Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.     

Demonstration of neutralizing autoantibodies against IL-1 alpha in sera from patients with rheumatoid arthritis

We have recently reported the presence of IgG which has a potent inhibitory activity against IL-1 alpha in some sera from patients with rheumatoid arthritis. The mechanism of this inhibition by IgG against IL-1 alpha is now elucidated. IgG with IL-1 alpha-inhibitory activity inhibited the binding of 125I-IL-1 alpha to receptors on rheumatoid synovial cells. In addition, preincubation of synovial cells with the inhibitory IgG did not block the binding of 125I-IL-1 alpha to receptors, suggesting a direct interaction between IgG and IL-1 alpha. To examine which region of the IgG, namely Fab or Fc region, has the inhibitory activity, the IgG was digested with papain, and Fab and Fc fragments were purified. Fab fragments, but not Fc fragments, inhibited both IL-1 alpha-induced thymocyte-proliferation and the binding of 125I-IL-1 alpha to receptors. We further demonstrated that the inhibitory IgG which was bound to protein A Sepharose could bind a significant amount of 125I-IL-1 alpha, whereas only a negligible binding of the radiolabeled ligand was detected when IgG without the inhibitory activity was used as control. Moreover, the binding of 125I-IL-1 alpha to IgG with the inhibitory activity was clearly blocked by Fab fragments of IgG having the inhibitory activity. Finally, affinity-purified IgG over an IL-alpha affinity column showed approximately 100-fold more potent inhibitory activity on IL-1 alpha-induced thymocyte proliferation compared with untreated IgG. From these results, we conclude that IgG molecules with IL-1-alpha-inhibitory activity are neutralizing autoantibodies against IL-1 alpha.     

Purification and characterization of recombinant human interleukin 4. Biological activities, receptor binding and the generation of monoclonal antibodies.

A synthetic gene coding for human interleukin 4 (IL-4) was cloned and expressed in Saccharomyces cerevisiae (baker's yeast) as a C-terminal fusion protein with the yeast prepro alpha-mating factor sequence, resulting in secretion of mature IL-4 into the culture medium (0.6-0.8 micrograms/ml). A protocol was developed for purification of this protein. Crude cell-free conditioned medium was passed over a concanavalin A-Sepharose affinity column; bound proteins were eluted and further purified by S-Sepharose Fast Flow cation exchange and C18 reverse-phase h.p.l.c. Highly purified IL-4 was obtained by this method (0.3-0.4 mg per litre of culture) with a recovery of 51%. Thermospray liquid chromatography-mass spectrometry showed the C-terminal N-glycosylation site to be largely unmodified, and also showed that the N-terminus of the purified recombinant IL-4 (rIL-4) was authentic. Thiol titration revealed no free cysteine residues, implying that there are three disulphide groups, the positions of which remain to be determined. We have characterized the biological activities of the purified rIL-4. This material is active in B-cell co-stimulator assays, T-cell proliferation assays and in the induction of cell-surface expression of CD23 (the low-affinity receptor for IgE) on tonsillar B-cells. Half-maximal biological activity of the rIL-4 was achieved at a concentration of 120 pM. We have radioiodinated rIL-4 without loss of biological activity and performed equilibrium binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding site with high affinity (Kd = 100 pM) and revealed 1100 receptors per cell. Receptor-ligand cross-linking studies demonstrated a single cell-surface receptor with an apparent molecular mass of 124 kDa. Two monoclonal antibodies have been raised to the human rIL-4, one of which blocks both the biological activity of rIL-4 and binding to its receptor.     

