritis

OBJECTIVE: We studied the functions of peripheral blood (PB) and synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA), focusing the molecular basis for the activated state and the functional responsiveness of RA neutrophils to inflammatory cytokines. METHODS: Paired samples of PB neutrophils and SF neutrophils from the inflamed knee joint were obtained from 18 RA patients (5 males and 13 females). RESULTS: RA neutrophils exhibited increased spontaneous superoxide (O2-) release and adherence, increased basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase, accelerated spontaneous apoptosis, and enhanced O2- release in response to N-formyl-methionyl-leucyl-phenylalanine as compared with healthy normal PB neutrophils. When challenged with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) or tumor necrosis factor alpha (TNF-alpha), RA neutrophils exhibited reduced responses to these cytokines, which included O2- release, adherence, priming for enhanced O2- release, and phosphorylation of ERK and p38. The functional alterations were greater in SF neutrophils than in PB neutrophils from RA. Reduced responsiveness to cytokines in RA neutrophils was closely associated with increased serum and SF levels of GM-CSF and TNF-alpha. RF and RAHA titers were closely correlated with increased TNF-alpha level in SF. CONCLUSION: These findings indicate that RA neutrophils are in the activated state with increased basal phosphorylation of ERK and p38, and exhibit reduced responsiveness to inflammatory cytokines (G-CSF, GM-CSF and TNF-alpha) and accelerated spontaneous apoptosis.     

Demonstration of fibronectin associated with platelets in synovial fluid from patients with rheumatoid arthritis

Paired samples of peripheral blood and synovial fluid (SF) aspirated from inflamed knee joints from 15 adult patients with classical rheumatoid arthritis, as well as peripheral blood obtained from 15 healthy subjects, were anticoagulated with ACD. Peripheral blood platelets were separated from other plasma constituents by gel filtration of platelet-rich plasma on Sepharose 2B. When using this technique on SF, it was found that platelets could be isolated from other SF constituents, and that each of the SF's gave a high yield of eluted platelets. Direct immunofluorescent staining for human fibronectin was performed with isolated and suspended platelets obtained from the three different sources. Aliquots of both intact and permeabilized platelets were stained. Intact peripheral platelets from all patients and normal subjects revealed a weakly positive staining, whereas the staining of intact SF platelets from all patients was clear and bright. The fluorescence of intact cells was surface-located and speckled. For permeabilized platelets, the staining had a punctate intracellular pattern, with a varying number of discrete and bright fluorescent foci per cell. Counting of the foci in each platelet specimen revealed that this number, which was also found in peripheral platelets from all patients and normal subjects, was distinctly greater than the number of foci in all the SF platelet specimens. No relationship was found between the various staining results and medical treatment or Waaler-Rose serology of the patients. The findings indicate that the SF platelets had large amounts of surface-bound and small amounts of intracellular fibronectin, whereas the converse was found in the case of peripheral platelets from the patients and normal subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of neutrophil activating peptide-1 (NAP-1/IL-8) by blood and synovial fluid mononuclear cells from patients with arthritis.

As polymorphonuclear leukocytes (PMN) are predominant in inflammatory synovial fluids, we investigated the production of neutrophil-activating peptide-1 (NAP-1) by mononuclear cells (MC) from 15 synovial fluids and matched peripheral blood. MC were cultured for 24 h alone or with stimulants (ConA, LPS). NAP-1 was determined in the supernatants by a bioassay (elastase release from normal human PMN) and an immunoassay (sandwich ELISA with a mouse anti-NAP-1 mAb and an alkaline phosphatase labelled goat anti-NAP-1 pAb). The results showed a significant increase in NAP-1 production by synovial fluid MC when compared to peripheral blood MC. Both cell types produced more NAP-1 in the presence of added stimuli. The results obtained with the two methods of detection were in close agreement. No relationship was found between the amount of NAP-1 produced in 24 h and the number of synovial fluid leukocytes, the erythrocyte sedimentation rate, the diagnosis of the underlying arthritis or the treatment of the patients.