Rheumatoid factors.

Rheumatoid factors (RFs) have been studied for over 50 years and are probably the most written about of any antibody. Nevertheless, the etiology of these RFs and the precise role they play in the pathogenesis of rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) remain a major interest. When RFs participate in the generation of inflammation in RA and JRA, they probably do so by forming immune complexes (IC) or are themselves able to bring about the inflammatory response. Their presence has been associated with more severe disease, vasculitis, and systemic symptoms. The present review summarises the literature over the last few years on new and interesting findings on RF. This review covers an update on RF assays, RF cross-reactivity, specificity studies, immune complex formation, RF lymphocyte studies, and RF binding.

In vitro effects of methotrexate on peripheral blood monocytes: modulation by folinic acid and S-adenosylmethionine.

The mechanism of action of low dose methotrexate in rheumatoid arthritis has not been established. It has been shown to have an anti-inflammatory effect and to inhibit neutrophil chemotaxis, but the effect on monocytes has not been widely studied. Normal donor peripheral blood monocytes were incubated with methotrexate in vitro and their superoxide production, chemotaxis, and phagocytosis subsequently assessed. Additionally, the influence of different culture media, and of folinic acid, and the methyl donor S-adenosylmethionine, and spermidine on the methotrexate mediated effects were evaluated. It was found that methotrexate in low concentrations inhibited in vitro monocyte chemotaxis and superoxide production but only after prolonged incubation. This inhibition was augmented by incubation in medium containing a low methionine concentration and was abolished by folinic acid and S-adenosylmethionine, suggesting that methotrexate may interfere with specific methylation reactions.

Production of IgM rheumatoid factor by normal lymphocytes after stimulation with preparations containing IgM rheumatoid factor from patients with juvenile arthritis.

Preparations containing IgM rheumatoid factor (RF) and hidden IgM RF were isolated from the serum samples of nine patients with juvenile rheumatoid arthritis. Six of these preparations stimulated lymphocytes from normal donors to produce IgG and IgM, of which up to 11% had IgM RF activity. In contrast, the polyclonal activator pokeweed mitogen also stimulated IgM production, but only 1% had IgM RF activity. A relation between the activator and IgM RF or hidden IgM RF is suggested. This is based on the positive correlation between IgM RF concentration in these preparations and their ability to stimulate lymphocytes to produce IgG, IgM, and IgM RF. These data indicate that preparations from patients with juvenile rheumatoid arthritis containing IgM RF and hidden IgM RF are potent stimulants of lymphocytes from normal donors, with specific production of IgM RF.

Antibody to streptococcal cell wall peptidoglycan-polysaccharide polymers in sera of patients with juvenile rheumatoid arthritis but absent in isolated immune complexes.

Sera of 88 children with juvenile rheumatoid arthritis (JRA) (10 seropositive, polyarticular onset, 29 seronegative, polyarticular onset, 32 pauciarticular onset, and 17 systemic onset) were evaluated for the presence of serum antibodies to streptococcal cell wall peptidoglycan-polysaccharide polymers (PG-PSP). Immune complexes (IC) isolated by the antihuman IgM (HIgM) affinity column method were also evaluated for the presence of antibodies to PG-PSP. Forty-one of 88 patients with JRA (7 of 10 seropositive, polyarticular onset, 11 of 29 seronegative, polyarticular onset, 16 of 32 pauciarticular onset, and 7 of 17 systemic onset) showed elevated levels of antibodies to PG-PSP in their sera. IgM rheumatoid factors (RF) were demonstrated in 70/88 isolated IC fractions of patients with JRA and IgG RF in 7; however, none of the patients demonstrated the presence of antibodies to PG-PSP in their isolated IC fractions from the anti-HIgM affinity column. These data indicate that antibodies are produced to PG-PSP in all JRA onset types, but they are not constituents of isolated IC by the anti-HIgM affinity column method.

Cross-reactive antiidiotypic antibodies against human rheumatoid factors from patients with juvenile rheumatoid arthritis.

