Role of CD8(+) lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment.

Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.

Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes.

Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.

Minimal tumor necrosis factor receptor binding protein: optimum biological activity of a truncated p55 soluble tumor necrosis factor receptor-IgG fusion protein.

We have previously demonstrated, using expressed deletion constructs, that the fourth membrane proximal cysteine-rich repeat of the p55 TNF receptor (TNF-R) is not required for binding of tumour necrosis factor-alpha (TNF) or lymphotoxin-alpha (LT; tumour necrosis factor-beta). We and others have also shown that the soluble p55 TNF-R, rendered dimeric by fusion to an IgG backbone is extremely effective at neutralizing the harmful effects of TNF overproduction, such as in toxic shock. Here we address the question of how the TNF binding properties of the truncated TNF-R comprising the three distal cysteine-rich repeats (delta4 TNF-R), when fused with an IgG backbone, compare with those of the full length soluble receptor. We constructed several versions of the soluble delta4 TNF-R, on a complete IgG heavy chain backbone and on an IgG lacking the CH1 (first constant region) domain. The constructs were expressed with an Ig or native TNF receptor leader sequence and altered or native N terminal sequence, to compare efficiency of expression. When compared with a full length, soluble receptor Ig fusion protein, the affinity of all for TNF was identical, as were their activities in in vitro binding and cytotoxicity assays. In vivo studies showed that the delta4 and wild type fusion proteins afforded equivalent protection against LPS-induced lethality. However, the delta4 proteins exhibited a significantly lower affinity for LT, and reduced activity in LT binding and cytotoxicity assays. We conclude that the truncated TNF receptor IgG fusion protein is as effective at neutralizing TNF activity as the full length soluble receptor fusion protein. Its lower affinity for LT may make it a more selective agent in blocking the action of TNF, while causing less interference with the action of LT. Also its smaller size may make it a more useful therapeutic agent as it may be less immunogenic than the full length receptor.

Neutralization of TNF by the antibody cA2 reveals differential regulation of adhesion molecule expression on TNF-activated endothelial cells.

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.

Inhibition of tumor necrosis factor activity minimizes target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffic to the retina.

Recent studies demonstrated that administration of a p55-tumor necrosis factor (TNF) receptor IgG-fusion protein (TNFR-IgG) prevented the clinical onset of experimental autoimmune encephalomyelitis but did not alter the number or tissue distribution of autoantigen-specific CD4+ effector T cells which trafficked into the central nervous system. To determine whether specific target tissues of autoimmune damage remain intact after TNFR-IgG treatment despite the presence of inflammatory cells within the tissues, we examined rats with experimental autoimmune uveoretinitis (EAU), as in this model, the main target of autoreactive CD4+ T cells, the retinal rod outer segments (ROS), can be examined readily by light microscopy. As judged by direct ophthalmoscopy, the onset of inflammation in the anterior chamber of the eye in EAU following administration of TNFR-IgG was delayed by 6 days compared to untreated controls, but the magnitude of the response was only slightly less than controls. Histological examination of the retinae and direct assessment of retinal inflammation revealed a disproportionate sparing of ROS in the TNFR-IgG-treated animals despite a level of retinal inflammation not substantially less than controls in which ROS damage was marked. Analysis of retinal leukocytes by immunofluorescence microscopy and flow cytometry indicated that approximately equal numbers of CD4+ alpha beta TCR+ lymphocytes were present in treated and control retinae, more than 30% of CD4+ cells in both experimental groups expressed the CD25 or MRC OX40 activation markers and most cells, which would include the CD4+ T lymphocytes, were activated as evidenced by MHC class II expression. Fewer activated macrophages and granulocytes were present in the treated retinae, possibly reflecting the lower level of tissue damage and subsequent accumulation of these inflammatory cells. The results demonstrate directly that a tissue specifically targeted for autoimmune destruction can be protected despite the influx of fully activated CD4+ T cells.

Functional comparisons of different tumour necrosis factor receptor/IgG fusion proteins.

