In vitro immunoglobulin production by mononuclear cells in rheumatoid arthritis

Since patients with rheumatoid arthritis (RA) exhibit serum hypergammaglobulinemia and autoantibody (rheumatoid factor) production, we compared elaboration and control of in vitro RA mononuclear cell (MNC), Ig assayed by enzyme-linked immunoassays or by hemolytic plaque formation, in 37 RA patients and 17 normal subjects. We found (1) RA spontaneous plaque-forming cells were significantly reduced (RA 344 vs normal 627 PFC/10(6) MNC, P less than 0.002); (2) RA spontaneous IgG and IgM (but not IgA) elaboration was significantly diminished (IgG RA 339, normal 776; IgM RA 255, normal 869 ng/ml, P less than 0.001; IgA RA 87, normal 124); (3) RA stimulated IgG and IgM production (but not IgA) was also decreased (IgG RA 2434, normal 3862, P less than 0.06; IgM RA, 1676, normal 3323, P less than 0.005; IgA RA 1859, normal 2315); (4) reduced RA Ig elaboration was not clearly due to altered numbers of T or non-T cells, age, medications, clinical features of disease, or response kinetics; (5) relative improvement of RA in vitro IgG, but not usually IgM, secretion followed removal of adherent cells, addition of indomethacin or addition of mitomycin C-treated T cells; (6) MNC from synovial fluids, but not bone marrows, exhibited spontaneous Ig production in excess of stimulated synovial fluid cellular or peripheral blood Ig elaboration. These observations indicate selective impairment of peripheral blood MNC IgG and, particularly, IgM secretion in RA. This defect appears to reflect accessory cell influences which differ from normal as well as the sequestration of primed or activated cells in the synovial fluid.     

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.     

Humoral and cellular immune parameters in untreated and phenytoin- or carbamazepine-treated epileptic patients

The peripheral blood lymphocyte subsets, serum immunoglobulins (Ig A, G, M), and C3 and C4 complement protein concentrations were determined in 40 healthy subjects, 30 phenytoin-treated, 22 carbamazepine-treated and 38 untreated epileptic patients. The levels of B-lymphocytes, IgM and C3 complement proteins were found to be significantly higher in untreated epileptics than in healthy controls (P<0.01, P<0.02 and P<0.05, respectively). The absolute number of B-lymphocytes appeared to be unaffected by phenytoin or carbamazepine treatment; however, IgM levels were significantly lower in carbamazepine-treated patients than both epileptic (P<0.01) and healthy (P<0.05) controls. Phenytoin-treated patients had a significant reduction in the mean IgA and IgG levels compared to healthy and epileptic controls (P<0.05). With both drug treatments, significantly lower T-suppressor lymphocyte counts and thus higher T-helper to T-suppressor lymphocyte ratios were observed with respect to healthy and epileptic controls. Our results demonstrate that while phenytoin decreases serum IgA and IgG levels, carbamazepine reduces IgM levels significantly, and untreated epileptics show immune profiles significantly different to those of healthy subjects, suggesting that epilepsy per se may be associated with certain immune aberrations induced by antiepileptic drugs.     

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.     

Selective enhancement of human IgE production in vitro by synergy of pokeweed mitogen and mercuric chloride

IgE production in vitro was investigated in cultures of human peripheral blood mononuclear cells (MNC) from non-atopic donors with pokeweed mitogen (PWM), mercuric chloride (HgCl2), or both. PWM alone induced a few IgE immunoglobulin secreting cells (ISC) detected by reverse plaque forming cells (PFC) and many IgG, IgM, and IgA PFC. HgCl2 alone failed to produce significant numbers of ISC of any class. PWM plus HgCl2 caused a selective increase of IgE PFC without affecting IgG, IgM, and IgA PFC. Co-cultures of B cells plus mitomycin C (MMC) treated T cells stimulated by PWM alone produced more IgG, IgM, IgA and IgE PFC than those of B cells plus T cells. However, PWM plus HgCl2 produced significantly more IgE PFC selectively in those cultures. This effect was observed in the cells of most of the donors, but a few donors showed different responses.     

