Treatment with tumour necrosis factor alpha antagonists in patients with rheumatoid arthritis induces anticardiolipin antibodies.

OBJECTIVE: To determine the frequency and clinical impact of anticardiolipin antibodies (aCL) in patients with rheumatoid arthritis treated with infliximab and etanercept. METHODS: 121 patients from the Stockholm tumour necrosis factor alpha (TNFalpha) follow up registry (STURE) treated with infliximab or etanercept were studied. RESULTS: At baseline 9/65 (14%) infliximab and 10/56 (18%) etanercept treated patients had positive aCL. After 3 months the frequencies of aCL positivity were 29% (p<0.05 compared with baseline) and 27%, respectively, and after 6 months 28% and 25%. Increases were seen for both IgG and IgM aCL. Increasing age, a higher number of prior DMARDs, and higher DAS28 were predictors for the development of aCL. In the infliximab treated patients, 26/30 (87%) aCL(-) but only 7/14 (50%) aCL(+) patients met the ACR20 criteria (p<0.05), and the frequency of treatment limiting infusion reactions in the aCL(+) patients was higher than expected (17%). aCL positivity in the etanercept treated patients did not show such a clinical correlate. Four patients had thromboembolic events, of whom two were aCL(+) and two aCL(-). CONCLUSION: Frequencies of both IgM and IgG aCL positivity increase in patients treated with these TNFalpha antagonists for 3 months or longer. Increasing age, a greater number of prior DMARDs and a greater disease activity at baseline are predictors for the development of aCL. The development of aCL during treatment with infliximab, but not etanercept, is associated with worse clinical results and more frequent serious infusion reactions. aCL are an important class of autoantibodies associated with TNFalpha blocking therapy.

Antineutrophil cytoplasmic antibodies induce decreased CD62L expression and enhanced metabolic activity in monocytes.

Monocyte in vitro activation by antimyeloperoxidase (anti-MPO)- and antiproteinase-3 (anti-PR3)-positive sera, corresponding immunoglobulin G (IgG) fractions and monoclonal antibodies against MPO and PR3 was evaluated. The expression of adhesion molecules, l-selectin (CD62L) and CR3 (CD11b), involved in leucocyte endothelial adhesion, and metabolic activity, measured as the production of hydrogen peroxide, were analysed. Decreased expression of CD62L was demonstrated in monocytes after incubation with antineutrophil cytoplasmic antibody (ANCA)-positive sera. This finding was not accompanied by changes in CD11b expression. Metabolic activity was increased in monocytes after incubation with ANCA-positive IgG fractions as well as after incubation with monoclonal anti-MPO and anti-PR3. These findings support the concept that the pathophysiological effect of ANCA is partly mediated through the action on crucial events in monocyte activation, such as CD62L downregulation and oxygen radical production.

Longitudinal study of interleukin-10, tumor necrosis factor-alpha, anti-U1-snRNP antibody levels and disease activity in patients with mixed connective tissue disease.

OBJECTIVE: To investigate the levels and relationship between IL-10, TNF-alpha, anti-U1snRNP antibodies and disease activity in longitudinally collected serum samples from patients with mixed connective tissue disease (MCTD). METHODS: Six patients followed for 17-138 months were investigated with ELISA for estimation of cytokine levels and antibodies to the different epitopes of the U1snRNP. Disease activity was assessed by systemic lupus activity measure (SLAM). RESULTS: IL-10 and TNF-alpha levels fluctuated with time in at least half of the patients. Three patients had increased IL-10 levels and two had increased TNF-alpha in all samples. There was no correlation between cytokine levels and disease activity or clinical manifestations. All patients had increased levels of antibodies to the main components of the U1snRNP. Both antibody levels and disease activity decreased with time. A correlation between TNF-alpha and U1snRNP antibody levels were observed in five patients. CONCLUSIONS: Increased and fluctuating levels of IL-10 or TNF-alpha without correlation to disease activity were observed in MCTD patients. In some patients increased cytokine levels were observed over several years irrespective of disease activity indicating that they could be constitutively increased in these individuals.

