Inhibitory effect of anti-class II antibody on the spontaneous activation of B cells in patients with systemic lupus erythematosus. Analysis with IL-1 production and IL-1 receptor expression

The mechanism of the spontaneous activation of B cells in patients with SLE was analyzed from the standpoint of the production of IL-1 from B cells and the expression of IL-1R on B cells. SLE B cells spontaneously produced IL-1-like factors which stimulated murine thymocyte proliferative responses. Their m.w. was about 17,000 and their isoelectric point was 4.8. The IL-1-like activity produced by B cells was absorbed with rabbit anti-IL-1 alpha antibody, but not with anti-IL-1 beta antibody. The differentiation of SLE B cells was enhanced by rIL-1 alpha, beta or IL-1-like factors produced by SLE B cells in a concentration-dependent manner. SLE B cells expressed large number of IL-1R detected by FITC-conjugated IL-1 alpha. By a Percoll gradient density centrifugation, IL-1-producing cells and B cells responsive to IL-1 were enriched in a higher density fraction, but were reduced in a lower density fraction. IL-1R-positive B cells were enriched in the lower density fraction, but were depleted in the higher density fraction. However, the expression of IL-1R on the lower density B cells was reduced by 2-day culture. The expression of IL-1R on the higher density B cells was increased during a 2-day culture. Anti-class II antibody inhibited the production of IL-1R on the higher density B cells. These results suggest that the cellular interaction among B precursor cells mediated by class II Ag induces the production of IL-1 and the expression of its receptors on their surface and the interaction between IL-1 and its receptors stimulates B precursor cells to spontaneously differentiate into Ig-producing cells as an autocrine mechanism in patients with SLE.     

Mechanism of spontaneous activation of B cells in patients with systemic lupus erythematosus. Analysis with anti-class II antibody

The mechanism of spontaneous activation of B cells in patients with systemic lupus erythematosus (SLE) was analyzed by using anti-class II monoclonal antibodies in vitro. B cells from SLE patients showed enhanced proliferation and Ig production by in vitro culture without any stimulation. The number of Ig-producing cells increased during a 5-day culture period, but the addition of anti-class II antibodies such as anti-HLA-DR, DQ, or DP monoclonal antibodies inhibited these B cell responses in a dose-dependent manner. Anti-class I and anti-B1 antibody gave no effect. The inhibitory effect of anti-class II antibodies on B cell responses became more remarkable when B cells were cultured on a longer period. By a Percoll gradient density centrifugation, Ig-producing cells were enriched in the lower density fraction, but became depleted in the higher density fraction. However, B cells of the higher density fraction developed into Ig-producing cells after 5 days of culture and anti-class II antibodies inhibited this development. When mitomycin C- and cycloheximide-treated B cells were added to the in vitro culture of B cells as a stimulator, B cell responses were enhanced in a dose-dependent manner. T cells treated with mitomycin C and cycloheximide had no enhancing effect on B cell responses. Furthermore, the enhancing effect of the stimulator B cells was inhibited by the pretreatment of stimulator B cells with anti-class II antibodies. These results suggest that in patients with SLE the abnormality exists in B precursor cells which are easily activated by interacting with other B cells to differentiate into Ig-producing cells and anti-class II antibodies inhibit the B cell activation by interfering with this cellular interaction.     

Responses of B cells from autoimmune mice to IL-5

Three strains of mice (NZB/W F1 X NZW (NZB/W), BXSB, and MRL/Mp-lpr/lpr (MRL/lpr] develop an autoimmune disease that is clinically and immunologically similar to human SLE. A characteristic of these mice is polyclonal B cell hyperactivity. To explore whether this may be related to hyper-responsiveness to B cell stimulatory factors, we investigated the proliferative and secretory responses of B cells from these mice to semi-purified natural and rIL-5, a major regulator of B cell development in the mouse. As this lymphokine stimulates growth and differentiation of activated B cells, attention was focused on in vivo-activated B cell populations, obtained from the interface of 50/65% Percoll density gradients, from normal or autoimmune mice. This cell population from NZB/W mice secreted IgM and incorporated [3H]TdR at significantly higher levels in response to IL-5, and was more sensitive to IL-5, than a comparable population from several normal murine strains. NZB/W female and male mice displayed heightened responses to IL-5, indicating that this is characteristic of the strain in general and is not associated with the accelerated severe disease of the females. Small resting B cells from NZB/W and normal mice were insensitive to IL-5 stimulation. In contrast to NZB/W mice, no difference was observed in the magnitude of either proliferative or Ig secretory responses between in vivo-activated B cell populations from autoimmune BXSB and MRL/lpr or normal mice. Thus, B cell hyper-responsiveness to IL-5 is a characteristic of NZB/W mice but not of two other lupus-prone murine strains. As one unique feature of NZB/W mouse B cells compared to normal and other autoimmune B cells is an elevated proportion of Ly-1+ B cells, the possibility of IL-5 hyper-responsiveness being associated with this B cell subpopulation was investigated. Fluorescence-activated cell sorter sorted Ly-1+ and Ly-1- B cells both responded to IL-5, however Ly-1+ B cells consistently showed a higher stimulation index in both proliferative and Ig secretory responses to this lymphokine.     

