Excessive production of interleukin 6/B cell stimulatory factor-2 in rheumatoid arthritis

High levels of interleukin 6 (IL 6/B cell stimulatory factor-2) were detected in synovial fluids from the joints of patients with active rheumatoid arthritis (RA). The cells found in freshly isolated synovial fluid constitutively expressed IL 6 mRNA. The synovial tissues obtained by joint biopsy were also found to produce IL 6 in vitro. Immunohistochemical analysis demonstrated that CD2+ T cells as well as CD20+ blastoid B cells in the synovial tissues produce IL 6. The data indicate that IL 6 is generated constitutively in RA and its overproduction may explain the local as well as the generalized symptoms of RA, since IL 6 can function as B cell growth and differentiation factor as well as hepatocyte-stimulating factor.     

八株小鼠胸腺基质细胞系产生白细胞介素1及白细胞介素6的水平

应用IL-1诱导LBRM33-IA5细胞产生IL-2、及支持IL-6依赖细胞系KD-83细胞的增殖,分别测定了本室所建8株小鼠胸腺基质细胞系自发分泌IL-1及IL-6的能力.8株MTSC 分泌IL-1水平不同,>100U/ml者,1株;30～40U/ml 者,2株.8株细胞均能产生IL-6,其中7株的分泌水平>80U/ml;1株为38U/ml.比较各株分泌IL-1及IL-6的水平,本文显示,在MTSC 各系中,尚存在IL-1非依赖性IL-6分泌途径.本文尚比较了两种测定IL-1活性的方法,支持胸腺细胞增殖法测得者,实为IL-1+IL-6的效应,不能反映IL-1的实际水平.     

Establishment of an interleukin 6 (IL 6)/B cell stimulatory factor 2-dependent cell line and preparation of anti-IL 6 monoclonal antibodies

Murine hybridomas producing monoclonal antibodies (mAb) specific to human interleukin 6 (IL 6/BSF-2) were established. One of these hybridomas (MH60.BSF2) was found to be dependent on IL 6 for its in vitro growth. None of the known biological factors tested, such as recombinant (r) human (Hu) IL 1 alpha, rHuIL 1 beta, rHuIL 2, rHuIL 3, rHuIL 4, rHu interferon (IFN)-gamma, HuIFN-beta, rHuG-CSF, or recombinant murine (Mu) IL 3, MuIL 4, rMuIL 5, could induce the in vitro growth of MH60.BSF2 cells. The half-maximum tritiated thymidine uptake by MH60.BSF2 cells could be achieved by picogram amounts of rIL 6, making this hybridoma clone an indicator cell for specific and sensitive detection of the IL 6 activity in test samples. The MH166.BSF2 clone was found to produce IgG1,chi type mAb (alpha BSF2-166) capable of neutralizing IL 6 activity. The other clone, MH60.BSF2, produced IgM,chi type mAb (alpha BSF2-60) unable to neutralize IL 6 activity. Both mAb specifically reacted with IL 6 as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis. An enzyme-linked immunosorbent assay (ELISA) utilizing alpha BSF2-166 and rabbit anti-IL 6 antibodies was established which could detect as low as 50 pg/ml of IL 6. Since both the ELISA and MH60.BSF2 hybridoma could detect small amounts of IL 6 in biological fluids, they constitute powerful tools in exploring the presence or the role of IL 6 in various immunological disorders.     