We prepared antiidiotypic (anti-Id) antibody to 2 polyclonal IgM rheumatoid factors (IgM-RF) and 2 polyclonal "hidden" IgM-RF. The anti-Id antibodies were isolated by chromatography on Sepharose 4B, to which was bound rabbit anti-human IgG Fc fragments. F(ab')2 fragments from the anti-Id antibodies were generated by pepsin digestion and isolated by gel filtration. The anti-Id antibodies directed against RF from 4 patients with juvenile rheumatoid arthritis (JRA) were tested by an inhibition hemolytic assay for cross-reactivity with IgM-RF from 4 adult patients with rheumatoid arthritis, 6 patients with JRA, and 13 JRA patients with hidden RF. The 4 anti-Id antibodies had variable cross-reactivity with the isolated adult RA RF, JRA RF, and JRA hidden RF. Similar results were obtained by a direct-binding enzyme-linked immunosorbent assay for the anti-Id antibodies. The broad pattern of cross-reactivity was apparently unrelated to a particular amino acid sequence, but was associated with the antigen-binding site of IgM-RF. These results suggest the possibility that the anti-Id antibodies prepared against isolated RF obtained from JRA patients bear the "internal image" of antigen; that is, the Fc region of human IgG. These anti-Id antibodies may be generated in JRA patients and may possess specific immunomodulatory properties.

Enzyme linked (ELISA) immunoabsorbent assay for the detection of hidden 19S IgM rheumatoid factors in juvenile rheumatoid arthritis.

Determination of hidden IgM rheumatoid factors (RF) in juvenile rheumatoid arthritis (JRA) offers advantages for diagnosis and in following disease activity. Sera from 30 patients with JRA were assayed for RF by latex fixation test (LFT), sensitized sheep cell agglutination test (SCAT), nephelometry, and by ELISA. IgM containing fractions were prepared by chromatography and assayed for hidden RF by the hemolytic method and ELISA. Ten patients were seropositive by LFT. All of them gave positive tests on the serum for RF with the SCAT, nephelometry, and ELISA, and on the IgM containing fractions by the hemolytic assay and ELISA. Seventeen patients seronegative by LFT were positive for hidden RF by the hemolytic test and ELISA on the IgM containing fraction. When unfractionated serum was used, 15 were positive by ELISA. Three patients were seronegative and also negative for hidden RF by the hemolytic assay and ELISA. Thus, only 2 of 30 patients had discordant results between the hemolytic assay on the IgM containing fraction and the ELISA on the serum. Our results indicate the ELISA on the serum in conjunction with the LFT offers a simple, rapid, alternative test for hidden 19S IgM RF in JRA patients.

Glycosaminoglycan composition of tight skin and control mouse skins.

Tight skin (TSK) mouse skin has previously been shown to contain increased amounts of collagen and glycosaminoglycans (GAG). We now report on the GAG composition of TSK mouse skin. Using an electrophoretic and an enzymatic HPLC method, skin from 99 TSK and control mice (newborn, 1 month-old, and 6 months-old) were analyzed for GAG composition. There was no significant difference between TSK and control GAG.

IgM rheumatoid factor plaque-forming cells in juvenile rheumatoid arthritis.

Using a direct, plaque-forming cell (PFC) assay with sensitized sheep erythrocytes, lymphocytes that secrete IgM rheumatoid factors (RF) have been detected in the peripheral blood of patients with juvenile rheumatoid arthritis. Of 15 juvenile rheumatoid arthritis patients tested, 8 were seropositive and 7 were seronegative, but 6 of the seronegative patients had hidden 19S IgM-RF. Ten patients (5 seropositive and 5 with hidden 19S IgM-RF) demonstrated RF-PFC in their peripheral blood (range 15 to greater than 200 RF-PFC/10(6) mononuclear cells). Of 11 patients who had active disease, 10 had RF-PFC, and the 4 patients who had inactive disease had no PFC in their peripheral blood. HLA typing of all 15 patients revealed no correlation between the presence of RF-PFC and HLA type.