Recombinant murine IL-5 induces high rate IgA synthesis in cycling IgA-positive Peyer's patch B cells

Recent studies have shown that purified IL-5 from T cell lines and clones enhances IgA synthesis in LPS-triggered splenic B cell cultures, and that this effect is augmented by IL-4. In this study we have examined the ability of rIL-5 and rIL-4 to support spontaneous Ig synthesis in normal Peyer's patch (PP) B cell cultures. The rIL-4 supported proliferation of the HT-2 and in vivo adapted BCL-1 cell lines, increased Ia expression on normal spleen B cells, co-stimulated splenic B cell proliferation in the presence of anti-mu and enhanced IgG1 synthesis in LPS triggered splenic B cell cultures. The rIL-5 supported BCL-1 proliferation, co-stimulated splenic B cell proliferation in the presence of dextran sulfate, and increased IgA synthesis in LPS-stimulated splenic B cell cultures. Markedly enhanced IgA responses occurred in PP B cell, but not splenic B cell cultures supplemented with rIL-5 in the absence of an added B cell trigger. However, rIL-4 alone did not enhance IgA synthesis or increase the IgA synthesis of PP B cell cultures stimulated with rIL-5. The rIL-5 receptive PP B cells were present in the blast cell subpopulation, inasmuch as a low density fraction isolated on Percoll gradients accounted for the enhanced IgA synthesis. Further, cell cycle analysis of whole PP B cells using propidium iodide in conjunction with staining for surface B220, demonstrated that approximately 12 to 16% of the B cells were in the S and G2/M stages of cell cycle, the remainder being in Go + G1. The surface IgM+ B cells were predominantly in Go + G1, whereas the sIgA+ B cell subpopulation was enriched for cells in the S and G2/M compartments. The PP B cell subset responsible for enhanced IgA synthesis in the presence of rIL-5 was sIgA-positive because FACS-depletion of the sIgA+ B cells resulted in the total loss of rIL-5 enhanced IgA synthesis. Further, when PP B cells were enriched for sIgA+ B cells by cell sorting, these cells responded to rIL-5 with increased IgA synthesis in a dose-dependent manner. When the actual numbers of IgA secreting cells were assessed in PP B cell cultures with supplemental rIL-5, no significant increase in total IgA-producing cells was noted when compared with B cells cultured without rIL-5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proliferation of phenotypically immature human thymocytes with and without interleukin 2 receptors

Previous studies have indicated that the human thymus is composed of several discrete compartments. Cortical thymocytes are reactive with the monoclonal antibody anti-T6, whereas most medullary cells, unreactive with anti-T6, stain brightly with anti-T3, which defines mature T cell populations. Only a minor thymocyte population lacks both T3 and T6 but expresses T11 antigens. Within the thymus, several proliferating lymphoblasts are present. In addition a distinct subset shows the capacity to proliferate in response to mitogens. By continuous Percoll density gradient centrifugation, we have obtained a cell fraction comprising the vast majority of cells able to proliferate spontaneously or after PHA stimulation. By a panning procedure performed with anti-T3 and anti-T6 antibodies, three phenotypically distinct thymocyte subsets were separated from this fraction, and their functional capabilities were tested. The spontaneous proliferating activity was found to be mainly attributable to thymocytes unable to respond to mitogen, expressing the cortical T6 marker and lacking receptors for IL 2. T3-positive cells are able to respond to mitogen. However, these thymocytes are incapable of producing the adequate amount of IL 2 required to fully saturate their intrinsic proliferative capability. Surprisingly, the phenotypically least mature intrathymic T lymphocytes (T3 and T6 negative) respond to phytomitogen, at least in part, in an interleukin-dependent manner. It is noteworthy that a large proportion of these T3- and T6-negative thymocytes express IL 2 receptors and class II MHC antigens without in vitro activation. These novel findings have potential implications in the context of current models of differentiation pathways within the human thymus.     