Use of anti-idiotypic antibodies as probes for the interaction of TSH subunits with its receptor

TSH is a heterodimeric glycoprotein hormone, whose dissociated subunits are without biological activity. This has precluded the assessment of the relative contribution of each subunit to hormone action. We have raised anti-idiotypes to monoclonal antibodies specific, respectively, for the alpha and beta hTSH subunits. The anti-beta anti-idiotype inhibited 125I-hTSH binding to the beta subunit-specific monoclonal quantitatively, whereas 125I-hTSH binding to the alpha subunit-specific monoclonal was not inhibited by anti-alpha anti-idiotypes, suggesting that only the former is an "internal image" anti-idiotype. Neither of the two anti-idiotypes nor equimolar mixtures thereof inhibited 125I-bTSH binding to thyroid membranes, even though radiolabelled anti-idiotypes showed saturable binding to thyroid plasma membrane which was inhibited 41-65% by bTSH. Each anti-idiotype alone caused 9% inhibition (compared to 50% by NRIgG) of thyroid plasma membrane adenylate cyclase. Equimolar mixtures (125 micrograms/ml IgG of each anti-idiotype) induced enzyme activity equivalent to 85% of that of 250 mU/ml of TSH. The TSH-like action of the two anti-idiotypes was also reflected in their capacity to increase (450% by 250 micrograms/ml IgG compared to normal rabbit IgG) the uptake of 131I into isolated thyrocytes and to promote the organization of such cells into follicular structures. At 250 micrograms/ml, anti-beta anti-idiotype promoted the organization of small follicles and only at a concentration of 500 micrograms/ml did it enhance 131I uptake.     

Expression of IL-1 receptors on human peripheral B cells

The expression of IL-1R on human peripheral B cells was analyzed by the binding assay with 125I-labeled human rIL-1 alpha and by the flow cytofluorometry by the use of FITC-conjugated IL-1 alpha. The proliferation and the differentiation of B cells stimulated with Staphylococcus aureus Cowan I (SAC) in the presence of T cell-derived factors were dependent on IL-1. By the binding experiment with 125I-labeled IL-1 alpha, B cells expressed only few IL-1R without any stimulations. When they were stimulated with SAC, IL-1R on B cells began to increase by only 1 h, reached the maximum level at 6 h. The binding of 125I-labeled IL-1 alpha to B cells was inhibited by the addition of either cold IL-1 alpha or IL-1 beta suggesting that IL-1R on B cells reactive for IL-1 alpha and IL-1 beta were identical. By Scatchard plot analysis, the existence of two classes of IL-1R on B cells was found. A major class of IL-1R (320 molecules/cell) has a lower affinity (Kd = 3.8 x 10(-10) M) and a minor class of IL-1R (70 molecules/cell) has a higher affinity (Kd = 4.4 x 10(-12) M). When B cells were stimulated with SAC, both lower and higher affinity IL-1R were increased to 1960 molecules/cell and 300 molecules/cell, respectively. Furthermore, IL-1R on B cells were also detected with FITC-conjugated IL-1 alpha by a flow cytofluorometer. Only 3 to 5% of B cells expressed IL-1R without any stimulations. When B cells were stimulated with SAC, IL-1R-positive B cells were increased to 20%. The addition of anti-class II antibodies inhibited B cell proliferation and differentiation induced with SAC, IL-1, and T cell-derived factors. Anti-class II antibodies also inhibited the number of IL-1R on B cells. These results suggest that the expression of IL-1R was induced as the initial stage of B cell activation and that class II Ag play an important role for the expression of IL-1R on B cells.     

Interaction of recombinant monocyte-derived interleukin 1 receptor antagonist with rheumatoid synovial cells.

