IgM rheumatoid factor elaboration by blood, bone marrow, and synovial mononuclear cells in patients with rheumatoid arthritis

IgM rheumatoid factor (RF) elaboration by rheumatoid arthritis (RA) synovial, bone marrow, and blood mononuclear cells (MNC) is reported. IgM RF was prepared from RF-positive sera by sequential euglobulin precipitation, Sephacryl S300 gel filtration, and IgG-Sepharose affinity chromatography. Purified material, which contained no detectable IgG or IgA, was used in an enzyme-linked immunosorbent assay (ELISA) to quantitate cellular elaboration of IgM RF. Excellent standard curves (r2 = 0.98) were obtained without nonspecific binding of samples or antisera to IgG-coated microtiter plates and without cross-reactivity of standards with antisera other than anti-IgM. We found RA blood MNC (11 patients) spontaneously averaged 15 ng/ml IgM RF (6% of total IgM produced), but elaborated 254 ng/ml IgM RF following pokeweed mitogen (PWM) stimulation (22 patients), exceeding that of 13 normal controls. Bone marrow MNC spontaneously (4 patients) produced 71 ng/ml IgM RF and secreted 78 ng/ml IgM RF with PWM stimulation (9 patients). In contrast synovial fluid MNC (5 patients) spontaneously elaborated 6652 ng/ml IgM RF, significantly (P less than 0.05) more than blood or bone marrow MNC; PWM-stimulated synovial fluid MNC (5 patients) produced 5472 ng/ml IgM RF. These observations confirm selective localization of activated, IgM RF-producing cells to the rheumatoid synovial space.     

High Incidence of Spontaneous Ig-Producing Lymphocytes in Peripheral Blood and Synovial Fluid of Patients with Active Seropositive Rheumatoid Arthritis

Numbers of in vitro spontaneous IgG, IgM and IgA plaque-forming cells (PFC) as assessed by a modification of the protein A haemolytic plaque assay were determined in the blood and synovial fluid of patients with seropositive rheumatoid arthritis (RA) and compared with those of control groups. The total numbers of PFC were significantly higher in the peripheral blood of patients with active seropositive RA than in that of normal controls. In addition, most B lymphocytes in the synovial fluid of patients with active seropositive RA were active immunoglobulin (Ig) producers, whereas synovial fluid lymphocytes from patients with inactive seropositive RA and seronegative arthritis were not. In general, IgA PFC were relatively high in blood, whereas IgG PFC dominated in the synovial fluid. IgM PFC appear to be relatively low in blood and synovial fluid. However, a relative increase of IgG PFC was noted in the peripheral blood of patients with active RA. To test for polyclonality of the increased Ig synthesis, we tested the sera of patients and controls for the presence of polyclonal antibodies against sheep erythrocytes (SRBC) and SRBC modified by fluorescein isothiocyanate (FITC) and trinitrophenyl (TNP). No differences were observed with SRBC and TNP-SRBC agglutinin titres between patients and controls, but patients with RA had higher titres of FITC-SRBC agglutinins than normal sera. This finding supports the concept of a polyclonal nature of antibody production in RA patients.     

T-Cell Helper Activity and B-Cell Function of Synovial and Blood Lymphocytes from Patients with Rheumatoid Arthritis or Other Forms of Chronic Arthritis

The helper effect of T cells on B-cell immunoglobulin (Ig) responses induced by pokeweed mitogen (PWM) or purified protein derivative of tuberculin (PPD) was studied in lymphocytes from synovial fluid (SF) and blood of nine patients with rheumatoid arthritis (RA) and eight patients with other forms of chronic arthritis. In PWM cultures the helper effect of SF T cells on Ig responses (IgG, IgM, IgA) of autologous and allogeneic blood B cells was lower than that of blood T cells (P less than 0.01). This decrease was more pronounced in patients with RA than in patients with non-RA. In PPD cultures no significant difference was found between the helper effect of SF T cells and blood T cells on the Ig responses of allogeneic blood B cells or on the IgG response of autologous blood B cells, whereas the helper effect of SF T cells on the IgM and IgA responses of autologous blood B cells was decreased. The Ig responses to PWM or PPD in cocultures of autologous blood B and T cells were not significantly different between patients and healthy controls. The PWM- and PPD-induced Ig responses of SF B cells were lower than those of blood B cells when cocultured with autologous blood T cells. SF B cells produced IgG but usually little IgM and IgA. Thus there was a dysfunction of SF B cells and of SF T cells in a PWM-driven system, but a fairly good helper function of SF T cells in a PPD-driven system.     