Polyclonal activation of xid B cells by auto-Ia-reactive T cell clones

The mechanism of T cell-dependent activation of xid B cells into Ig-producing cells was studied by employing H-2-restricted, antigen-specific T cell clones. Helper factors (B cell stimulatory factors, BSF) released from KLH-specific T cell lines could induce polyclonal Ig production in B cells from (CBA/N X BALB/c)F1 (NBF1) female mice but not from CBA/N or NBF1 male mice. Direct addition of helper T cell lines induced Ig production in xid B cells from CBA/N or NBF1 male mice. A T cell clone, MK6, which was derived from NBF1 male mice and specific against Iad determinant, could activate NBF1 male but not CBA/N B cells. Another clone, CK4, derived from CBA/N mice and having specificity against KLH plus I-Ak determinant could activate both CBA/N and NBF1 male B cells into IgM- and IgG-producing cells in the absence of KLH, and monoclonal anti-I-Ak antibody specifically blocked such activation. These results suggest that xid B cells are able to be activated by the signal provided by the recognition of Ia molecules on B cells by auto-Ia-reactive T cells. Xid B cells from CBA/N mice that had been co-cultured with a T cell line specific against I-Ak determinant for 24 hr became reactive to BSF and capable of differentiating into Ig-producing cells in the presence of BSF. The results showed that even xid B cells could be responsive to BSF if they were in a certain activation stage.     

Cloned allospecific human helper T cell lines induce an MHC-restricted proliferative response by resting B cells

To analyze helper T (Th) cell-induced B cell proliferation in man, we have cloned allospecific Th cells, grown them as long-term IL 2-dependent T cell lines (TCL), and analyzed their phenotypic and functional properties. The two TCL described in this report, A-7 and A-57, are both composed exclusively of T3+, T4+, T8- T cells blasts. In proliferative assays, with a panel of x-irradiated allogeneic stimulator cells, A-7 was found to proliferate in response to DR3-bearing cells, whereas A-57 responds to DR2-positive stimulators. Both TCL are capable of providing MHC-restricted polyclonal help for allogeneic B cells, as measured in the reverse hemolytic plaque assay. Of greater interest, x-irradiated A-7 and A-57 cells are capable of inducing a proliferative response by allogeneic B cells that is absolutely MHC restricted at the inductive (Th-APC) level. Thus, x-irradiated A-7 cells only trigger proliferation by DR3+ B cells, whereas A-57 cells selectively activate DR2+ B cells. In contrast, after antigen-specific activation, x-irradiated A-7 and A-57 cells can recruit a significant proliferative response by allogeneic B cells bearing "irrelevant" DR antigens. The possibility that Th-induced B cell proliferation may be restricted at the effector (Th-B cell) level was addressed by fractionating B cell populations into "activated" and "resting" subsets by discontinuous Percoll density gradient centrifugation and further purification by employing a monoclonal antibody directed against an antigen expressed on activated B cells (4F2). These studies demonstrate that activated B cells are readily and nonspecifically recruited to proliferate by activated Th cells, whereas optimal proliferative responses by resting B cells require MHC restricted Th-B cell interaction.     

Proliferation of human peripheral blood lymphocytes induced by recombinant human interleukin 2: contribution of large granular lymphocytes and T lymphocytes