Specificity of immediate skin reaction with streptokinase-streptodornase (SK-SD) in humans.

Five patients with no detectable serum IgA (less than 20 mug/ml) and one patient with low serum IgA were compared to normal subjects. The number of circulating E-RFC was normal as was the lymphocyte DNA synthesis induced by PHA, Con A, and streptokinase-streptodornase. The patients had normal numbers of IgA-bearing lymphocytes and normal or increased numbers of B cells. Purified anti-immunoglobulin antibodies specific for IgG, IgA and IgM induced a normal lymphocyte DNA synthesis as did PWM. The patients' lymphocytes were able in vitro to transform into actively secreting IgA plasmocytes. This transformation was determined by counting the IgA and immunoglobulin-containing cells and then measuring the IgA and IgG secretion in the cultures. In some patients PWM was selectively suppressive in IgA B-cell transformation into IgA secreting cells; in the other patients PWM had no effect on the IgA B-cell differentiation. PWM enhanced the IgG secretion in the patients' cultures as well as IgA and IgG secretion in the normal controls.     

Fc IgM receptors on human lymphoblastoid B cell lines.

Five human lymphoblastoid cell lines have been investigated for their ability to secrete immunoglobulins (IgG, IgA, IgM) and for the presence of different cell surface markers, with special emphasis on the Fc IgM receptor, using a rosette technique with IgM-coated bovine red blood cells (EA-IgM). Four cell lines (Hu, 8432, SB, PA3) were characterized as having as cell origin due to the presence of surface immunoglobulins, complement receptors, mouse red blood cell rosette formation, low avidity Fc IgG receptors and absence of sheep red blood cell rosette formation. Two of these B cell lines (Hu and PA3) secreted IgM, two cell lines (8432 and SB) secreted IgG, while the human T cell line Molt 4 did not secrete Ig. All four B cell lines exhibited Fc IgM receptors (17–35%) while the T cell line Molt 4 had no detectable Fc IgM receptor. The receptor was specific for IgM. Neither aggregated IgG, soluble IgG immune complexes nor EDTA could abrogate the rosette formation, IgM immune complexes, pure human IgM, as well as normal human serum had an inhibitory effect on EA-IgM rosette formation. The receptor was trypsin-sensitive and required protein synthesis for expression. There was no correlation between the expression of Fc IgM receptors and secretion of any given Ig class, indicating that B cells may express Fc IgM receptors independently of their commitment to produce IgM or IgG.     

IgM- and IgD-bearing peripheral blood lymphocytes differentiate to IgM but not IgG or IgA immunoglobulin-secreting cells

Human peripheral blood mononuclear cells were depleted of surface IgM⁺ or IgD⁺ cells and assayed for mitogen-induced differentiation to immunoglobulin-secreting cells (ISC) of IgM, IgG and IgA classes. Stimulatory agents included T cell-dependent poke weed mitogen, B cell mitogen Staphylococcus aureus bacteria strain Cowan I, and a combination of the two which gives uniform, high levels of ISC from all normal donors. Depletion of either IgM- or IgD-bearing B lymphocytes resulted in loss of cells bearing the other Ig class and blocked most of the mitogenic reactivity to anti-IgM and anti-IgD. Proliferative responses to Cowan I in these depleted populations were about 20% that of unfractionated mononuclear cells. Depletion of T cells increased the mitogenic response to Cowan I and to the two antibody preparations, showing that they are T-independent mitogens. Depletion of IgD⁺ cells caused partial loss of mitogen-induced IgM ISC (22%-60% of unseparated controls) but no loss of IgG or IgA ISC. Depletion of IgM-bearing cells caused complete loss of IgM ISC, but no loss of IgG or IgA ISC. We previously demonstrated that anti-IgM antibody blocked mitogen induction of Ig secretion of these three classes in spleen cells, but only IgM secretion in blood mononuclear cells. Together, the results suggest that the majority of cells in normal blood responding to mitogens to mature to IgG or IgA production belong to IgM^?, IgD^? B cell subsets, in contrast to precursors of secreting cells for these isotypes in the spleen. Thus, these blood precursors appear to be more mature than the corresponding spleen cells.     