Effect of human intestinal macrophages on immunoglobulin production by human intestinal mononuclear cells isolated from patients with inflammatory bowel disease.

The effect of macrophages on spontaneous immunoglobulin production by isolated human intestinal mononuclear cells (MNC) is unknown. Depletion of macrophages by adherence to fibronectin or by panning with macrophage-specific monoclonal antibody 3C10 lead to a significant reduction in IgA. IgG and IgM production by intestinal MNC from both normal (n = 10) and inflammatory bowel disease (IBD) (n = 13) mucosa. The reduction in immunoglobulin produced by macrophage-depleted intestinal MNC was greater in IBD patients than in normal controls. There was a significant correlation (r = 0.816, P less than 0.001) between the percentage of macrophages depleted by panning with 3C10 and the reduction in IgG produced by macrophage-depleted intestinal MNC. Addition of either fibronectin-adherent cells or the supernatant from these macrophage-enriched cells enhanced immunoglobulin production in a dose-dependent fashion. A greater increase in IgG production by macrophage-depleted cells was seen when cultured with supernatant from inflamed IBD mucosal cells, compared with that from normal mucosal cells. The soluble factor(s) responsible in the supernatant was acid and heat susceptible but was not affected by freezing and thawing. Addition of recombinant human interleukin-1 beta or human interferon-gamma to cell cultures did not influence immunoglobulin production. Thus, human intestinal macrophages enhance spontaneous immunoglobulin production by isolated intestinal MNC by secreting soluble factor(s) which remain to be fully characterized.     

The effect of phenytoin in vitro on normal human mononuclear cells and on human lymphoblastoid B cell lines of different Ig isotype specificities

The anticonvulsant drug phenytoin, in less than cytotoxic concentrations, caused significant reductions in Ig secretion by unstimulated or EBV-stimulated normal MNC, as measured by PFC or secretion of Ig into the culture medium. Isotype-specific LBL varied in their sensitivity, the secretion of IgA (1 line) and IgG (3 lines) being reduced by phenytoin near therapeutic concentrations, whereas that of IgM (1 line) was resistant. Six-day exposure of MNC to phenytoin caused no selective depletion of or enrichment for B cells, monocytes or T cell subsets. The results suggest that the reduction in serum Ig levels reported in phenytoin-treated epileptic patients is, at least in part, due to a direct effect of the drug on the B lymphocyte. However, among EBV-activated normal MNC, those secreting IgA were no more sensitive to the drug than those secreting IgG or IgM, and other factors may, therefore, operate to cause the preferential reduction in serum IgA in phenytoin-treated patients.     

A quantitative assay for IgA rheumatoid factor

We have developed a solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgA rheumatoid factor (RF) in biological fluids. Human IgM RF, IgG RF, IgG, IgA, IgM and whole serum did not significantly interfere with the IgA RF assay. Patients with sero-positive rheumatoid arthritis (RA) had significantly higher concentrations of IgA RF than sero-negative RA patients or healthy adult controls. Concentrations of IgA RF in paired sera and synovial fluids from sero-positive RA patients were comparable. Levels of IgA RF demonstrated a moderately good correlation with levels of IgM RF in sero-positive RA sera (r = 0.673). However, the ratio of IgA RF concentration to IgM RF concentration in sero-positive RA sera varied widely.     

Increased spontaneous secretion of rheumatoid factor by intestinal lamina propria mononuclear cells from Crohn's disease but not ulcerative colitis patients.