Development of lupus-related side-effects in patients with early RA during sulphasalazine treatment-the role of IL-10 and HLA.

OBJECTIVE: The development of systemic lupus erythematosus (SLE)-related syndromes during treatment with sulphasalazine has been described and demonstrated to be influenced by genetic factors. The prevalence of this drug-induced condition and the immunological mechanisms involved are less known. The aim of this study was to determine the prevalence of sulphasalazine-induced lupus-like reactions in a well-defined early rheumatoid arthritis (RA) cohort and to analyse the roles of HLA haplotypes, autoantibodies and the B-cell stimulating cytokine interleukin-10 (IL-10) as possible underlying risk factors. Patients and methods. Forty-one consecutive patients with early RA, in whom sulphasalazine was used as the first disease-modifying anti-rheumatic drug in single therapy and was maintained for at least 6 months, were investigated for the occurrence of lupus-related events. Longitudinal analyses of rheumatoid factor (RF), antinuclear antibodies (ANA), anti-double-stranded DNA antibodies and serum IL-10 (ELISA) and the typing of HLA DR and DQ alleles were performed. RESULTS: Four of the 41 patients developed lupus-like disease. Three of four patients who had lupus-related events vs four of 37 patients without side-effects had an HLA DR 0301 haplotype. The patients developing lupus-related side-effects had increased levels of serum IL-10 and a high frequency of ANA in speckled patterns before the onset of therapy. CONCLUSION: The development of SLE-like symptoms and SLE-related autoantibody production was observed more commonly than expected, with an increased risk in patients with SLE-related HLA haplotypes, increased serum IL-10 levels and ANA in speckled patterns. The data suggest that immunomodulation associated with sulphasalazine treatment may contribute to the development of lupus-related reactions in genetically predisposed individuals.

Enhanced generation of interleukins 1 beta and 6 may contribute to the cachexia of chronic disease.

The cachexia of disease may be promoted by proinflammatory cytokines, eg, interleukin (IL) 1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6. These, as well as some antiinflammatory cytokines, eg, IL-1 receptor antagonist (IL-1ra), IL-10, and transforming growth factor beta 1 (TGF-beta 1), were analyzed in serum (IL-6, IL-1ra, IL-10, TGF-beta 1) and stimulated blood monocytes (IL-1 beta, TNF alpha, IL-6) obtained from elderly patients with protein-energy malnutrition (PEM). Twenty-one uninfected malnourished patients aged 75 +/- 1 y (mean +/- SD), with a body mass index (BMI; in kg/m2) of 17.2 +/- 0.5 and various noncancer disorders, and 22 healthy matched control subjects aged 72 +/- 1 y, with a BMI of 25.4 +/- 0.7 (significantly different from patients, P < 0.001), were included. Fifteen patients and their corresponding control subjects were reexamined 3 mo later. Isolated monocytes were stimulated with lipopolysaccharide (LPS) and concentrations of IL-1 beta, TNF-alpha, and IL-6 were determined. Serum concentrations of IL-6, IL-1ra, IL-10, TGF-beta 1, and acute-phase reactants were analyzed. Serum concentrations of orosomucoid and IL-6 were higher in the malnourished subjects than in the control subjects (1.14 +/- 0.1 compared with 0.8 +/- 0.3 g/L, P < 0.001; and 5 ng/L compared with undetectable concentrations, P < 0.01, respectively). Higher generation of IL-1 beta (2.7-fold; P < 0.05) and IL-6 (3.7-fold; P < 0.05) was found in monocytes from patients with PEM relative to the control subjects when monocytes were stimulated with 0.1 microgram LPS/L. Monocyte TNF generation and serum concentrations of IL-10, IL-1ra, and TGF-beta 1 did not differ. Similar results were obtained at follow-up. IL-1ra was negatively correlated with delayed cutaneous hypersensitivity (r = -0.34, P < 0.05). We conclude that enhanced generation of proinflammatory cytokines such as IL-6 and IL-1 beta in malnourished patients may contribute to the PEM often encountered in chronic nonmalignant disorders.