Recombinant human interleukin 2 (rH IL-2) in the presence or absence of additional stimuli, was found to be able to induce and support the proliferation of human peripheral blood lymphocytes (PBLs). These proliferative effects were observed at low doses (less than or equal to 10 U/ml) of interleukin 2 (IL-2) only when additional signals (antigen, mitogen) were provided. However, higher doses (greater than or equal to 100 U/ml) of rH IL-2 significantly stimulated the proliferation of PBL even in the absence of exogenous lectin, antigen, or allogeneic serum. The subpopulation of lymphocytes most responsive to these higher doses of rH IL-2 was the large granular lymphocyte (LGL), the morphologic homologue of natural killer activity. After the separation of human PBLs on discontinuous Percoll gradients, cells from fraction 2 (greater than 90% LGLs) responded in a dose-dependent manner to rH IL-2 alone, whereas cells from fraction 6 (greater than 90% T cells) were only slightly responsive to rH IL-2 alone. A portion of the proliferation of cells from fraction 2 was dependent on the expression of the TAC receptor, because the prior removal of TAC-positive cells significantly reduced IL-2-induced lymphocyte proliferation. These results demonstrate that human LGL that have not been exogenously stimulated can proliferate in direct response to IL-2, and suggest that LGL are the major cellular phenotype in the proliferative response that has been observed clinically.     

Human recombinant IL-4 induces activated B lymphocytes to produce IgG and IgM

In this report, we describe a novel biologic activity of IL-4 namely, its ability to induce activated human B cells to produce IgM. Staphylococcus aureus Cowan I-activated blasts prepared from high density tonsil B cells were found to secrete IgG and IgM, but no IgE, when cultured in the presence of rIL-4. The differentiating activity of rIL-4 was totally blocked by a neutralizing anti-IL-4 antiserum, therefore demonstrating that the IgG/IgM-inducing activity of rIL-4 was an intrinsic property of IL-4. rIL-4 was only minimally inducing Ig production of blasts prepared from low density B cells, whereas it induced B cell blasts prepared from high density B cells to secrete a high amount of Ig. Delayed additions of the neutralizing anti-IL-4 antiserum demonstrated that a 48-h contact between IL-4 and B cell blasts was required for optimal Ig production. The IL-4-mediated IgG and IgM production was neither suppressed by IFN-gamma nor by anti-CD23 mAb 25, whereas these agents have been shown earlier to inhibit IgE production of enriched B cells cultured in the presence of IL-4. These data indicate that the IgG/IgM-inducing activity of IL-4 is not regulated like the IL-4-induced IgE production by enriched B cells.     

Enhancement of human B cell proliferation and differentiation by tumor necrosis factor-alpha and interleukin 1

The role of tumor necrosis factor-alpha (TNF-alpha) in human B cell responses was examined and compared with that of interleukin (IL) 1 by assessing the ability of each cytokine to support proliferation and differentiation. Recombinant TNF-alpha (rTNF-alpha) and recombinant IL-1 (rIL-1) each enhanced the generation of immunoglobulin-secreting cells (ISC) in cultures of pokeweed mitogen-stimulated B cells incubated with T cells. To examine the direct effect of rTNF-alpha and rIL-1 on the responding B cell, highly purified peripheral blood B cells were stimulated with Cowan I Staphylococcus aureus (SA). In the absence of T cell factors, proliferation was minimal and there was no generation of ISC. Recombinant IL-2 (rIL-2) supported both responses. Although rTNF-alpha alone did not support SA-stimulated generation of ISC, it did increase SA-stimulated B cell DNA synthesis by two- to eightfold. In addition, rTNF-alpha augmented B cell proliferation in rIL-2 supported SA-stimulated cultures. Moreover, rTNF-alpha enhanced the generation of ISC stimulated by rIL-2 alone or rIL-2 and SA. rIL-1 also augmented DNA synthesis and generation of ISC by B cells stimulated with SA and rIL-2. However, rTNF-alpha enhanced proliferation and ISC generation in SA + rIL-2-stimulated cultures even when they were supplemented with saturating concentrations of rIL-1. Utilizing a two-stage culture system, it was found that the major effect of rTNF-alpha was to enhance responsiveness of SA-activated B cells to rIL-2, whereas it exerted only a minimal effect during initial stimulation. These results indicate that TNF-alpha as well as IL-1 augment B cell responsiveness. Moreover, the ability of rTNF-alpha to enhance B cell responsiveness was not an indirect effect resulting from the induction of Il-1 production by contaminating monocytes, but rather resulted from the delivery of a signal by rTNF-alpha directly to the responding B cell that promoted both proliferation and differentiation after initial activation. The data therefore indicate that human B cell responsiveness can be independently regulated by the action of two separate monocyte-derived cytokines.     

Functional properties of human germinal center B cells.