Interleukin 1 receptor antagonist (IL-1ra) is a newly described cytokine that is produced by human monocytes cultured on adherent immunoglobulin G (IgG). These studies have characterized the binding of IL-1ra to receptors on human rheumatoid synovial cells in comparison to binding of IL-1 alpha. The human synovial cells bound 35S-IL-1ra with a Kd of 213 pM and a Ki of 134 pM. 125I-IL-1 alpha bound to the synovial cells with similar values, showing a Kd of 205 pM and a Ki of 58 pM. Cross-inhibition studies were performed to examine whether IL-1ra and IL-1 alpha interacted with the same receptors and in an identical fashion. At the highest concentrations of inhibitory proteins, the binding of each ligand was inhibited 100% by the same or opposite ligand. This result indicated that IL-1ra and IL-1 alpha bound to the same receptors and not to overlapping subsets of receptors. In addition, the binding of 35S-IL-1ra was inhibited in an identical fashion by equimolar amounts of IL-1ra or IL-1 alpha. However, twofold or greater amounts of IL-1ra in comparison to IL-1 alpha were required to offer comparable inhibition of binding of 125I-IL-1 alpha. These results suggest that IL-1ra and IL-1 alpha bind with equal avidity to IL-1 receptors but may not bind identically. Additional experiments are necessary to establish whether these two ligands may bind to different regions of the extracellular portion of the IL-1 receptor.     

Tumor necrosis factor-alpha enhances inhibitory effects of interleukin-1 beta on Leydig cell steroidogenesis

Human recombinant tumor necrosis factor-alpha (rTNF alpha) alone (up to 1000 units/ml) did not alter either basal or human chorionic gonadotropin (hCG)-induced testosterone formation in primary culture of rat Leydig cells. However, concomitant addition of rTNF alpha with human recombinant interleukin-1 beta (rIL-1 beta) enhanced the inhibitory effects of rIL-1 beta. The rIL-1 beta dose response curve was shifted to the left (IC50 changed from 1 ng/ml to 0.3 ng/ml). Even though rTNF alpha had no effect on testosterone formation, hCG-stimulated cyclic AMP formation was inhibited by rTNF alpha in a dose dependent manner. In the presence of both rTNF alpha and rIL-1 beta, hCG-induced cyclic AMP formation and binding of [125I]-hCG to Leydig cells were further inhibited. Testicular macrophages represent about 20% of the interstitial cells. TNF alpha and IL-1 may be produced locally by interstitial macrophages and have paracrine effects on Leydig cell function.     

A synergistic interaction of IL-6 and IL-1 mediates the thymocyte-stimulating activity produced by recombinant IL-1-stimulated fibroblasts

We characterized the ability of normal human lung fibroblasts to elaborate thymocyte-stimulating activity, spontaneously, and in response to rIL-1. Supernatants from unstimulated fibroblasts did not contain thymocyte-stimulating activity, whereas supernatants from fibroblasts incubated with rIL-1 alpha or rIL-1 beta contained more thymocyte-stimulating activity than could be accounted for by passively transferred rIL-1 alone. This heightened thymocyte-stimulating activity was mediated by fibroblast-derived IL-6 inasmuch as it was neutralized by anti-serum against human rIL-6, and rIL-1-stimulated fibroblasts to accumulate messenger RNA for IL-6 and produce soluble IL-6 protein. However, IL-6 alone could not account for the intensity of this effect because rIL-6 only weakly stimulated thymocyte proliferation. In addition, antisera against the rIL-1 moiety that was used to prepare the supernatant had different effects on supernatants that contained and did not contain active IL-6. In the presence of IL-6 these antisera caused a greater decrease in thymocyte-stimulating activity than could be accounted for by passively transferred rIL-1 alone. When the IL-6 was neutralized the remaining thymocyte-stimulating activity could be quantitatively accounted for and neutralized by antisera against the rIL-1 that was passively transferred. Furthermore, rIL-6 and rIL-1 (alpha or beta) synergized in stimulating thymocyte proliferation. Thus, rIL-1 stimulates fibroblasts to produce a thymocyte-stimulating activity that is largely mediated by a synergistic interaction of fibroblast-derived IL-6 and IL-1. These findings suggest that fibroblast production of IL-6 may mediate or amplify some of the tissue effects of IL-1. In addition they suggest that biologic effects previously attributed to IL-1 may be due to IL-6 alone or the concerted action of IL-1 and IL-6.     