Constitutive interleukin 2 production by the JURKAT human leukemic T cell line

Interleukin 2 (IL2) is a lymphokine produced from phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells and characterized biologically by its ability to maintain the in vitro proliferation of activated T cells. In a search for a convenient alternative source of biologically active human IL2, cells from the five established T cell lines, MOLT4, HSB2, CCRF-CEM, RPMI1301 and JURKAT were cultured at high concentrations for 18-36 h (induction cultures), and their cell-free supernatants thereafter screened on IL 2-dependent cultured human and mouse T cells. MOLT4, HSB2, RPMI1301, and CCRF-CEM all failed to produce detectable levels of IL2. Of the three JURKAT cell lines obtained from different sources, one, designated JMN, produced high levels of IL2 activity. A second, JM, failed to produce any IL2, while the third, JHAN, produced intermediate levels. Stimulation of the IL2-producing JMN or JHAN variants with PHA, the phorbol diester 12-0-tetradecanoyl-phorbol-13-acetate (TPA), or both PHA and TPA together, resulted in an apparent increase of IL2 activity in the culture supernatant when assayed by a short-term tritiated thymidine incorporation test. However, both PHA and TPA added directly to the test cells caused substantial thymidine incorporation. Moreover, the nonproducer line JURKAT-JM could not be converted to an IL2 producer by stimulation with PHA, TPA, or both. When JMN supernatants were used to support actual long-term growth and cloning of T cells in limiting dilution, the constitutively produced IL2 was superior to that produced after PHA and/or TPA stimulation. Addition of TPA, but not of PHA, to lectin and TPA-free JMN IL2 resulted in a decreased ability of such supernatants to support clonal T cell growth, suggesting that TPA had a growth-inhibiting effect. These results show that the continuously growing JURKAT-JMN cell line could provide a suitable source of mitogen-free human IL2.     

Recombinant human interleukin 6 (B cell stimulatory factor 2) enhances immunoglobulin secretion by single murine hapten-specific B cells in the absence of cell division

We have assessed the role of recombinant human IL-6 (r-hu-IL-6)In promotion of early actlvation, proliferation, and immunoglobulin(lg)secretion amongst single hapten-specific murine splenic B cellsin vitro it was found that r-hu-IL-6 acting alone was able toinduce early B cell activation in a proportion of B cells, asmeasured by a significant increase in cell diameter within 24h. An enhanced effect was seen in the concomitant presence ofa ‘T-Independent’ antigen. None of the B cells activatedby r-hu-IL-6 appeared to divide, as the frequencies of proliferatingclones Induced by either medium alone or antigen alone werevirtually identical whether r-hu-IL-6 was present or absent.However, assay of the culture supernatants for the presenceof Ig by ELlSA revealed that r-hu-IL-6 effected a significant2-fold increase in the frequency of B cells secreting Ig. Thus,the prlme effect of r-hu-IL-6 appears to be to recruit moreprecursor B cells into Ig secretion, rather than to promoteproliferation or to enhance the amount of Ig secreted by pre-committedbut non-cycling B cells. Delayed addition experiments showedthat r-hu-IL-6 enhanced Ig secretion late in the activationpathway. Kinetics studies demonstrated detectable Ig secretionas early as day 2, and when taken with its apparent abilityto Induce early activation, these flndings suggest that IL-6is not exclusively a late-acting Interleukin. Studies with sizefractionated hapten-spectflc B cells showed the larger B cellsto be preferentially responsive to r-hu-IL-6. The data presentedstrongly suggest that IL-6 may play a role In B cell maturationas a polyclonal stimulator of low rate Ig secretion. The bioactivityof IL-6 In this slngle cell system thus differed from that ofIL-4 and/or IL-5. These findings are discussed with relevanceto B cell activation in vivo.     

Defective production of interleukin 6 by macrophages from C3H/HeJ mice: differential stimulation of interleukin 1 and interleukin 6 induction

Resident macrophages from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, in contrast with macrophages from other mouse strains, produced neither interleukin 6 (IL-6) nor interleukin 1 (IL-1) spontaneously. The stimulation of C3H/HeJ macrophages with poly(I:C), LPS or, to a lesser extent, muramyl dipeptide (MDP), induced the production of both cytokines. High levels of intracellular IL-1 activity were produced which were not released in surrounding media unless silica was added to stimulated cells. On the other hand, IL-6 was secreted in the absence of any additional stimulus, hence also in the absence of extracellular IL-1. The kinetics of IL-1 and IL-6 production by LPS-stimulated macrophages were studied. Intracellular IL-1 produced by stimulated C3H/HeJ macrophages reached the levels produced by macrophages from C3H/HeN mice. IL-6-induced secretion, however, was about 5-fold less important than in C3H/HeN macrophages, suggesting a quantitative defect rather than an intrinsic defect in IL-6 production.     