Germinal centers (GCs) are histologically defined areas where B cells undergo extensive proliferation and maturation, or die of apoptosis. GC B cells isolated from human tonsils can be phenotypically identified by expression of peanut agglutinin (PNA)-binding sites and can be further divided into subpopulations based on their expression of CD77. To assess the functional potential of GC B cells, we studied CD77+ PNA+ B cells isolated from tonsils by examining their differentiation status and their ability to proliferate in vitro to various cytokines and costimulants. We found that CD77+ GC B cells are less differentiated than CD77- GC B cells; GC B cells less frequently express cytoplasmic IgG and IgM, and spontaneously secrete less Ig compared to CD77- GC B cells. To identify conditions capable of inducing GC B cell proliferation, we examined IL-4, IL-2, IFN-gamma, low molecular weight BCGF (LMW-BCGF), and an MLR supernatant along with costimulants such as anti-IgM antibody, Staphylococcus aureus Cowan I (SAC), PMA, and pokeweed mitogen (PWM). While non-GC B cells proliferate strongly in response to these stimuli, GC B cells did not proliferate. However, CD77+ as well as CD77- GC B cells mounted a rapid and strong proliferative response upon stimulation with IL-4, but only in the presence of anti-CD40 antibody. Moreover, although nine additional cytokines were examined, only IL-4 was capable of supporting CD77+ GC B cell proliferation in the presence of anti-CD40 antibody. When cells were stimulated with IL-4 and anti-CD40 antibody, we also found that IFN-gamma consistently decreased the proliferative response of CD77+ GC B cells without affecting the response of non-GC B cells. Taken together, these data indicate that GC B cells have characteristic growth requirements and that IL-4 may be important for GC B cell growth in vivo.     

Inhibitory influence of IL-4 on human B cell responsiveness

The role of IL-4 in human B cell activation, proliferation, and differentiation was examined. rIL-2, but not rIL-4, was able to promote maximum proliferation and generation of Ig-secreting cells in cultures of highly purified B cells stimulated with Cowan I Staphylococcus aureus (SA). Addition of rIL-4 to rIL-2-supported cultures of SA-stimulated peripheral blood, spleen, or lymph node B cells dramatically suppressed both proliferation and differentiation. Results from experiments in which rIL-4 was added to culture at progressively later times indicated a requirement for rIL-4 to be present during the first 2 days of a 5-day incubation to cause inhibition of responsiveness. When a two-stage culture system was utilized, rIL-4 was found to support proliferation or differentiation of B cells initially activated with SA for 2 days only minimally. However, rIL-4 did not inhibit responses of SA preactivated B cells supported by IL-2. The presence of rIL-4 during the initial 48-h activation of B cells with SA and rIL-2 resulted in a profound inhibition of the ability of the activated B cells to respond subsequently to rIL-2 or lymphokine-rich T cell supernatants. A similar 48-h incubation with rIL-4 alone without SA had no effect on subsequent B cell responsiveness. The presence of rIFN-gamma during B cell activation decreased the inhibitory effect of IL-4. Other cytokines including IFN-alpha, IL-1, and commercially available low m.w. B cell growth factor also diminished the inhibitory effect of IL-4. These results indicate that IL-4 inhibits the capacity of human B cells to be activated maximally by SA and rIL-2 and therefore suggest a new immunomodulatory role for this cytokine.     

Both B151-T cell replacing factor 1 and IL-5 regulate Ig secretion and IL-2 receptor expression on a cloned B lymphoma line

In the present study, we have demonstrated that both B151-T cell-replacing factor 1 and rIL-5 are responsible for the activity to partially induce CL-3 cells into IgM-synthesizing cells and also to synergize with IL-2 to augment IL-2R expression on and IgM synthesis in CL-3 cells. These actions of rIL-5 on a homogeneous cloned line (BCL1-CL-3 cells) allow us to identify and characterize the two alternated B cell developmental pathways. One is an IL-2-independent, IL-5-driven differentiation pathway without preceding up-regulated IL-2R expression, and the other is an IL-5 plus IL-2-dependent augmented differentiation pathway with preceding up-regulated IL-2R expression. We have also demonstrated the functional difference of two distinct B cell growth-promoting factors, B cell-stimulating factor 1 (rIL-4) and rIL-5. CL-3 cells are equally stimulated to grow by rIL-4 and rIL-5, whereas only rIL-5 can render CL-3 cells responsive to rIL-2, indicating that these two lymphokines affect B cells in a strikingly different manner.     