Increased levels of rheumatoid factors (RF) have been observed in the serum of Crohn's disease but not ulcerative colitis patients, and have been proposed to relate to an increased state of intestinal lymphocyte activation. We have therefore examined the spontaneous in vitro secretion of RF by intestinal lamina propria mononuclear cells (MNC) isolated from specimens from control and inflammatory bowel disease (Crohn's disease, ulcerative colitis) patients. Normal intestinal lamina propria MNC spontaneously secrete rheumatoid factors of different isotypes during 14 days of in vitro culture (9.7 ng/ml IgA RF, 11.6 ng/ml IgM RF and 64.6 ng/ml IgA anti-Fc (IgG)). In matched studies intestinal MNC isolated from normal large bowel exhibited significantly greater levels of RF synthesis and secretion in vitro than normal small bowel intestinal MNC. A large increase in spontaneous RF secretion was observed from Crohn's disease intestinal MNC (21.4 ng/ml IgA RF, 21.4 ng/ml IgM RF, and 108.15 ng/ml IgA anti-Fc (IgG)), when compared with normal controls. The amount of RF secreted was dependent on the amount of inflammatory activity of the bowel specimens, from which the MNC were isolated (198.3 ng/ml of IgA anti-Fc(IgG) from involved versus 50.0 ng/ml from matched non-involved tissue). Ulcerative colitis MNC released decreased amounts of RF (7.1 ng/ml IgA RF, 6.2 ng/ml IgM RF, and 42.3 ng/ml IgA anti-Fc(IgG)). These observations using isolated intestinal MNC may explain the findings of RF changes in the sera of inflammatory bowel disease patients. Our observations support the hypothesis of a heightened state of activation in normal intestinal lamina propria MNC, which is further increased in active Crohn's disease. The dissimilarities observed between Crohn's disease and ulcerative colitis may indicate fundamental differences in disease pathophysiology and will lead to further studies exploring intestinal immunoregulatory properties of RF.     

Analysis of IgG subclass production in cell cultures from IgA deficient patients and in normal controls as a function of age

IgA deficient individuals may also have low serum levels of IgG subclasses, especially IgG2. In the present study we examined the development of plasma cells producing IgM, IgA or IgG, and the IgG1 and IgG2 subclasses, following lipopolysaccharide (LPS) and pokeweed mitogen (PWM) stimulation of mononuclear cells (MNC) from normal and IgA deficient individuals as a function of age. Studies of blood MNC from 38 normal donors (age range 2-44 years) revealed an age-related distribution pattern of mu, gamma, alpha, gamma 1 and gamma 2 plasma cells produced in mitogen-stimulated and control cultures. Decreased IgA responses to both LPS and PWM were consistently observed in cultures of MNC from all of the nine children with IgA deficiency. When compared with age-matched controls the IgG response was also diminished in PWM stimulated cultures, whereas the IgM responses were normal. The IgG deficit was due to reduced responses for the gamma 1 and gamma 2 subclasses, and was most pronounced for IgG2; IgG2 plasma cell differentiation was particularly depressed in LPS cultures. In contrast to normal adult cells, blood MNC from the nine children with IgA deficiency and age-matched controls (2-17 years) yielded more IgG1 than IgG2 plasma cells in both control and LPS cultures, while the pattern of response to PWM was similar in all groups (gamma 1 greater than gamma 2). A good concordance was found between the level of secreted Ig in the culture supernatants and the relative number of IgM or, IgG and IgA plasma cells identified by immunofluorescence staining of cytoplasmic immunoglobulins.     

Quantitation of Cells Secreting Immunoglobulins after Elution from Rheumatoid Synovial Tissue

Mononuclear cells (MNC) eluted from rheumatoid synovial tissue of 16 patients with rheumatoid arthritis were examined for immunoglobulin-secreting cells (ISC). Immediately after elution and separation synovial tissue MNC contained considerable numbers of ISC. IgG and IgA ISC were more abundant than IgM ISC. At the same time there were low numbers of ISC in blood. Synovial tissue ISC were lost during incubation with pokeweek mitogen (PWM), possibly because tissue MNC were already maximally triggered in vivo. This was in contrast to blood MNC, in which the number of ISC increased significantly during incubation with PWM.     