Constitutive production of B cell differentiation factor-like activity by human T and B cell lines

A lymphokine demonstrating human B cell differentiation factor (BCDF)-like activity was isolated from immature (MOLT-4f, CCRF-CEM and CCRF-HSDB-2) and mature (HUT-78) malignant human T lymphoid cell lines and from human B lymphoblastoid cell lines (BJAB and ALL-7031-B). All the cell lines were grown long term in serum-free medium. This BCDF-like activity has a molecular mass in the range of 40-60 kDa and stimulates immunoglobulin synthesis of cell lines capable of producing IgA (GM-1056), IgG (GM-1500 and CESS) and IgM (CBL#3). It was not produced by a myeloid cell line. We were only able to identify the differentiation activity produced by the T and B cell lines by using appropriate molecular mass fractions from the serum-free medium as controls. This BCDF-like activity is different from that of the human BCDF so far described as it has a higher molecular mass and is constitutively produced by malignant T lymphoid cell lines which are human T cell leukemia virus-I negative and by B lymphoblastoid cell lines.     

High-affinity binding sites for human 26-kDa protein (interleukin 6, B cell stimulatory factor-2, human hybridoma plasmacytoma growth factor, interferon-beta 2), different from those of type I interferon (alpha, beta), on lymphoblastoid cells

The human 26-kDa glycoprotein (26K) is a cytokine produced by lymphoid as well as nonlymphoid cells. So far it is active as (a) a potent hybridoma and plasmacytoma growth factor on mouse cells, (b) a B cell differentiating factor on human cells, and (c) (for some authors) an interferon (IFN). Internally labeled recombinant human 26K, obtained by translation of mRNA in Xenopus oocytes, was used to investigate the presence of specific receptors for this new cytokine and to analyze its binding to responsive cells. The results indicate that (a) the 26K-responsive human lymphoblastoid CESS cells express about 1500 high-affinity (Kd = 30 pM) binding sites for this cytokine, (b) this binding is not competed for by interleukin (IL)1, IL2, tumor necrosis factor (TNF), IFN-alpha 2, IFN-beta or IFN-gamma, and (c) these 26K-binding sites are different from the classical type I (alpha-beta) IFN receptors by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Human B cell proliferation is stimulated by interleukin 2

The proliferation of human B cells was studied for response to interleukin 2 (IL-2) produced in Escherichia coli using recombinant DNA technology. The IL-2 was found to be an homogenous preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the anti-IL-2 monoclonal antibody DMS-1. IL-2 was found to stimulate B cell proliferation. Activation of the B cells using anti-IgM antibodies increased this response. Resting T cells from the same donors were found to be less reactive to IL-2. The results suggest that human B cell proliferation can be stimulated by IL-2 alone.     

Implantation: Stimulation of human endometrial epithelial cell interleukin 6 production by interleukin 1 and placental protein 14

The effect of interleukin 1 (ILI) and placental protein 14 (PP14)on the production of interleukin 6 (IL6) by cultured human endometrialepithelial cells prepared from endometrial biopsy material obtainedat different stages in the menstrual cycle was investigated.Basal IL6 production by cells prepared from proliferative endometriumwas greater than that produced by cells prepared from secretoryendometrium (7.3 ± 0.3 and 1.1 ± 0.2ng/well/24h respectively, P < 0.001). IL1 (0.025–2.5 ng/ml) causeda dose-dependent increase in IL6 production by cells preparedfrom both proliferative and secretory endometrium, but cellsprepared from secretory endometrium responded to a lower concentrationof ILI than those prepared from proliferative endometrium. ILI-stimulatedIL6 production by epithelial cells prepared from secretory endometriumtypically reached 10 times basal values, while in cells preparedfrom proliferative endometrium stimulated levels were approximatelytwice the basal values. PP14 (1–50 µg/ml) also causeda dose-dependent increase in IL6 production by epithelial cellsprepared from secretory endometrium, but had no effect on IL6production by cells prepared from proliferative endometrium.Even in secretory cells PP14 was less effective than IL1 atstimulating IL6 production, with stimulated levels only reachingtwice the basal values. This suggests that PP14 and IL1 actvia different mechanisms in the stimulation of IL6 production.The results show that IL6 production by human endometrial epithelialcells is stimulated by other immunomodulatory peptides and thismay be part of the network of such peptides in the endometriumwhich may influence embryo implantation.     