IFN-gamma-mediated inhibition of human IgE synthesis by IL-21 is associated with a polymorphism in the IL-21R gene

IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit Ag-induced IgE production by mouse B cells. In the present study, we show that human rIL-21 strongly enhances IgE production by both CD19+ CD27- naive, and CD19+ CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4 and that it promotes the proliferative responses of these cells. However, rIL-21 does not significantly affect anti-CD40 mAb and rIL-4-induced Cepsilon promoter activation in a gene reporter assay, nor germline Cepsilon mRNA expression in purified human spleen or peripheral blood B cells. In contrast, rIL-21 inhibits rIL-4-induced IgE production in cultures of PBMC or total splenocytes by an IFN-gamma-dependent mechanism. The presence of a polymorphism (T-83C), in donors heterozygous for this mutation was found to be associated not only with lower rIL-21-induced IFN-gamma production levels, but also with a lower sensitivity to the inhibitory effects of IL-21 on the production of IgE, compared with those in donors expressing the wild-type IL-21R. Taken together, these results show that IL-21 differentially regulates IL-4-induced human IgE production, via its growth- and differentiation-promoting capacities on isotype-, including IgE-, committed B cells, as well as via its ability to induce IFN-gamma production, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.     

Human trophoblast and JEG choriocarcinoma cells are sensitive to lysis by IL-2-stimulated decidual NK cells.

Freshly isolated decidual large granular lymphocytes (LGL) show natural killer (NK) activity against K562 cells but not against normal human trophoblast. We now show that these decidual LGL proliferate in vitro in response to recombinant interleukin-2 (rIL-2) and that these rIL-2-stimulated cells acquire a broad cytolytic potential that is characteristic of lymphokine-activated killer (LAK) cells. Both fetal fibroblasts and JEG-3 choriocarcinoma cells are resistant to lysis by freshly isolated decidual effectors but are readily killed by IL-2-stimulated decidual LGL. The ability to kill these target cells is acquired after only 18 hr exposure to rIL-2. rIL-2-activated decidual LGL also kill cultured normal trophoblast cells but much lower levels of cytolysis were seen even after the effectors had been stimulated with rIL-2 for 4-6 days. The preferential killing of malignant over normal human trophoblast cells raises questions about the potential role of IL-2-activated decidual LGL in the control of unduly invasive or malignant trophoblast populations in vivo.     

IL-6/BSF-2 functions as a killer helper factor in the in vitro induction of cytotoxic T cells

rIL-6/B cell stimulatory factor 2 was found to augment CTL generation from mature as well as immature human T cells stimulated with UV-treated allogeneic cells. rIL-6 also acted on peanut agglutinin-positive murine thymocytes and Lyt-2-positive splenic T cells to give rise to CTL. rIL-6 alone could not induce CTL generation, the presence of IL-2 during the early phase of culture period was found to be essential for the IL-6 activity in the induction of CTL. The effect of rIL-6 was not mediated by the induction of IL-2 inasmuch as rIL-6 did not augment IL-2 production in MLC and anti-IL-2 antibody could not neutralize IL-6 activity. rIL-6 augmented CTL generation even when added 72 h after the initiation of cultures. The enhancing activity of rIL-6 could be neutralized with anti-IL-6 antibodies even when added 72 h after the initiation of cultures. The present data indicate that IL-6 acts in the late phase of CTL generation.     

Demonstration that human B cells respond differently to interleukin 2 and B cell differentiation factor based on their stages of maturation