Studies of neutrophil and monocyte oxidative responses in polycythaemia vera and related myeloproliferative disorders.

We have previously reported that polymorphonuclear granulocyte (PMN) chemiluminescence (CL) and superoxide anion production are abnormally low in patients with polycythaemia vera (PV) after simulation with n-formyl-methionyl-leucyl-phenylalanine (fMLP), but normal when elicited by phorbol myristate acetate (PMA). This study documents that both fMLP and PMA induced CL was normal in PMN from patients with chronic myelogenous leukaemia (CML) and essential thrombocythaemia (ET). Furthermore, we monitored intracellular hydrogen peroxide (H2O2) production in PMN and monocytes from patients with PV, CML and ET by flow cytometry. H2O2 production in resting and PMA-stimulated cells was normal in all diseases. So also was fMLP induced H2O2 generation in ET PMN and monocytes. In contrast, fMLP-induced H2O2 production was significantly lower both in PV PMN (1.8 +/- 0.7 mean fluorescence intensity units in PV compared to 8.4 +/- 3.4 in healthy controls; P < 0.02), and in PV monocytes (0.3 +/- 0.5 compared to 2.5 +/- 0.7 in controls; P < 0.02). A less pronounced reduction of fMLP stimulated H2O2 production was noted in CML PMN (3.8 +/- 3.1 compared to 8.4 +/- 3.4 in controls; P < 0.05), and monocytes (1.3 +/- 0.6 compared to 2.5 +/- 0.7 in controls; P < 0.05). The reduction of H2O2 generation in PV and CML PMN was not attributed to subpopulations of less responsive cells. However, one ET and one CML patient showed a subpopulation of less responsive PMN. Thus intracellular H2O2 (as well as extracellular release of superoxide ions) is reduced in fMLP-stimulated PV PMN and monocytes but normal after PMA stimulation, a phenomenon that is not consistently found in other myeloproliferative disorders.

The prevalence of antinuclear antibodies in healthy young persons and adults, comparing rat liver tissue sections with HEp-2 cells as antigen substrate.

The prevalence of antinuclear antibodies (ANA) was studied in 290 healthy adults, aged 20-88 years, and in 219 children, aged 1 month to 15 years. Two antigen substrates, rat liver tissue sections and HEp-2 cells, were compared at different serum dilutions. At titre 1/40, the number of positive adult samples was 6.9% with HEp-2 cells and 6.2% with rat liver. Using a lower serum dilution, HEp-2 cells were shown to be significantly more sensitive than rat liver. However, 25% of the positive samples at serum titre > or = 1/40 could only be found on HEp-2 cells, as compared to 17% for rat liver, even after extended investigation. In children, 7.3% of the samples were positive using HEp-2, as compared to 2.3% with rat liver tissue, at titre 1/10. None of the samples were positive for nDNA or other specific antigens as measured by immunodiffusion. One sample had antibodies against core histones and two samples showed antibodies against subcellular antigens by immunoblot. We conclude that the occurrence of ANA in healthy Swedish children and adults is similar to previous studies from other countries. Both rat liver tissue and HEp-2 cells were found to be suitable for screening purposes, if the increased sensitivity of HEp-2 cells is taken into consideration, particularly with regard to children. Furthermore, it may be necessary to use more than one substrate to exclude ANA positivity in clinical practice.

Metabolic activity in human alveolar macrophages increases after cessation of smoking.