Nine different IgG fusion proteins and one non-fusion protein, all containing sequences from the extracellular domain of either of two human TNF receptors, were compared for their ability to bind and inhibit human TNF-alpha or TNF-beta. The fusion proteins differed with respect to TNF receptor type (p55 or p75 TNF receptor), receptor valency (one, two or four receptor domains per molecule), the presence or absence of a CH1 domain in the IgG constant region, and the proportion of the extracellular domain included in the construct. In vitro TNF binding assays and cytotoxicity assays indicated that, of the constructs that bound TNF, the greatest difference in affinity and neutralizing capability was between monovalent and bivalent receptor constructs. Differences were also noted between tetravalent and bivalent versions of p55 fusion proteins, as well as between a p75 fusion protein comprising the complete extracellular domain and one lacking the C-terminal 53 amino acids of the extracellular domain. p55 constructs that included only the second cysteine-rich domain (CRD) or only the second and third CRDs showed no TNF binding activity. The presence or absence of an IgG CH1 domain made no difference in the ability of fusion proteins to neutralize TNF-alpha or TNF-beta. Animal experiments comparing the tetravalent and bivalent p55 fusions and the effects of the CH1 domain did not show significant differences in their ability to protect mice from endotoxin-induced lethality, although the p55 fusion proteins appeared to be more protective than the p75 fusion proteins. Thus, this study has identified structural modifications to TNF receptor/IgG fusion proteins which have differing effects on their neutralizing ability towards TNF-alpha or TNF-beta.

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.

Lipopolysaccharide (LPS) neutralizing peptides reveal a lipid A binding site of LPS binding protein.

Endotoxic shock follows a cascade of events initiated by release of lipopolysaccharide during infection with Gram-negative organisms. Two overlapping 15-mer peptides were identified, corresponding to residues 91-108 of human lipopolysaccharide binding protein that specifically bound the lipid A moiety of lipopolysaccharide with high affinity. The peptides inhibited binding of lipopolysaccharide to lipopolysaccharide binding protein, inhibited the chromogenic Limulus amebocyte lysate reaction, and blocked release of tumor necrosis factor alpha following lipopolysaccharide challenge both in vitro and in vivo. These results suggest lipopolysaccharide binding protein residues 91-108 form at least part of the lipopolysaccharide binding site. Moreover, derivatives of lipopolysaccharide binding protein residues 91-108 might modulate lipopolysaccharide toxicity in the clinical setting.

Chimeric anti-TNF-alpha monoclonal antibody cA2 binds recombinant transmembrane TNF-alpha and activates immune effector functions.

Results of clinical trials have indicated that cA2, a neutralizing mouse/human IgG1 chimeric anti-human TNF-alpha monoclonal antibody, may have therapeutic benefit for rheumatoid arthritis patients. Arthritic joints contain, in addition to elevated levels of soluble TNF-alpha, high numbers of CD4+ T cells and macrophages, cells known to express transmembrane TNF-alpha upon activation. For that reason, we sought to determine if cA2 binds to transmembrane TNF-alpha and what effects such binding may have on TNF-alpha-expressing cells. A cell line expressing a cell-surface, mutant form of transmembrane TNF-alpha was prepared for these studies. Analysis of these TNF+ cells by flow cytometry, direct binding, and competitive binding assays showed that cA2 binds to the transmembrane form of TNF-alpha with high avidity. Binding of the IgG1 isotype of cA2, but not an IgG4 version of cA2, resulted in efficient killing of the TNF+ cells by both antibody-dependent cellular toxicity and complement-dependent cytotoxicity effector mechanisms. These findings indicate that, in addition to blocking soluble TNF-alpha activity, cA2 can bind to transmembrane TNF-alpha in vitro and suggest that cA2 binding may lead to lysis of TNF-alpha-expressing cells in vivo.

Successful therapy of collagen-induced arthritis with TNF receptor-IgG fusion protein and combination with anti-CD4.

We have previously shown that anti-tumour necrosis factor (TNF) monoclonal antibody (mAb) ameliorates established collagen-induced arthritis and that the efficacy of this form of treatment can be enhanced by concurrent anti-CD4 treatment. Here we assess the efficacy of a human p55 TNF receptor-IgG fusion protein (p55-sf2), given alone or with anti-CD4 mAb. TNF receptor-IgG fusion protein (100 micrograms) suppressed paw swelling and limb recruitment in established arthritis and reduced the incidence of erosions in the proximal interphalangeal joints from 92% to 50%, which was comparable to 41% erosions using anti-TNF mAb. Methylprednisolone acetate (4.2 mg/kg/week) reduced clinical signs of inflammation in a manner comparable to TNF blockade but had little effect on the incidence of erosions. Co-administration of anti-CD4 and TNF receptor-IgG led to an even greater therapeutic effect than TNF receptor-IgG alone, with the incidence of erosions being reduced from 100% to 17%. Serological analyses showed that the beneficial effects of anti-CD4 and TNF receptor-IgG could be partly explained by the ability of anti-CD4 to prevent a neutralizing antibody response. These results confirm the importance of TNF in destructive inflammatory arthritis and demonstrate the feasibility of therapeutically targeting TNF with a form of TNF receptor. Finally, the findings confirm the beneficial effects of TNF-targeted therapy coupled with anti-CD4 therapy.