Effects of immunoglobulin G from patients with systemic lupus erythematosus on human B cell function

We examined the effect of systemic lupus erythematous (SLE) sera and Ig fractions on IgG and IgM release by cultured normal peripheral blood mononuclear cells (PBMC) when these cells were preincubated with serum dilutions or Ig fractions. Increases in both IgM and IgG (P less than 0.001 and less than 0.01) in cultured cell supernatants were recorded when PBMC were preincubated with SLE serum dilutions. IgG but not IgM from SLE was found to stimulate PBMC to release IgG (P less than 0.01). Similar results were obtained when SLE IgG was preincubated with adherent cell depleted cells (ADC) or isolated normal B cell fractions. When normal PBMC were preincubated with SLE serum or IgG and subsequently stimulated with pokeweed mitogen (PWM), a relatively blunted IgG release was observed (P less than 0.05); however, IgM release was significantly increased (P less than 0.001). This effect was not observed when PBMC were preincubated with SLE IgM, normal serum dilutions, or normal Ig fractions. Relative blunting of PWM response after PBMC were preincubated with SLE IgG was not reversed in PBMC depleted of adherent cells, OKT8+, or OKT9+ cells. Depletion of PBMC of LeuM1 cells increased IgG release in response to PWM when cells had been preincubated with SLE IgG. SLE serum or Ig fractions did not induce B cell growth factor release by T cells. SLE IgG appeared to act directly on B cell enriched populations to release IgG; this was not associated with significant increase in thymidine uptake, or apparent lysis of cells.     

Abnormal B cell function in haemophiliacs and their relationship with factor concentrates administration.

The present study was performed to evaluate B cell function in haemophiliacs. Spontaneous and pokeweed mitogen (PWM)-induced immunoglobulin (Ig) production was determined by ELISA in the supernatants of cultured peripheral blood lymphocytes (PBL) from 14 haemophiliacs and 17 normal donors. Spontaneous IgM, IgA and IgG production was three times higher in patients than normal controls, while PWM-induced IgM, IgA and IgG production was markedly reduced in patients compared to normal donors (P less than 0.025). Allogeneic co-cultures of haemophiliacs and normal B plus T cell fractions revealed that these results are due to a defect of the patients' T cell depleted fraction. These abnormalities were not found in three patients who had received no clotting factor concentrates for at least 1 year prior to the study. Additionally, the annual amount of clotting factor concentrates received by treated patients correlates well with the enhancement of spontaneous Ig production (r = +0.688, P less than 0.02), the decrease of PWM-induced Ig secretion (r = -0.655, P less than 0.02), and the elevation of serum IgG levels (r = +0.610, P less than 0.05). These findings suggest that the administration of clotting factor concentrates play an important role in the altered B cell function in haemophiliacs.     

IgA and IgM rheumatoid factors in serum, saliva and other secretions: relationship to immunoglobulin ratios in systemic sicca syndrome and rheumatoid arthritis.

Paired serum and saliva samples from seven patients with systemic sicca syndrome (SSS), 15 patients with rheumatoid arthritis and a positive Schirmer's test (RA+), 15 patients with rheumatoid arthritis and negative Schirmer's test (RA-) and 14 normal individuals were analysed for albumin and immunoglobulin concentration as well as IgA and IgM rheumatoid factor (RF) activity. Protein levels in saliva were higher in SSS and RA+ but, when corrected for serum concentration and salivary flow rate, only the IgG ratio remained significantly elevated in SSS (P less than 0.01) and RA- (P less than 0.05) and the IgM ratio was reduced in RA- (P less than 0.05) compared to controls. Although IgM RF activity in serum and saliva was strongly correlated (P less than 0.001) in all three patient groups, the activity in saliva was considerably lower than serum activity. In the two (RA) patients tested, IgM RF in saliva contained secretory component. Mean salivary IgA RF activity varied between 34% (RA-) and 84% (SSS) of serum activity and correlated with serum activity in SSS (P less than 0.001) and RA- (P less than 0.01). IgA RFs in saliva, but not in serum, contained secretory component. Additional demonstration of IgA RF activity in nasal and duodenal secretions in SSS may be related to involvement of the common mucosal immune system.     