Alveolar macrophages (AMs) were recruited by bronchoalveolar lavage (BAL) from human smokers before and one, three, and six months after smoking cessation. The metabolic activity of the AM was quantified as luminol-enhanced chemiluminescence (CL) both at rest and after in vitro stimulation with phorbol myristate acetate (PMA). The resting CL values did not differ before and after smoking cessation. The activity after PMA stimulation was unaltered at one and three months. However, the maximal metabolic response, as well as the rate, were significantly (P < 0.02 and P < 0.01, respectively) higher at six months, compared to prior smoking cessation. In addition, the time to reach the maximal peak was reduced after six smoke-free months, indicating a more rapid cell activation. The cell concentration in the BAL-fluid decreased (P < 0.001) as soon as after one smoke-free month and remained low at the following lavages. The lower metabolic response one and three months after smoking cessation, and the increased response six months after, together with a rapid normalization of the cell concentration in the BAL fluid, may be explained by the persistence of tobacco-smoke particles in the alveolar space, which could influence cell activity.

Increased expression of adhesion proteins (MAC-1) and altered metabolic response in human leukocytes exposed to surfactant in vitro.

In this study the modulatory effects of a well-defined surfactant preparation on blood leukocytes were investigated. The expression of the cell surface receptor MAC-1 was analyzed by flow cytofluorometry, and the metabolic response was measured by a chemiluminescence technique. An increase (p less than 0.05) in the MAC-1 receptor expression was observed in the granulocytes but not in the monocytes. There was a decrease in the metabolic response of the leukocytes after stimulation with phorbol myristate acetate (PMA) and a delay (p less than 0.01 for both) in the peak activity. Formyl-methionyl-leucyl-phenylalanine (fMLP) caused an increased peak (p less than 0.01). Thus, the surfactant preparation had a modulatory effect on blood leukocytes with regard to the expression of the cell surface receptor MAC-1 and the metabolic response.

The inhibition of CR1 mobilization of human granulocytes by the presence of erythrocytes. A possible mechanism for intravascular regulation of granulocyte modulation.

In this study we show a spontaneous mobilization at 37 degrees of the complement receptor for C3b (CR1) of granulocytes prepared by a method in which erythrocytes were removed by specific lysis, as well as a method where granulocytes were prepared by dextran sedimentation at low temperature without using centrifugation. This increase of CR1-expression was not obtained when erythrocytes were present during the incubation. This inhibitory effect of erythrocytes was maximal at an erythrocyte:granulocyte ratio of 600:1 or more and was not caused by interference with the fluorescence of the immunoassay. EDTA plasma had no inhibitory effect on CR1 mobilization, indicating that the phenomenon was not due to plasma proteins, nor the used anti-coagulant. An increased CR1 mobilization was, however, obtained in the presence of erythrocytes if the granulocytes were simultaneously exposed to the chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (FMLP) at the concentration 10(-9) M or more. However, to obtain a CR1 expression comparable to systems without erythrocytes, a 10-fold higher concentration of FMLP was needed. These results suggest that the inhibitory effect of erythrocytes on the spontaneous receptor mobilization of granulocytes could be a mechanism to keep the complement receptors and other surface structures within the cells while circulating in the blood, to be expressed at the cellular surface only by the appropriate signal, such as propagated by soluble mediators from an inflammatory focus.

Quenching of intracellular autofluorescence in alveolar macrophages permits analysis of fluorochrome labelled surface antigens by flow cytofluorometry.

We report a new technique in which the autofluorescence of alveolar macrophages from smokers is quenched by crystal violet. This technique permits immunostaining of surface antigens of these cells and enables the stained cells to be analysed by flow cytofluorometry. The variable solubility of crystal violet makes it important to characterize the crystal violet solution by its quenching properties and not rely on the assumed concentration of dissolved dye. High concentrations of crystal violet lowered the number of cells and gave a decreased amount of surface antigen (CD11b). However, a lower concentration of crystal violet could be used if the cells were fixed with paraformaldehyde (4%) and the membranes were permeabilized with n-octyl-beta-D-glucopyranoside (0.6%). Using a phagocytic model with FITC-conjugated particles we were able to show that this treatment gave an efficient permeabilization of phagolysosomal membranes.