Soluble Mouse B7-H3 Down-Regulates Dendritic Cell Stimulatory Capacity to Allogenic T Cell Proliferation and Production of IL-2 and IFN-γ

B7-H3 is a recently identified member of the B7 gene family. Its ubiquitous expression in both lymphoid and nonlymphoid tissues suggests that it could play an important role in the maintenance of self-tolerance. However, the exact function of B7-H3 is still elusive. The purpose of current study is to demonstrate the possible function of soluble mouse B7-H3 for prevention of DC-mediated T cell activation. For this purpose, we established a soluble mouse B7-H3 fusion protein （mB7h3-hIg） eukaryotic expression vector （pmB7h3-hIg） with a C-terminal human IgG1 Fc. A C57BL/6 （B6）-derived dendritic cell line （DC2.4 cells） was used for the establishment of stable transfectants for generation of soluble mB7h3-hIg. Ectopic mB7h3-hIg expression was confirmed by RT-PCR, Western blot and ELISA analyses. A 49.7 kD protein was detected by Western blot from DC2.4 cells transfected with pmB7h3-hlg. It was found that soluble mB7h3-hIg expression has no effect on cell cycling and apoptosis and the expression of CD80 and CD86 of the DC2.4 cells. However, ectopic soluble mB7h3-hIg expression was found to significantly affect the allo-stimulatory capability for DC2.4 cells. DC2.4 cells expressing soluble mB7h3-hIg showed a significant reduced allo-stimulatory capability as compared with the controls determined by MLC. Further studies revealed that soluble mB7h3-hIg could also inhibit IL-2 and IFN-γ, production of allogenic T cells. These results suggested a great potential of soluble B7-H3 for treatment of graft rejection and autoimmume disease. Cellular ＆ Molecular Immunology. 2006;3（3）：235-240.     

Absence of antiviral activity in recombinant B cell stimulatory factor 2 (BSF-2)

Recombinant B cell stimulatory factor 2 (rBSF-2) did not display any detectable level of antiviral activity when using human diploid fibroblasts, DIP-2, FS-4, FS-7, amnion-derived WISH and FL cells with vesicular stomatitis virus (VSV) and Sindbis virus as challenging agents (less than 2.5 X 10(2) IU/mg). Furthermore anti-IFN-beta could not neutralize the immunoglobulin-inducing activity of BSF-2. Moreover anti-BSF-2 could not neutralize the antiviral activity of IFN-beta. The data indicate that BSF-2 is functionally and immunologically not related to IFNs.     

Suppression of interleukin 4 production from type 2 helper T cell clone by antisense oligodeoxynucleotide.

Type 2 helper T cell (Th2) clone has been reported to secrete interleukin (IL) 4 and IL5 in response to the specific antigen presented by syngeneic antigen-presenting cells. In the present report, we synthesized phosphorothioate analogue of an antisense oligodeoxynucleotide complementary to nucleotide 17-36 of IL4 mRNA (S-oligo), and tested its ability to inhibit IL4 production from a Th2 clone, D10.G4.1. (D10). D10 cells were cultured with mitomycin C-treated C3H spleen cells in the presence of 100 micrograms/ml conalbumin for 48-72 h. Secreted IL4 and IL5 were assayed biologically using HT2 cells and dextran sulfate-stimulated murine B cells, respectively. When 5-10 micrograms/ml S-oligo was added to the culture, IL4 production from D10 was suppressed by 70-90%. The same concentrations of S-oligo inhibited neither the antigen-induced proliferation of D10 nor the secretion of IL5 from the Th2 clone. These results suggest that this S-oligo is useful for inhibiting the production of IL4 preferentially without affecting other functions of Th2 cells.     