Humoral immune function in severe, active rheumatoid arthritis

Peripheral blood mononuclear cells (PBMC) from 30 patients with definite or classic active rheumatoid arthritis who were on no remittive drugs were studied for spontaneous and pokeweed mitogen (PWM)-stimulated immunoglobulin plaque-forming cell frequency (IgPFC), spontaneous IgM-rheumatoid factor (IgM-RF) secretion, and in vitro proliferative responses to soluble recall antigens. Rheumatoid spontaneous total (IgG + IgM + IgA) IgPFCs were higher than those of normal controls when assayed after 7 days in culture. Spontaneous and PWM-stimulated IgM-PFCs, in contrast, were significantly less than normal regardless of when assayed. Spontaneous synthesis of IgM-RF was observed in 56% of the RA patients, but absolute amounts produced were widely heterogeneous. Spontaneous IgM-RF production by RA PBMC was associated with low or absent spontaneous IgM-PFC production. Moreover, a strong association was found between the median amount of IgM-RF secreted and depressed proliferative responses to soluble recall antigens. Our results define several abnormalities of immunoglobulin production in a clinically homogeneous and highly active rheumatoid population and delineate methodologic variations that can complicate the interpretation of similar data in the literature. In addition, our findings suggest that subgroups of rheumatoid patients that show distinct cellular and humoral immune abnormalities can be identified.     

Decreased suppressor T cell activity in patients with hepatic cirrhosis (HC).

Hypergammaglobulinaemia (HGG) is frequently found in patients with hepatic cirrhosis (HC). Using an assay system of in vitro PWM-stimulated immunoglobulin (Ig) production, the amounts of IgG, IgA, and IgM produced by peripheral blood lymphocytes (PBL) from 15 HBs Ag-negative patients with HC and from 16 age-matched healthy subjects were quantitated by radioimmunoassay. We found that PBL from patients with HC produced significantly greater amounts of IgG (P less than 0.05) but not IgA or IgM than did those from control subjects. This increased IgG production by PBL from patients with HC was attributed to enhanced T helper activity and not to enhanced B cell function. We also searched for defects in naturally occurring suppressor T cell activity which is sensitive to irradiation. Irradiation-induced enhancement for IgG production was significantly lower in patients with HC compared with age-matched control subjects (P less than 0.01). Similarly, we examined the effect of Con A-induced suppressor T cells on the in vitro PWM-stimulated IgG production by allogeneic PBL and observed the decrease of Con A-induced suppressor T cell activity in patients with HC (P = 0.01). We conclude, therefore, that the increased serum levels of Ig, particularly IgG in patients with HC may result from in part on the basis of depressed ability of naturally occurring suppressor T cells or Con A-induced suppressor T cells to suppress Ig production.     

Lacrimal and salivary immunoglobulins in Sj?gren's syndrome

The diagnosis of Sj?gren's syndrome is sometimes difficult. We have previously demonstrated the diagnostic value of salivary immunoglobulins IgG and IgM in Sj?gren's syndrome. In the present study, we assessed both lacrimal immunoglobulins (1 Ig) and salivary Ig (s Ig) in Sj?gren's syndrome. We studied 112 patients: 71 had rheumatoid arthritis (57 sero positive), 19 had connective tissue diseases or vasculitis (six Sj?gren's syndrome alone, six systemic sclerosis, three mixed connective tissue diseases, three polyarteritis, one relapsing polychondritis), and 22 patients had other inflammatory, metabolic or degenerative joint diseases. Lacrimal Ig and salivary Ig were assessed by double immunodiffusion with antisera specific for IgG, IgM and IgA. Each Ig class was scored on a scale ranging from 0 to 3 plus without knowledge of the patient's diagnosis. The results of factorial analysis demonstrated a strong relationship between xerophtalmia, positive Schirmer's test, s IgG, s IgM, 1 IgG, and 1 IgM in patients with seropositive rheumatoid arthritis or other connective tissue diseases. Analysis of individual parameters showed a significant relationship between 1 IgG and ocular complaints (P less than 0.01), positive Schirmer's test (P less than 0.05), positive rose bengal dye test (P less than 0.05), 1 IgM (P less than 0.01), s IgG (P less than 0.01) and s IgM (P less than 0.05). A significant relationship was also found between s IgG and ocular complaints (P less than 0.02), positive rose bengal dye test (P less than 0.01), positive minor salivary gland biopsy (P less than 0.05), s IgM (P less than 0.01), and 1 IgM (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormalities in the glycosylation of IgG in spouses of patients with rheumatoid arthritis. A family study.