19S IgM rheumatoid factor-7S IgG rheumatoid factor immune complexes isolated in sera of patients with juvenile rheumatoid arthritis.

19S IgM rheumatoid factors (RF) and hidden 19S IgM RF have been associated with increased disease activity in juvenile rheumatoid arthritis (JRA). Recently, immune complexes (IC) were isolated from JRA sera by several methods which demonstrated the presence of 19S IgM RF. The present study evaluates 25 JRA patients' sera by separation on a Sepharose 4B column to which were bound F(ab')2 fragments of goat IgG antihuman IgM antibody to separate IgM-containing IC. The columns were sequentially eluted with 1 M ammonia and 0.1 M glycine-HCl buffer, pH 3.0. The isolated fractions were assayed for 19S IgM RF and 7S IgM RF by ELISA, IgG levels by immunodiffusion, and by preparative isoelectric focusing. The ammonia eluate from the alpha HIgM column revealed IgG, 19S IgM RF in six patients, and IgM RF in four patients. All were polyarticular-onset JRA patients. In the glycine-HCl eluate of sera, 19S IgM RF and IgG were also detected in 15 patients, all six seropositive, polyarticular-onset, six seronegative, polyarticular-onset, and three pauciarticular-onset patients. Significant 7S IgG RF titers were demonstrated in the glycine-HCl eluates of six patients, five seropositive, polyarticular-onset patients, and one seronegative, polyarticular-onset patient. Analysis by preparative isoelectric focusing of the IgM RF and IgG RF positive ammonia and glycine-HCl eluates showed IgM RF throughout the pH range (4-10), but the highest amount of IgM RF was in the pH range 4.0-5.5. Significant IgG RF titers were detected only in this restricted spectrotypic area of pH 4.0-5.5.(ABSTRACT TRUNCATED AT 250 WORDS)

19S IgM rheumatoid factor-7S IgG rheumatoid factor immune complexes isolated in patients with rheumatoid arthritis.

Sera of 12 patients with rheumatoid arthritis (RA) who had positive IgM-rheumatoid factor (RF) tests were separated by use of immunoabsorbent columns (goat anti-human C3 [alpha HC3] and rabbit anti-human C1q [alpha HC1q]) and polyethylene glycol (PEG) precipitation-protein A affinity chromatography to isolate their immune complexes (IC). The isolated fractions were assayed for 19S IgM RF and 7S IgG RF by enzyme-linked immunosorbent assay (ELISA). The sera were further analyzed by preparative isoelectric focusing (IEF). The alpha HC1q and alpha HC3 columns were sequentially eluted with barbital buffer, 0.02 mol/L EDTA, 0.5 mol/L NaCl, and 1 mol/L propionic acid. All 12 patients had IgM RF and IgG RF in the EDTA fractions from both immunoabsorbent columns, but only IgM RF in the NaCl fractions. The PEG precipitation-protein A preparations were eluted with 0.5 mol/L glycine HCl and 3.5 mol/L MgCl2. All 12 patients had significant titers of 19S IgM RF (greater than or equal to 1:192) and 7S IgG RF (greater than or equal to 1:96) in the acid-eluted fraction. Analysis of the sera by preparative IEF revealed IgM RF with a polyclonal spectrotype pattern with pH of 3.0 to 10.0, but predominantly acidic proteins with isoelectric points of 4.0 to 5.5 IgG RF were found in the same restricted spectrotypic pattern. These studies demonstrated that IC can be detected in the sera of patients with RA by isolation with alpha HC1q and alpha HC3 immunoabsorbent columns and PEG precipitation-protein A affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation and characterization of complement-fixing immune complexes in juvenile rheumatoid arthritis patients.