Comparison between autoantibodies arising during Trypanosoma cruzi infection in mice and natural autoantibodies

The autoantibodies induced in (C57BL/6 x BALB/c)F1 mice during Trypanosoma cruzi (CL strain) infection were analyzed and compared with natural autoantibodies present in healthy mice. Mice were killed at intervals after infection and their sera were tested by enzyme immunoassay against a panel of self- and non-self-Ag: actin, myoglobin, myosin, tubulin, DNA, and TNP-OVA. The level of IgM and IgG autoantibodies against all Ag started to increase from day 15 until 6 wk after the parasite infection. The high level of all autoantibodies persisted 3 mo postinfection, and 1 yr later, half of the mice still had elevated levels of IgM and IgG autoantibodies, particularly antitubulin IgG antibodies. IgM and IgG were isolated from pools of normal and infected mouse sera and their binding capacity to all Ag was compared. The titers of infected mouse sera were increased and the slopes of both IgM and IgG binding curves of autoantibodies to actin, myosin, and tubulin were greater than those of control mouse sera, indicating higher affinities. The average dissociation constant of the IgG2a autoantibody to mouse tubulin was 5 times lower than that of natural antitubulin IgG2a antibodies. Furthermore, absorption of the IgG from infected mouse sera onto a tubulin immunoadsorbent removed half the reactivity with tubulin and also with myosin, actin and parasite extracts. The eluted antibodies bound the same Ag. When IgG were further analyzed by Western blot on proteolytic fragments of tubulin, we found that antibodies from both groups bound to the same broad spectrum of polypeptide bands. However, additional fragments were recognized by antibodies from infected mice. All these results indicate that the autoantibodies naturally present in mice are significantly affected after infection with T. cruzi, in quantity as well as in specificity and affinity.     

Monoclonal antibodies against the complement control protein Factor H (β1 H)

Two mouse monoclonal antibodies against the human complement control protein, Factor H (beta 1H), are described. The antibodies are both IgG - gamma 1 - subclass and are directed against different epitopes on the human Factor H molecule. One of the antibodies, MRC OX 24, increases the cofactor activity of Factor H in Factor I-mediated cleavage of soluble C3b. The second antibody, MRC OX 23, which has no effect alone, reduces the increase in cofactor activity observed in the presence of the first antibody. However, MRC OX 24 inhibits the binding of 125I-labelled Factor H to surface-bound C3b (EAC3b). Again MRC OX 23 alone does not have an effect but decreases the inhibition in 125I-labelled Factor H binding to EAC3b observed with MRC OX 24. These studies show clearly that the interaction of Factor H with soluble C3b is different to its interaction with surface-bound C3b. In an indirect immunoprecipitation system using these monoclonal antibodies, single-chain molecules of 150 000 mol.wt. are specifically precipitated from human serum and also from the sera of other primates - rhesus monkey, cynomolgus monkey, and African green monkey. There was no precipitation from sera of cow, pig, sheep, chick, or rabbit. Using a radioimmunoassay with radiolabelled monoclonal MRC OX 23, the concentration of Factor H in human plasma was determined.     

Localization of human mononuclear cell interleukin 1

The detection and localization of interleukin (IL) 1 in human monocytes was carried out by flow cytometry using monoclonal antibodies to IL-1 alpha and IL-1 beta proteins. IL-1 alpha was detected on the surface of monocytes and the surface expression increased following lipopolysaccharide activation. No demonstrable IL-1 beta protein could be observed on the cell surface by antibody staining, while both IL-1 alpha and IL-1 beta could be visualized intracellularly by the appropriate monoclonal antibodies following acetone permeabilization of the monocytes. Further experiments with cell associated IL-1 revealed that most of the biological activity of human monocytes could be inhibited by affinity purified polyclonal antibodies to IL-1 alpha protein, whereas no inhibitory activity was observed with IL-1 beta specific antibodies. These data support the hypothesis that a differential localization of IL-1 alpha and IL-1 beta exists within human blood-derived monocytes.     