Forty-seven members of eight families with a rheumatoid proband were analysed for abnormal glycosylation of IgG. The results (%G(o) which is the percentage of oligosaccharide chains lacking galactose) were corrected for age and expressed as SD units about the mean for the normal population. Seven of 8 probands, 3/8 spouses, 3/5 RA relatives and 4/26 non-RA relatives had %G(o) values greater than 1SD above the age corrected mean for the normal control population (P less than 0.001, less than 0.01, less than 0.005 and greater than 0.5 respectively). A further 13 spouse pairs were studied. Ten of 13 probands and 8/13 spouses had %G(o) values greater than 1SD above the mean (P less than 0.001 and less than 0.001 respectively). Thus in total, a strikingly high number of unaffected spouses had high %G(o) values (11/21). IgM, IgA and IgG rheumatoid factors were studied. While RA patients' sera showed a correlation between IgM and IgA rheumatoid factors and %G(o), (IgM, r = 0.41 0.05 greater than P greater than 0.02, IgA, r = 0.36, P = 0.05), no correlation between IgG RF and %G(o) was noted in the RA patients. No correlation was found between any of the RF classes and %G(o) in spouses and non-RA relatives.     

Immunoglobulin G and A antibody responses to Bacteroides forsythus and Prevotella intermedia in sera and synovial fluids of arthritis patients

The objective of the present study was to investigate immunoglobulin G (IgG) and IgA antibody immune responses to Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, and Candida albicans in the sera of patients with rheumatoid arthritis (RA), the synovial fluid (SF) of patients with RA (RA-SF samples), and the SF of patients without RA (non-RA-SF samples). An enzyme-linked immunosorbent assay was used to determine IgG and IgA antibody levels in 116 serum samples from patients with RA, 52 RA-SF samples, and 43 non-RA-SF samples; and these were compared with those in SF samples from 9 patients with osteoarthritis (OA-SF samples) and the blood from 100 donors (the control [CTR] group). Higher levels of IgG antibodies against B. forsythus (P < 0.0001) and P. intermedia (P < 0.0001) were found in non-RA-SF samples than in OA-SF samples, and higher levels of IgG antibodies against B. forsythus (P = 0.003) and P. intermedia (P = 0.024) were found in RA-SF samples than in OA-SF samples. Significantly higher levels of IgA antibodies against B. forsythus were demonstrated in both RA-SF and non-RA-SF samples than in OA-SF samples. When corrected for total Ig levels, levels of IgG antibody against B. forsythus were elevated in RA-SF and non-RA-SF samples compared to those in OA-SF samples. Lower levels of Ig antibodies against B. forsythus were found in the sera of patients with RA than in the plasma of the CTR group for both IgG (P = 0.003) and IgA (P < 0.0001). When corrected for total Ig levels, the levels of IgG and IgA antibodies against B. forsythus were still found to be lower in the sera from patients with RA than in the plasma of the CTR group (P < 0.0001). The levels of antibodies against P. gingivalis and C. albicans in the sera and SF of RA and non-RA patients were comparable to those found in the respective controls. The levels of IgG and IgA antibodies against B. forsythus were elevated in SF from patients with RA and non-RA-SF samples compared to those in OA-SF samples. Significantly lower levels of IgG and IgA antibodies against B. forsythus were found in the sera of patients with RA than in the plasma of the CTR group. This indicates the presence of an active antibody response in synovial tissue and illustrates a potential connection between periodontal and joint diseases.     