Immune complexes (IC) in sera from patients with juvenile rheumatoid arthritis (JRA) were isolated by the use of immunoabsorbent columns. Sera from 14 JRA patients (four seropositive for 19S IgM RF and 10 seronegative, but nine having hidden 19S IgM RF) were analyzed by the anti-human Clq (alpha HClq) and anti-human C3 (alpha HC3) columns. The columns were sequentially eluted with veronal buffer, 0.02 M EDTA, 0.5 M NaCl, and 1 M propionic acid. By the alpha HClq column, IgM RF were detected in at least one of the separated IC fractions of 13 of 14 patients and IgG RF in three patients. By the alpha HC3 column, only five patients demonstrated IgM RF and only one IgG RF in the eluted fractions. On sucrose density gradient analysis (SDGA), all IC were demonstrated in the peaks greater than or equal to 19S.19S IgM RF were demonstrated by ELISA in all 14 patients, but IgG RF in only three. These studies demonstrate that complement-fixing 19S IgM RF, IgG, and IgG RF containing IC can be detected in the serum of JRA patients.

Hidden 19S IgM rheumatoid factor in adults with juvenile rheumatoid arthritis onset.

Forty-eight adult patients with juvenile rheumatoid arthritis (JRA) (onset before age 16 years) were evaluated at the age of 17 years or more for the presence of hidden 19S IgM rheumatoid factors (RF), i.e., 19S IgM RF that can be detected by the complement-dependent haemolytic assay in the IgM-containing fraction after separation of the serum by acid gel filtration. The average age of the patients was 25.3 years. The mean duration of disease was 16.5 years. Thirty-two of 48 patients (67%) showed the presence of hidden 19S IgM RF in their serum. Disease activity correlated with hidden RF titres in 62% (55/88) of the evaluations. The results indicate that patients with seronegative JRA onset continue to have significant titres of hidden 19S IgM RF in their sera into early adulthood.

Hidden and classical 19S IgM rheumatoid factor in a juvenile rheumatoid arthritis patient.

Hidden 19S IgM rheumatoid factors (RF), i.e., 19S IgM RF which can be detected in the IgM containing fraction after acid gel filtration of serum, are found in 59-68% of patients with juvenile rheumatoid arthritis (JRA). Their presence generally correlates with disease activity. We describe a 12 year-old female with a polyarticular onset of JRA who, during her first 15 months of disease, was seronegative but had hidden RF titers of 1:128----1:256. Inhibition studies on her hidden RF showed specificity for HIgG greater than RIgG and equal specificity for the human IgG subclasses (IgG1 = IgG3). In the second and third year of disease, she became seropositive with RF titers varying from 1:40 to 1:320 while her hemolytic titers on her IgM fractions were decreased from 1:128 to 1:32. Inhibition studies now demonstrated a higher avidity for RIgG; and HIgG3 inhibited more than HIgG1. These studies documented for the first time a JRA patient who early in the disease was negative for RF and positive for hidden RF, and who later became seropositive.

Autoantibodies in juvenile arthritis.

Sera from 104 children with JA with different onset-types of disease were evaluated for 19S IgM RF by the LFT , hidden 19S IgM RF by the hemolytic assay, ANA by HEp-2 cell substrate, and levels of IC by the C1qSPA . Their relationship to active disease was determined. Classical 19S IgM RF were detected by the LFT in only seven patients. All were late-onset polyarticular females. Hidden 19S IgM RF were detected by the hemolytic assay in the separated IgM-containing fraction in 55 patients of all onset-types. Clinical activity correlated with the presence of hidden 19S IgM RF in 82% of cases. ANA, using the HEp-2 cell substrate, were found in 61 patients, the majority showing a speckled, immunofluorescent pattern. ANA were noted in all RF positive patients and in nine of 10 patients with iridocyclitis. IC were found in 39 patients, and correlation with clinical activity occurred in 54% of cases. A search for positive associations among the four parameters showed no statistically significant correlations except for the concordance of ANA positivity in all seven RF positive patients. The presence of hidden RF correlated more closely with disease activity (P less than 0.001) than did that of ANA or IC. The significance of these data and previous studies remains to be determined. We have demonstrated that in the average JA population 7% have 19S IgM RF and about 60% have hidden RF, ANA, or elevated levels of IC. The present findings of 98 of 104 patients with at least one of the abnormal immunoproteins , the association of ANA in patients with iridocyclitis or with RF positivity, of hidden RF with disease activity, and the presence of 19S IgM RF in isolated IC suggest a possible immunologic etiology for JA.