Human insulin autoantibody fine specificity and H and L chain use

Fine specificity and H and L chain isotypes of insulin autoantibodies in sera from 11 subjects were examined. None of these 11 subjects was treated with exogenous insulin. Two patterns of fine specificity were found. In one, the autoantibodies were specific for human insulin, with a requirement for threonine at B30. The conservative substitution in pork insulin (threonine to alanine) abrogated IgG binding by these sera. Insulin autoantibodies in other sera cross-reacted with beef, pork, and human insulin; not requiring threonine at B30. Reciprocal competitive inhibition experiments showed that epitopes recognized by the human specific insulin autoantibodies were exclusively on the B chain, whereas the cross-reactive sera contain autoantibodies that recognize both the B chain and combinatorial (A and B chain) epitopes. The fine specificity of cross-reactive insulin autoantibodies are thus similar to insulin antibodies from insulin-treated subjects. When IgG subclasses and L chains of insulin autoantibodies were examined, however, restricted C region usage was found. The hierarchy was IgG3 greater than G1 greater than G2 greater than G4; with one subclass dominant in each serum, although others were used. L chain use was similarly restricted. There was no correlation between isotype and fine specificity or between H and L chain type. It is concluded that heterogeneity of insulin autoantibodies is restricted. The response is probably more oligo- or pauciclonal than insulin antibody from insulin-treated subjects.     

A monoclonal antibody recognizing 68- to 75-kilodalton protein(s) associated with the human IL-1 receptor

We produced an IgM mAb termed 4.9 against an EBV-containing lymphoblastoid cell line, termed 3B6. This mAb reacted with both various B and T cell lines such as HSB2 cells, with an NK-like cell line YT-C3 cells, and with human fibroblast MCR-5 cells. It also reacted with normal resting peripheral B lymphocytes, monocytes, and anti-CD2- or anti-CD3-activated T lymphocytes. The 4.9 mAb immunoprecipitated two bands estimated to be of Mr 68 and 75 kDa from iodinated 3B6 cells. The 4.9 mAb inhibited the proliferation of peripheral T lymphocytes induced either by anti-CD3 mAb or anti-CD2 mAb. The 4.9 mAb inhibited also the proliferation of murine thymocytes both in the presence of PHA and IL-1 and the proliferation of human fibroblasts in the presence of IL-1. Radiolabeled IL-1 binding on 3B6 cells revealed two types of IL-1 binding sites with high and low affinity for IL-1 (300 sites/cell with a Kd of 6 x 10(-11)M and 6000 sites/cell with a Kd of 3 x 10(-9)M). On both 3B6 and YT-C3 cells, mAb 4.9 inhibited specifically the binding of 125I-labeled rIL-1, alpha or beta, whereas the irrelevant IgM mAb did not. Conversely, rIL-1, alpha or beta, could inhibit specifically the binding of radioiodinated 4.9 mAb to 3B6 or YT-C3 cells, whereas rIL-2, rIFN, or the irrelevant IgM mAb were ineffective. 125I-4.9 mAb bound 3B6 cells with an association constant (Ka) of 2 x 10(8)/M and demonstrated 6000 binding sites/cell. We thus conclude that mAb 4.9 recognizes a protein complex (68 to 75 kDa) closely associated with the IL-1R.     

Purification and characterization of the immunostimulatory properties of recombinant human interleukin-1 beta

In this study we have used a new method for human recombinant IL-1 beta (rIL-1 beta) purification and investigated its immunostimulatory biological activity. The IL-1 beta gene was cloned using a novel mRNA preparation from activated human blood monocytes. The purification protocol consists of extraction and two chromatographic steps using the new Soloza cation exchange resin. The purified protein was characterized electrophoretically, by amino acid analysis and reverse phase chromatography. The protein migrated on SDS-PAGE with a molecular weight of 18.200 but demonstrated the minor presence of aggregates (dimers and trimers). Specific activity of purified rIL-1 beta in comitogenic assay on mouse thymocytes was 10(8) U/mg protein. rIL-1 beta increased in a dose dependent manner proliferation of Con A-stimulated murine thymocytes, splenocytes, PHA-stimulated human peripheral blood lymphocytes and transformed B-cell lines. Comitogenic activity depended on the degree of lymphocyte preactivation and was similar to that of natural human IL-1 beta. rIL-1 beta enhanced IL-2 production by murine spleen cells and EL-4 cell line and IL-2 receptor expression by human peripheral blood mononuclear cells. It induced PGE2 release from human blood monocytes but had no effect on human neutrophil chemotaxis, phagocytosis and respiratory burst.     