The mechanisms whereby interleukin 2 (IL 2), interferon-gamma (IFN-gamma), and B cell differentiation factor (BCDF) alone or in combination modulate human B cell differentiation are currently under intensive study. To dissect out the effects of individual lymphokines contained in mixed lymphocyte reaction-culture supernatants (MLR-CS) on B cell differentiation, we employed pure factors that possessed the same activity as factors contained in MLR-CS (IL 2: 50 U/ml, IFN-gamma: 7 U/ml, BCDF-Nal: 5 pM/ml, BCDF-YA2: 12.5% v/v) singly and in combination to human B cells. By activating purified human B cells with Staphylococcus aureus Cowan I (SAC) for 3 days, separating B blast cells by the Percoll centrifugation method, and then either using these B blast cells as B cells in the earlier stage after SAC-activation, or further culturing these B blast cells for 4 more days without any stimuli and using these B cells as B cells in the later stage after SAC-activation, we could define two different populations of cells. Disparity in the populations could be demonstrated by the observation that B cells in the earlier stage were 81.2% Tac-antigen+, 23.2% B2+, 68.9% transferrin receptor+, and 90.5% HLR-DR+, whereas B cells in the later stage were observed to be less positive for each surface antigen: 36.1% Tac-Ag+, 8.3% B2+, 45.3% transferrin receptor+, and 58.7% HLR-DR+. By adding each factor to both B cell fractions, we also demonstrated functional differences in the two populations. B cells in the earlier stage of activation only differentiated in response to IL 2 or IL 2 + IFN-gamma but not to BCDF, which was in contrast to B cells in the later stage that did not differentiate in response to IL 2 but did differentiate to BCDF. However, B cells in both stages proliferated in response to IL 2 but not to BCDF. Finally, we separated B cells in the later stage into two populations by the Percoll discontinuous gradient centrifugation. Lower density (larger) B cells were observed to proliferate but not to differentiate in response to IL 2, whereas higher density (smaller) B cells were observed to differentiate in response to BCDF. Therefore, we conclude that activated B cells initially become large and gain Tac-Ag and differentiate in response to IL 2 alone as well as the combination of IL 2 and IFN-gamma, whereas later in the more mature stage they become smaller again and differentiate into Ig-secreting cells only in response to BCDF.     

Interleukin 2 induces proliferation of normal "resting" human T cells in the absence of other known external stimulation

Interleukin 2(IL-2) is known to stimulate the progression of activated T cells from G1 through the rest of the cell cycle. We have demonstrated that addition of purified recombinant human IL-2 (rIL-2) to fresh normal human peripheral blood mononuclear cells (PBM), which were IL-2 receptor (Tac) negative by FACS analysis, stimulated marked proliferation of the PBM. IL-2-induced proliferation was also observed with umbilical cord blood mononuclear cells. Monocyte depletion of PBM resulted in a marked reduction of rIL-2-induced proliferative response which could be restored by adding back autologous irradiated monocytes but not by interleukin 1. The T cells preincubated with rIL-2 showed a five to six times enhanced autologous mixed-lymphocyte reaction (AMLR) compared to controls. The rIL-2-induced proliferative response of PBM was inhibited in a concentration-dependent fashion by preincubation of PBM with an anti-HLA-DR framework monoclonal antibody. The proliferating cells were shown by two-color flow cytometric analysis to be primarily Leu-1+ and Leu-4+ T cells (both leu-3+ and Leu-2+ subsets); however, 6 to 19% of responding cells had surface markers for B cells or NK cells. The data demonstrate that rIL-2 can induce proliferation of "resting" human T cells. The phenomenon may be related to a monocyte-dependent AMLR which induces IL-2 receptors and IL-2 responsiveness in a subset of T cells.     

Hyperreactivity of activated B cells to B cell growth factor in patients with systemic lupus erythematosus

Inasmuch as B cell function is in large part determined by lymphokine-derived accessory signals, we studied the effects of recombinant IL-2 and low-molecular-weight B cell growth factor (BCGF) on peripheral blood B cells activated with Staphylococcus aureus Cowan I to explain the B cell hyperfunction in patients with SLE. When S. aureus Cowan I-activated normal B cells were separated into Tac-antigen (Tac-Ag)+ and Tac-Ag- cells by employing a rosette technique, IL-2 induced only the Tac-Ag+ cells to proliferate, whereas both the Tac-Ag+ and Tac-Ag- cells responded to BCGF. The Tac-Ag+ and Tac-Ag- fractions of activated SLE B cells behaved like respective fractions of activated normal B cells for the pattern of response to these growth factors. It should be pointed out, however, that although the Tac-Ag+ B cells of SLE patients and those of normal controls responded to IL-2 to almost the same degree, both the Tac-Ag+ and Tac-Ag- B cells of SLE patients exhibited markedly enhanced proliferative responses to BCGF. The selectively enhanced responsiveness of a broader range of activated SLE B cells may lead to B cell hyperactivity in this disease.