Analysis of high and low responses toStaphylococcus aureus and interleukin 2 in human B lymphocytes

Fixed protein A-bearing staphylococci (SAC) stimulate human B cells via surface Ig, whereas IL-2 has been reported to provide a sufficient second signal for proliferation and differentiation. Using an ELISPOT assay to count cells secreting IgM, IgA, and IgG and flow cytometry with acridine orange to assess cell cycle progress, we have found that the purified B lymphocytes of a substantial minority (5/13) of healthy volunteers with normal serum Ig levels failed to differentiate to Ig secreting cells (ISC) in response to SAC + IL-2 (IgM, IgA, or IgG secreting cells, <5% of input B cells). High-responders generally formed 10–35% ISC. The proportions of B cells expressing IgG, IgA, IgM, or IgD were not different in the two groups. By average linkage cluster analysis, SAC/IL-2 high- and low-responders were shown to fall into two separate populations with respect to ISC. High- and low-responders tended to remain in the same group with repeated testing over several months, although some convergence was seen. The low-responders also showed significantly less advancement to late G1 and S phase than the high-responders, in the presence of SAC ± IL-2. Induction of IL-2 receptors on B cells by SAC + IL-2 was much greater in high-responders than in low-responders, as shown by flow cytometry with phycoerythrinconjugated IL-2. However, SAC + IL-2 induced transferrin receptors normally in low-responders, showing that some early activation steps occur in these cells. Low-responder B cells often improved their responses in the presence of macrophages and T cell supernatants. Finally, bypassing the surface Ig pathway using anti-CD3-activated T cells to stimulate B cells produced normal differentiation in low-responder B cells. Thus a subset of clinically normal individuals possesses B cells which fail to express IL-2 receptors, proliferate, and differentiate normallyin vitro in response to SAC + IL-2 yet can respond well to alternative activation pathways via T cells, monocytes, and their products.     

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.     

Enumeration of human peripheral blood lymphocytes secreting immunoglobulins of major classes and subclasses in healthy children and adults

The reverse enzyme-linked immunospot assay was modified to enumerate peripheral blood mononuclear cells (PBMC) secreting IgG1–4, IgA1–2, and IgM. Anti-human IgG F(ab')₂ and mouse monoclonal antibodies specific to each of the isotypes were used as solid-phase capture antibodies and developing antibodies, respectively. Although attempts were also made to detect IgD- and IgE-secreting cells (SC), their numbers in the peripheral blood were too few to be reliably estimated. The assay was applied to study healthy subjects including 21 neonates within 3 days of birth, 44 1- to 48-month-old children, and 32 adults. Sixty percent of these neonates had IgM SC in small numbers (<20 per 10⁶ PBMC), but only three neonates had IgSC of other isotypes. In contrast, by 1–2 months of age children had IgSC of all isotypes, including IgA2 and IgG4, often in higher numbers than adults. The relative frequencies of IgSC were IgA1 > IgG1 > IgM > IgG2 > IgG3 > IgG4 > IgA2 in the children and IgA1 > IgG1 > IgA2 > IgM > IgG4 > IgG2 > IgG3 in the adults. The order of the serum concentrations in the adults was IgG1 > IgG2 > IgA > IgM > IgG4 > IgG3. No correlation was found between the serum level and the IgSC number of individual isotypes (except for serum IgA and IgA1-SC). This new methodology should facilitate investigating the current status of immunoglobulin synthesis and the Ig repertoire in adults and children, in health and in disease.Dedicated to the memory of Dr. Charles Reimer.     

Spontaneous plaque forming cells in the peripheral blood of patients with systemic lupus erythematosus.