IL-1 and TNF transmodulate epidermal growth factor receptors by a protein kinase C-independent mechanism

The effect of the human rIL-1 alpha and rTNF-alpha on the binding of 125I-labeled epidermal growth factor ([125I]EGF) to its receptor (EGF-R) has been studied in human gingival fibroblasts (HuGi). Incubation of these cells with recombinant cytokines at 37 degrees C caused a rapid, dose-dependent decrease in their ability to subsequently bind subsaturating levels of [125I]EGF at 4 degrees C. Inhibition was evident at 5 min after addition of cytokines, reached a maximal level (60-70% reduction) after 15 to 30 min, and declined thereafter. Normal EGF binding was attained by 2 h. Half-maximal inhibition of EGF binding occurred at 10 pM IL-1 and 50 pM TNF. The two cytokines were not additive in their effect. Competition experiments at 4 degrees C showed that the cytokines did not interact directly with EGF-R; Scatchard analysis of binding of [125I]EGF to HuGi after treatment with IL-1 and TNF revealed an increase in EGF-R Kd from 0.75 nM to 2.9 nM with no change in receptor number. The effect of IL-1 and TNF on EGF-R was compared with that of the tumor-promotor PMA which is known to "transmodulate" EGF-R affinity by activating protein kinase C which then phosphorylates EGF-R. PMA caused a greater inhibition of EGF binding to HuGi (80 to 85% inhibition; ED50 = 500 pM), and recovery of binding was much slower. Importantly, in HuGi made deficient in protein kinase C by prolonged incubation with PMA, addition of fresh PMA no longer affected EGF binding, while the response to IL-1 and TNF was intact. Cytokine- but not PMA-mediated EGF-R transmodulation was partially reversed by treatment of the cells with millimolar concentrations of the kinase inhibitor amiloride. HuGi were incubated with H3 32PO4, stimulated with PMA or cytokines, and EGF-R were immunoprecipitated; IL-1 and TNF, like PMA, caused a 2- to 5-fold increase in receptor phosphorylation. We conclude that occupation of IL-1 and TNF-R activates a protein kinase, distinct from kinase C, for which EGF-R is a substrate.     

IL-2 stimulates the production of IL-1 alpha and IL-1 beta by human peripheral blood mononuclear cells

Human PBMC were cultured in medium containing human rIL-2, and the supernatants and cell lysates were analyzed for IL-1 alpha and IL-1 beta using specific RIA. IL-2, but not the excipient detergents included in the rIL-2 preparation, induced the synthesis of both cytokines. The concentrations of IL-1 alpha and IL-1 beta in the cell lysates and supernatants depended on both the concentration of rIL-2 in the culture medium and the duration of the incubation. After 24 h of stimulation, IL-2-induced IL-1 alpha remained almost entirely cell-associated. In contrast, IL-1 beta was present in both cell lysates and supernatants and was more abundant in the latter. SDS-PAGE analysis after radioimmunoprecipitation with anti-IL-1 antibodies indicates that cell-associated IL-1 resulting from IL-2 stimulation was in the form of the 35 kDa IL-1 precursor whereas secreted IL-1 was almost entirely in the form of the mature 18 kDa product. Depletion of monocytes from the PBMC culture substantially reduced IL-2-induced IL-1 production. In addition, Leu M3+ monocytes obtained through FACS, but not CD16+ NK cells, produced both IL-1 alpha and IL-1 beta in response to IL-2. The low level of endotoxin present in the IL-2 preparation used in our studies and the selective inhibition by polymyxin B of LPS-induced, but not IL-2-induced, IL-1 production by PBMC indicate that IL-2-induced IL-1 production was not due to endotoxin contamination. Furthermore, an anti-IL-2 antiserum selectively inhibited IL-1 production in response to IL-2 stimulation. We conclude that IL-2 is a potent inducer of IL-1 synthesis and secretion in vitro and propose that IL-1 may be generated in vivo in patients undergoing IL-2 immunotherapy.     