B cell growth and differentiation factors interact with receptors distinct from the interleukin 2 receptor

Human B lymphocytes preactivated with Staphylococcus aureus Cowan strain I can proliferate and differentiate to Ig-secreting cells when cultured in the presence of recombinant interleukin 2 (IL2). We have compared 2 different B cell growth factors (BCGF) and a B cell differentiation factor (BCDF) to IL2 in the regulation of human B lymphocyte growth and differentiation. Utilizing a competitive binding assay, neither a high molecular weight BCGF (HMW-BCGF) nor a low molecular weight BCGF (LMW-BCGF) competitively inhibited the binding of radiolabeled IL2. Blocking studies with the anti-Tac monoclonal antibody demonstrated that B cell proliferation to IL2 was inhibited while a crude supernatant containing BCGF and IL2 was only partially inhibited. B cell Ig secretion induced by IL2 was also inhibited by anti-Tac while a crude supernatant and partially purified BCDF were not. Furthermore, IL2 plus BCGF was shown to enhance B cell proliferation better than either factor alone and IL2 plus a BCDF enhanced B cell Ig secretion better than either factor alone. By separating activated B cells into Tac-positive and Tac-negative fractions, much of the previously noted enhancement with the 2 factors was found to be secondary to the differential sensitivity of the 2 populations to BCGF and IL2 or BCDF and IL2. Thus, LMW-BCGF, HMW-BCGF and a partially purified BCDF appear to interact with receptors distinct from the IL2 receptor in mediating their effects on B cell growth and differentiation.     

Heterogeneity of B cell responsiveness to interleukin 4, interleukin 6 and low molecular weight B cell growth factor in discrete stages of B cell activation in patients with systemic lupus erythematosus.

To investigate the differential stages of B cell activation in patients with systemic lupus erythematosus (SLE), the effects of recombinant interleukin-4 (rIL-4) interleukin-6 (rIL-6), and low mol. wt BCGF (1-BCGF) on B cell proliferation and differentiation were evaluated. In a co-stimulatory assay with anti-IgM, proliferative response to rIL-4 and 1-BCGF showed no significant differences between SLE patients and healthy controls. In a restimulation assay, after pre-activation with anti-IgM for 3 days, proliferative response to rIL-4 and 1-BCGF was significantly decreased in SLE patients compared with normal healthy controls (P less than 0.01). With regard to rIL-6 induced B cell differentiation, SAC-pre-activated low density, large B cells from both SLE patients and healthy controls differentiated well, whereas high density B cells did not differentiate at all. Of particular interest, low density B cells from active patients directly differentiated into Ig secreting cells in response to rIL-6 without SAC activation. These data indicate that heterogeneity of B cell responsiveness to B cell-tropic interleukins resulted from the discrete stages of B cell activation in SLE.     

Autoreactive T cell hybridoma-derived B cell stimulatory factor(s) governing IgA isotype immunoglobulin production by murine Peyer's patch B cells

In the present study we investigated whether autoreactive T cells derived from murine Peyer's patches (PP) have the capacity to regulate mucosal B cell differentiation to IgA-producing plasma cells in vitro. We also examined whether B cell development is mediated by lymphokines from immunoregulatory T cells - that is, B cell stimulatory factors (BSF) and cofactors (coBSF) - which include B cell growth factor (BCGF), putative alpha B cell immunoglobulin (Ig) heavy chain switch factor (BSWF alpha), and B cell differentiation factor (BCDF), as well as interleukin-2 (IL-2). To this purpose we developed in vitro a variety of BSF (especially putative BSWF alpha)-producing autoreactive (self-class II molecules responsive) T cell hybridoma cell clones from murine PP, and studied the functional activity of the BSF, especially a putative alpha Ig heavy chain switch (mu----alpha) factors(s). These T hybridome cell lines possessed the surface phenotypes of Thy 1.2, CD4+, CD5+ and CD8- and produced a variety of BSF, including two kinds of BCGF (IL-5, and a BCGF that did not require additional costimulators to induce proliferation of preactivated B cells), putative BSWF alpha/gamma, BCDF, and/or IL-2. The results strongly support the view that the autologous T cell plays an important role not only in B cell proliferation and terminal maturation, but also in alpha heavy chain switching in PP. This T-B cell interation is mediated at least in part through BSF lymphokines elaborated by the autoreactive T cell, probably activated in situ in the lymphoid tissue microenvironment.