Erythrocyte-mediated scavenging of reactive oxygen metabolites generated by human polymorphonuclear leukocytes during phagocytosis: comparison between normal and Down's syndrome blood cells.

The phagocytosis of complement-opsonized yeast particles by polymorphonuclear leukocytes from normal and Down's syndrome subjects was compared in the absence and presence of erythrocytes. Granulocytes from children with Down's syndrome showed a large increase of metabolic activity, as measured with chemiluminescence. This was not associated with autooxidative damage in the presence of erythrocytes. The results indicate that erythrocytes could exert an antioxidant effect on the granulocyte cell surface. This was reflected in an enhanced ingestion, which was more pronounced in the presence of Down erythrocytes with a higher superoxide dismutase and glutathione peroxidase content, than normal cells. Our conclusion is that the increase of reactive oxygen metabolites scavengers in Down's syndrome blood cells is of benefit for the homeostasis between generated reactive oxygen metabolites and their propagation.

The expression of CR1 and CR3 on non-modulated and modulated granulocytes of healthy blood donors as measured by flow cytofluorometry.

Complement receptors, CR1 and CR3, on neutrophils increase their cellular spontaneously at 37 degrees C or after mechanical stress during the cell preparation. We have established a cell preparation procedure and a cytofluorometric immunoassay method to evaluate the receptor expression in vivo in this study. The expression of CR1 and CR3 was studied after haemolysis in NH4Cl at different temperatures and incubation intervals. It was shown that cell preparation and receptor analysis must be performed at 15 degrees C or lower to avoid up-regulation of the receptor structures. Two minutes' incubation at 20 degrees C was sufficient to modulate the cells in this regard. Granulocytes from healthy blood donors were analysed and the mean fluorescence intensity (MFI), which reflects the number of receptors on the cell surface, showed a normal distribution for the CR1 (n = 158) and CR3 (n = 76) expression in the healthy population. The MFI of the two receptors showed a correlation (r = 0.71). Granulocytes from all donors could be modulated at 37 degrees C to a similar degree for both receptors (r = 0.76), despite the fact that they are supposed to be mobilized from different intracellular pools. A group of patients with localized inflammatory processes, such as sinusitis, differed from the healthy controls. In this group, the CR1 expression was not normally distributed, but 15 out of 26 patients (58%) had granulocytes with a CR1 expression exceeding the mean +/- SD of the normal population.

Hydrocortisone both decreases the up-regulation of complement receptors CR1 and CR3 and the ingestion process of human granulocytes.

We have studied the effect of hydrocortisone on the complement receptor expression (CR1 and CR3) of human granulocytes during the up-regulation phase and the following stable period when exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the medium alone at 37 degrees C. The receptor expression was also correlated to the C3bi-mediated phagocytosis with special reference to attachment and ingestion. The results showed that after incubation with fMLP the increased expression of CR1 and CR3 is accompanied by an increased ingestion, but not attachment, of complement-opsonized yeast particles. This increase was significantly lower with regard both to attachment and ingestion, as well as receptor expression if hydrocortisone was present during the up-regulation phase. However, the addition of hydrocortisone after the up-regulation of fMLP-treated granulocytes decreased the ingestion of particles but not the CR1 and CR3 expression. The results indicate a membrane effect of hydrocortisone that affects both the receptor mobilization and ingestion mechanism of the phagocytes.

Erythrocyte enhancement of C3b-mediated phagocytosis by human neutrophils in vitro: a combined effect of the erythrocyte complement receptors CR1 and erythrocyte scavengers to reactive oxygen metabolites (ROM).