Expression of CD5 regulates responsiveness to IL-1

The role of the CD5 surface molecule in T cell responsiveness to IL-1 was examined. A CD5-mutant Jurkat cell line was generated from a CD5+ parent cell line. This CD5- mutant subclone was infected with a defective retrovirus containing the CD5 cDNA and/or the neo gene encoding G418 resistance. The CD5+ wild type Jurkat produced IL-2 in response to anti-CD3 mAb, OKT3, cross-linked to a solid surface. IL-2 production was enhanced by co-culture with IL-1 or anti-CD5 Mab. Neither the CD5- mutant nor the CD5- G418-resistant infectant responded to anti-CD5 mAb or to IL-1. Responsiveness to IL-1 was restored by cell surface expression of CD5 in the CD5+ infectant. Both the CD5+ wild type Jurkat and the CD5+ infectant responded equivalently to purified IL-1, IL-1 alpha and rIL-1 beta. Optimal concentrations of IL-1 and anti-CD5 mAb had an additive effect on the enhancement of IL-2 production stimulated with cross-linked anti-CD3 mAb suggesting that IL-1 and CD5 act through distinct, complementary pathways to augment T cell activation. The correlation of CD5 expression and specific binding of rIL-1 beta was examined in these cell lines. Both the specific binding (at 4 degrees C) and subsequent internalization (at 37 degrees C) of 125I labeled rIL-1 beta was equivalent in the CD5+ infectant and the CD5+ wild type Jurkat cell, whereas specific binding of 125I-labeled rIL-1 beta was markedly decreased in the CD5-G418-resistant infectant. These observations strongly suggest that cell surface expression of CD5 regulates binding of and responsiveness to IL-1.     

Serum levels of autoantibodies against monomeric C-reactive protein are correlated with disease activity in systemic lupus erythematosus

This study was performed to investigate the relation between IgG autoantibodies against human C-reactive protein (anti-CRP) and disease activity measures in serial serum samples from 10 patients with systemic lupus erythematosus (SLE), of whom four had active kidney involvement during the study period. The presence of anti-CRP was analysed by enzyme-linked immunosorbent assay. The cut-off for positive anti-CRP test was set at the 95th centile of 100 healthy blood donor sera. Specificity of the anti-CRP antibody binding was evaluated by preincubating patient sera with either native or monomeric CRP. Disease activity was determined by the SLE disease activity index (SLEDAI), serum levels of CRP, anti-DNA antibodies, complement components and blood cell counts. Of 50 serum samples, 20 (40%) contained antibodies reactive with monomeric CRP, and 7 of 10 patients were positive on at least one occasion during the study. All patients with active lupus nephritis were positive for anti-CRP at flare. Frequent correlations between anti-CRP levels and disease activity measures were observed in anti-CRP-positive individuals. Accumulated anti-CRP data from all patients were positively correlated with SLEDAI scores and anti-DNA antibody levels, whereas significant inverse relationships were noted for complement factors C1q, C3 and C4, and for lymphocyte counts. This study confirms the high prevalence of anti-CRP autoantibodies in SLE and that the antibody levels are correlated with clinical and laboratory disease activity measures. This indicates that anti-CRP antibodies might have biological functions of pathogenetic interest in SLE. Further prospective clinical studies and experimental studies on effects mediated by anti-CRP antibodies are warranted.