Human mononuclear cells and neutral proteinases

We have been interested in contributions of certain cells and mediators to synovial inflammation rheumatoid arthritis (RA). The present studies were designed to determine (1) whether monocytes contained the neutral proteinase cathepsin G and (2) if neutral proteinase could induce or potentiate cellular IgM rheumatoid factor (RF) production. Monocyte-rich and monocyte-poor populations were isolated by Ficoll-Hypaque density sedimentation followed by glass adherence, and cellular lysates were obtained by repetitive freezing and thawing as we have reported for neutrophil-derived neutral proteinase. Cathepsin G was quantified immunochemically by an enzyme-linked irnmunoassay (ELISA) we developed utilizing commercially available anti-cathepsin G antibodies. Mononuclear and B-cel!-enriched cell cultures were prepared by standard methods and IgM RF measured by our ELISA. Cell-derived lysates from monocyte-enriched populations (84+3% monocytes, less than 1 % neutrophils) contained considerably greater amounts of measurable cathepsin G (OD₂₈₀=0.393±0.153) than lysates from equal numbers of monocyte (15±2% monocytes, less than 1% neutrophils) -depleted cells (OD₂₈₀=0.071±0.038;P<0.05). Eighteen patients with RA and three normal individuals did not have consistently increased cellular elaboration of Ig or IgM RF in vitro in response to proteinase (trypsin) stimulation; however, patients manifested 80% potentiation by trypsin of pokeweed-stimulated cellular IgM RF production in vitro (pokewee-dstimulated IgM RF 137±53 ng/mi, pokeweed/trypsin-induced IgM RF 246±100 ng/ml;P<0.02), changes being most striking for those patients seropositive by latex fixation test (84% increase,P<0.02). These data indicate that monocytes and/or macrophages localized to sites of inflammation are a source of cathepsin G and that neutral proteinases may potentiate cellular activation and auto antibody production in processes such as rheumatoid arthritis.Supported in part by funds from the Florida Chapter Arthritis Foundation and the Veterans Administration Medical Center.     

Prostaglandin-mediated immunoregulation: reduced sensitivity of in vitro immunoglobulin production to indomethacin in rheumatoid arthritis

Spontaneous and pokeweed mitogen (PWM) stimulated in vitro immunoglobulin production from peripheral blood mononuclear cells (PBMC) of control subjects and rheumatoid arthritis (RA) patients both receiving non-steroidal anti-inflammatory drugs (RA + NSAID) was measured by an enzyme linked immunosorbent assay (ELISA). Spontaneous IgG and IgM-RF production from the RA + NSAID was significantly higher than in control subjects. IgM production was also elevated but not significantly. Indomethacin (10(-6) - 10(-8)M) added to in vitro cultures failed to influence spontaneous production from either group. PWM stimulated IgG production was not significantly different between the two groups whilst IgM synthesis was significantly reduced in the RA individuals. IgM-RF production was observed only in the RA + NSAID group. Indomethacin inhibited PWM stimulated IgG and IgM production in control individuals but was significantly less potent on IgG, IgM and IgM-RF production from the RA + NSAID group. This reduction in the inhibitory effect of indomethacin correlated significantly with the high spontaneous immunoglobulin production and a low PWM stimulation index observed in the RA + NSAID group. Indomethacin had no significant effect on PWM stimulated PBMC proliferation in the rheumatoid individuals. These results suggest that B lymphocytes from some RA + NSAID are 'pre-committed' to produce immunoglobulins spontaneously in culture, possibly as a consequence of activation in vivo, and are therefore relatively insensitive to PWM stimulation. These B lymphocytes may have progressed beyond the immunoregulatory steps involving prostaglandins.     

T cell dependent differentiation of human B cells: direct switch from IgM to IgE, and sequential switch from IgM via IgG to IgA production.

Ig production by splenic human B cells that express different surface Ig isotypes were analysed in limiting dilution cultures. Therefore, FACS sorted IgM+, IgG+ and IgA1+ B cells were stimulated with PMA-activated EL4 thymoma cells as helper cells in the presence of IL-2 and IL-4. We found that at least every second B cell responded in vitro and secreted the antibody corresponding to its surface Ig isotype. IgE secreting cells developed from surface IgM+ D+ cells (1/31 to 1/167), but not from IgG+ or IgA1+ cells (much less than 1/5000). Negative signalling of the IgM+ B cells by addition of anti-IgM antibodies into the cultures reduced the number of single IgM producing cells by greater than 85%, and completely inhibited IgE switch. In contrast, anti-IgG and anti-IgA antibodies did not reduce the IgE response. The results indicate a direct switch from IgM to IgE secretion in vitro. In contrast to IgE, IgA secreting cells developed from IgM+D+ (1/30 to 1/51) and from IgG+ B cells (1/14 to 1/25). Negative signalling of the IgG+ B cell subset within total B cells by anti-IgG antibodies suppressed the development of IgG as well as IgA producing cells, but did not inhibit IgM and IgE responses. This indicates a sequential switch from IgM via IgG to IgA. Taken together, this study indicates that IgE secreting cells are derived directly from IgM+D+ B cells by non-sequential switching, whereas IgA producing cells preferentially develop by sequential switching via IgG+ B cells.