Precipitation of 19S IgM rheumatoid factor-IgG circulating immune complexes in patients with juvenile arthritis by polyethylene glycol and separation by immobilized protein A.

Immune complexes (IC) in sera and synovial fluid (SF) from patients with juvenile (rheumatoid) arthritis (JA) were isolated by making use of polyethylene glycol (PEG) to precipitate IC and then using staphylococcal protein A to separate the IC. The isolated IC were compared to IC in sera analysed by sucrose density gradients and measured by the C1q solid phase assay (ClqSPA). Isolated IC from sera of 10 of 16 JA patients demonstrated significant 19S IgM rheumatoid factor (RF) titres in their acid elutes utilizing the haemolytic assay. IgM and IgG were detected by low level radial immunodiffusion in the acid eluate of sera in all 10 patients who had significant RF titres. In isolated IC from SF, three of five JA patients had significant 19S IgM RF titres detected in their acid eluates. By sucrose density gradient analysis of these sera, elevated levels of IC detected by ClqSPA were found in the fractions of greater than or equal to 19S comparable to the previous isolated 19S IgM-IgG complexes. This detection of IC like material containing 19S IgM and IgG by the present method further supports the participation of classic and hidden 19S IgM RF in IC formation in JA patients.

HLA antigens in black and white patients with juvenile arthritis: associations with rheumatoid factor, hidden rheumatoid factor, antinuclear antibodies, and immune complex levels.

HLA typing for -A, -B, -C, -DR, and -MT antigens and simultaneous studies for the presence of 19S IgM rheumatoid factor (RF), hidden 19S IgM RF, antinuclear antibodies (ANA), and immune complexes (IC) were performed on 24 black and 80 white patients with juvenile arthritis (JA) of different onset types. HLA-DRW6 (p less than 0.05) was associated with pauciarticular onset and early onset pauciarticular black patients. HLA-DR4 was found in both blacks and whites with chronic disease (p less than 0.01) and with the presence of RF (p less than 0.05) and hidden RF (p less than 0.05). In the whites, HLA-DR5 (p less than 0.05) and DRW8 (p less than 0.001) were associated with pauciarticular onset and early onset pauciarticular patients. HLA- DRW8 was also associated with white JA patients with iridocyclitis (p less than 0.001) and black (p less than 0.01) and white patients (p less than 0.001) with the presence of ANA. HLA-MT2 was demonstrated in all 24 black patients (p less than 0.001) and in 54/80 white patients (p less than 0.001). HLA-MT2 was associated with black (p less than 0.01) and white (p less than 0.001) patients with early onset pauciarticular disease and the presence of iridocyclitis in white patients (p less than 0.001). The association of HLA antigens in black JA patients has not been reported before.

Glycosaminoglycan content in skin of the tight-skin mouse.

The tight-skin (TSK) mouse has cutaneous changes similar to those found in the skin of patients with progressive systemic sclerosis (PSS). Previous studies have shown that both have common abnormalities in skin thickness, dry weight, and hydroxyproline content. In this study, glycosaminoglycans (GAGs), major components of the ground substance, were quantitated in skins from TSK mice and compared with age-matched normal mice. Biochemical studies included determinations of hexosamines, uronic acids, and total GAGs by cetylpyridinium chloride precipitation. Dry weights and water-fat content of skin biopsy specimens from TSK mice were also compared with those of normal mice. Hexosamine, uronic acid, total GAGs, and dry weight were increased in TSK mouse skin when compared with normal mouse skin. The water-fat content did not differ significantly. These findings were similar to those known to occur in PSS skin, further suggesting that the TSK mouse might serve as an animal model for the skin changes found in PSS patients.