A reverse haemolytic plaque assay using staphylococcal protein A coupled to sheep red blood cells was set up in Cunningham chambers. Using this method, the numbers of Ficoll-Hypaque isolated peripheral blood lymphocytes (PBL) secreting IgG, IgA or IgM without preceding culture or mitogen stimulation were estimated in patients with systemic lupus erythematosus (SLE) and control subjects. Seven patients with clinically inactive SLE at the time of the study had values similar to those of the control subjects. In contrast, eight patients who had clinically active SLE had markedly increased numbers of PBL secreting IgG, IgA and IgM. Control experiments confirmed that the plaques were due to Ig secretion by lymphoid cells rather than to immune complexes adsorbed onto Fc receptor bearing cells or to passively adsorbed Ig. The results confirm the expected polyclonal B cell activation in patients with SLE and serial measurements showed that clinical relapses occurred only when the numbers of immunoglobulin secreting cells were high. Experiments in three patients with active SLE using native DNA prepared from T2 bacteriophage as the 'developing antigen' suggest that PBL secreting nDNA antibody can also be demonstrated by this method.     

Distribution of IgM, IgA and IgG secreting cells in the tissues of normal and tumour-bearing mice.

The levels of IgM, IgA and IgG secreting cells were examined in control, Corynebacterium parvum-stimulated and tumour-bearing, normal and athymic (Nu/Nu) mice. The percentage of IgA to IgM or IgG secreting cells is relatively higher in peripheral blood than in the spleen or peritoneum of normal mice. Within tumours, irrespective of their degree of vascularization and immunogenicity, the pattern of Ig secreting cells in similar to that seen in peripheral blood and different from that in spleen and peritoneum even in athymic mice. Intraperitoneal injection of C. parvum changes the relative percentages of Ig secreting cells in the peritoneal cavity to resemble that seen in the peripheral blood and tumours. It appears that Ig secreting cells extravasate from peripheral blood in a non-isotype specific manner into sites of chronic stimulation.     

Anti-IgA antibodies in epileptic patients with a low serum IgA concentration

Sera from 22 phenytoin-treated epileptic patients with a serum IgA less than 0.30 g/l were examined for anti-IgA antibodies using an ELISA. Only one patient had a complete IgA deficiency. Anti-IgA antibodies of restricted specificity were detected in serum from two of the patients. Their serum IgA concentrations were 0.03 and 0.27 g/l. The serum concentrations of IgM, IgG and the IgG subclasses were normal in both these patients. The frequency of this type of anti-IgA antibody is not higher in epileptic patients with a low serum IgA than in healthy controls, and class-specific anti-IgA antibodies are not a pathogenetic factor in the IgA deficiency occurring in epilepsy.     

Limiting dilution analysis of the B cell compartment in human bone marrow

Mononuclear cells (MNC) obtained from adult rib specimen were investigated for their capacity to produce immunoglobulin (Ig) in vitro. Using limiting dilution analysis 3 populations of B lineage cells could be distinguished. The first produces Ig in culture without any intentional activation. These cells are strictly nonproliferating and sustain extraordinary secretory rates of 5 X 10(7)-10 X 10(7) molecules IgG, IgA or IgM/cell/h for at least 2 weeks. They occur at frequencies of 1 X 10(-4)-10 X 10(-4) (marrow MNC) or represent 1-5% of marrow B cells. Following exposure to infectious Epstein-Barr virus (EBV), these cells do not (a) proliferate, (b) express EBV-determined nuclear antigens (EBNA), (c) enhance their secretory rates or (d) show secretory activity for prolonged time. These cells are therefore EBV resistant. If these cells produced their antibody at similar rates in vivo, then their frequencies would suggest that they could provide up to 2/3 of daily synthetic rates for IgG and IgA and 20-30% of the daily IgM production. The second population of marrow B cells is exclusively committed to IgM production. Following exposure to EBV these cells proliferate, express EBNA and begin to secrete IgM. The third population represents 90% of marrow B cells. In our hands, these cells are unable to produce Ig in vitro.     

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.     