Human erythrocyte CR1 receptors have been shown to bind complement-fixing immune complexes and, thus, facilitate their elimination from the circulation. The autotoxic effect of free radicals released from phagocytes during phagocytosis can be alleviated by scavengers like catalase and superoxide dismutase. Erythrocytes are known to contain these antioxidants. This study showed that 74% of opsonized yeast particles adhered to human erythrocytes. No difference was seen between yeast opsonized with C3b and yeast opsonized with both IgG and C3b. This adherence was due to the C3b receptor (CR1), as monoclonal antibodies against the CR1 receptor could abrogate the adherence. The yeast phagocytosis by neutrophils was increased by 15% when yeast-C3b was used, and by 34% when yeast-IgG/C3b was used in the presence of human red blood cells. The increase of phagocytosis was not seen when rat erythrocytes (lacking CR1) were present. The cytochrome c reduction decreased with the presence of human erythrocytes during phagocytosis, indicating a scavenging effect on the superoxide anions. The addition of scavengers or erythrocyte lysate, instead of erythrocytes, enhanced phagocytosis of yeast-IgG/C3b to at least the same extent as the erythrocytes. These observations suggest that human erythrocytes primarily enhance phagocytosis through the scavenging effect of those erythrocytes which are concurrently attached with the prey through its CR1 receptor, and then attached to the PMN.

The effect of reactive oxygen metabolites (ROM) on the attachment and ingestion phases of C3b- and IgG-mediated phagocytosis by macrophages.

The effects of enzymatically produced reactive oxygen metabolites (ROM) on the attachment and ingestion phases of C3b- and IgG-mediated phagocytosis by cultured mouse peritoneal macrophages (MPM) was investigated using a hypoxanthine-xanthine oxidase ROM-generating system. ROM-exposure at a dose which did not affect cell viability caused a slight decrease in the percentage of phagocytosing cells. The total number of cell-associated (attached and ingested) C3b- and IgG-coated particles initially decreased in relation to controls. After 120 min the number of attached and ingested C3b-particles had returned to the level of controls, while the corresponding value for IgG-particles lagged behind. The number of ingested particles decreased in both C3b- and IgG-groups at each time-point studied (30-150 min). A linear increase in the formation of lipid peroxidation products was measured during the period of observation, while transmission electron microscopical studies showed largely intact morphology. These results indicate that ROM species may induce membrane-related changes in inflammatory cells such as macrophages, probably due to lipid peroxidation; affecting the binding functions of the C3b- and Fc-receptors, without any obvious alteration in cellular fine structure.

In vitro effect of hydrocortisone on the attachment and ingestion phases of immunoglobulin G- and complement component 3b-mediated phagocytosis by human neutrophils.

The fluorescence quenching method (FQ method) was used to investigate the effect of hydrocortisone on the attachment and ingestion phases of immunoglobulin G (IgG)- and complement component 3b (C3b)-mediated phagocytosis by human neutrophils (PMNs). The results were compared with metabolic activity (O2- release) of the phagocytes. When the PMNs were treated with 5 X 10(-5) M hydrocortisone or more, both IgG-mediated and C3b-mediated interactions decreased. The number of intracellular particles decreased as the total number of PMN-associated particles decreased, indicating an effect mainly on particle attachment. This was substantiated by the fact that pretreatment of the PMN with cytochalasin B resulted in a hydrocortisone dose-related decrease of interacting particles. The FQ method made it possible to quantify the stimulus-phagocyte interaction in relation to the metabolic response. Superoxide anion release decreased at the highest concentration of hydrocortisone used (5 X 10(-3) M), which merely reflected the decreased number of interacting particles. No reduction in metabolic activation was obtained when the superoxide anion release was correlated with the number of interacting yeast-IgG particles. The results indicate that hydrocortisone primarily affects the binding capacity of Fc and C3b receptors, resulting in decreased metabolic activation. The effector functions, e.g., ingestion and metabolic activation, were not affected by hydrocortisone in this study.