Human interleukin-5 induces staphylococcal A Cowan 1 strain-activated human B cells to secrete IgM

Studies on the role of human interleukin (IL)-5 in B cell growth and differentiation have yielded conflicting results. To clarify this issue, we studied the role of purified recombinant IL-5 on activated human B cells which were depleted of Tcells and adherent cells. Human IL-5 augments IgM secretion, but not IgG or IgA secretion of purified human B cells activated with staphylococcal A Cowan 1 strain (SAC). However, the period of B cell activation with SAC is critical for the B cell to respond to IL-5. After 24 h of SAC activation, human B cells are responsive to the IL-5 signal, but with longer periods of activation, IL-5 responsiveness diminishes. This may explain some of the previous conflicting results. The IgM enhancement was not seen when B cells were activated with pokeweed mitogen. In addition, human recombinant IL-4 synergized with IL-5 in augmenting IgM secretion by SAC-activated B cells, while IL-5 synergized with IL-2 to augment IgM, IgG and IgA secretion by SAC-activated B cells. As the purified IL-5 was derived from a COS-1 cell supernatant, and COS-1 cells secrete IL-6, we examined whether a polyclonal IL-6 antibody blocked the IgM-enhancing activity of IL-5. IL-6 antibody did not block the IL-5 enhancement of IgM secretion, but a monoclonal antibody to IL-5 inhibited the human IL-5 activity on human B cells. These results demonstrate that human IL-5 augments IgM secretion of SAC-activated human B-cells. In addition, this lymphokine synergizes with IL-4 and IL-2 in supporting Ig secretion.     

Maternal Blood B-Cell (CD19+) Percentages and Serum Immunoglobulin Concentrations Correlate with Breast-feeding Behavior and Serum Prolactin Concentration

PROBLEM: Lactating women recover from pregnancy-induced immunosuppression while actively secreting immunologically active agents into milk. Few clinical studies have examined changes in postpartum maternal immune status or explored mechanisms. METHOD OF STUDY: We measured blood B-cell (CD19+) percentages and serum concentrations of immunoglobulin (Ig) G, IgM, and IgA at 1 to 2 weeks, 1 month, and 2 months postpartum in a longitudinal study of seven healthy, lactating women. RESULTS: More frequent or extended breast-feeding sessions were correlated with lower CD 19+ percentages, reduced serum IgG, and higher serum IgA and IgM concentrations. CD19+ percentages were correlated negatively with serum prolactin concentrations. Blood samples drawn before and 30 min after breast-feeding did not differ in CD 19+ percentages or serum Ig concentrations. CONCLUSIONS: These findings confirm our previous cross-sectional study showing a negative correlation between CD 19+ percentages and serum prolactin. Because lactation practices are modifiable, these findings suggest that women can influence the course of lactation-associated immunologic changes.     

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.     

Human immunoglobulin class and IgG subclass regulation: Dual action of interleukin-4

Epstein-Barr virus (EBV) was used as a polyclonal human B cell mitogen to investigate the regulation of immunoglobulin class and IgG subclass responses by interleukin-4 (IL-4). Activation of tonsillar B cells with EBV resulted in an early peak of polyclonal immunoglobulin secretion between days 13 and 14 consisting of IgM, IgA, and IgG1, IgG2, IgG3 and IgG4, but not IgE. Addition of IL-4 to EBV-activated B cells at concentrations of 100 U/ml or greater induced the production of IgE and enhanced IgG4 secretion, but had no effect, or more often inhibited the other isotypes. In contrast, low concentrations of IL-4 (1-5 U/ml) significantly increased the production of IgM, IgA, IgG1, IgG2and IgG3, but had no effect on IgG4 or IgE. The increase in immunoglobulin secretion obtained with low concentrations of IL-4 was found to occur only with high-density (resting) B cells, suggesting that IL-4 was not functioning simply as a late-acting differentiation factor. Low concentrations of IL-4 significantly increased IgG1, IgG2, IgG3, and IgA production by surface (s)IgM⁺ (sIgG^?/sIgA^?) B cells which is consistent with heavy chain switching. In some experiments, however, IL-4 enhanced IgM secretion by sIgM⁺ B cells, and IgA, IgG1, IgG2, IgG3 by sIgM B cells, suggesting that it may have an additional B cell differentiation factor activity which was not isotype specific. The different effect of IL-4 at high and low concentrations were similar to those observed in B cell activation experiments, and may be due to the existence of high- and low-affinity IL-4 receptors.