The mouse/human chimeric monoclonal antibody cA2 neutralizes TNF in vitro and protects transgenic mice from cachexia and TNF lethality in vivo.

The pleiotropic cytokine tumour necrosis factor-alpha (TNF) is thought to play a central role in infectious, inflammatory and autoimmune diseases. Critical to the understanding and management of TNF-associated pathology is the development of highly specific agents capable of modifying TNF activity. We evaluated the ability of a high affinity mouse/human chimeric anti-TNF monoclonal antibody (cA2) to neutralize the in vitro and in vivo biological effects of TNF. cA2 inhibited TNF-induced mitogenesis and IL-6 secretion by human fibroblasts, TNF-priming of human neutrophils, and the stimulation of human umbilical vein endothelial cells by TNF as measured by the expression of E-selectin, ICAM-1 and procoagulant activity. cA2 also specifically blocked TNF-induced adherence of human neutrophils to an endothelial cell monolayer. Receptor binding studies suggested that neutralization resulted from cA2 blocking of TNF binding to both p55 and p75 TNF receptors on the cells. In vivo, repeated administration of cA2 to transgenic mice that constitutively express human TNF reversed the cachectic phenotype and prevented subsequent mortality. These results demonstrated that cA2 effectively neutralized a broad range of TNF biological activities both in vitro and in vivo.

TNF receptor fusion proteins are effective inhibitors of TNF-mediated cytotoxicity on human KYM-1D4 rhabdomyosarcoma cells.

KYM-1D4 cells are a subline derived from a human rhabdomyosarcoma which are highly sensitive to TNF-mediated cytotoxicity. They were selected for this study because they express human TNF-R and are therefore a more relevant target for comparing the potential therapeutic value of human TNF-inhibitory agents than the usual murine cell lines. Two recombinant soluble TNF-R-IgG fusion proteins, one containing p55 TNR-R, the other containing p75 TNF-R, and a recombinant monomeric soluble p55 TNF-R were all found to block the cytotoxicity generated by human TNF-alpha and LT as well as also murine TNF. The p55 TNF-R-IgG fusion protein (p55-sf2) was the most effective of the antagonists tested, requiring an equimolar, (based on a monomeric configuration of TNF-alpha) or a 3-fold higher (based on a trimeric configuration of TNF-alpha) molar concentration to inhibit the cytotoxicity mediated TNF-alpha by 50%. p55-sf2 was also as effective at inhibiting the cytotoxicity mediated by LT or murine TNF in the KYM-1D4 assay. In contrast, the monomeric soluble p55 TNF-R was the least effective inhibitor, requiring a > 4000-fold higher molar concentration than p55-sf2 to achieve a similar degree of protection. The fusion proteins, particularly p55-sf2, may be useful as human therapeutic agents, as at low concentrations they can prevent both TNF-alpha-mediated and LT-mediated effects on human cells. As TNF-R-IgG fusion proteins also block the action of murine TNF in vitro, they may also be useful in the investigation of murine models of human inflammatory disease.

Expression and characterization of cM-T413, a chimeric anti-CD4 antibody with in vitro immunosuppressive activity.

Anti-CD4 monoclonal antibodies (mAbs) have shown considerable promise in the treatment of rheumatoid arthritis, psoriasis, and allograft rejection and may have potential use in blocking HIV-1 infection. One such anti-CD4 mAb we have developed, chimeric M-T412 (or cM-T412), has been used in clinical trials to treat rheumatoid arthritis, generalized postular psoriasis, and other autoimmune diseases. Here we report the cloning and expression of a second chimeric anti-CD4 mAb using M-T413, a murine mAb that blocks HIV-1 infection of H9 cells. We cloned the immunoglobulin light and heavy chain variable regions of M-T413, combined them with the human kappa (light chain) or G1, G2, G3 and G4 (heavy chain) constant regions in human expression vectors, and expressed these chimeric mAbs in 653 cells. Like chimeric M-T412 IgG1, the chimeric M-T413 mAbs inhibit T-cell proliferation in the mixed lymphocyte response and thus can act to immunosuppress CD4+ T-cell response. In contrast to M-T412, however, the M-T413 chimeric mAbs have reduced activity in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay using human CD4+ target and effector cells. We conclude that the chimeric M-T413 mAbs have potential utility in treating autoimmune disease and may be useful as prophylactics in preventing HIV-1 infection.

Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor alpha.

OBJECTIVE: To evaluate the safety and efficacy of a chimeric monoclonal antibody to tumor necrosis factor alpha (TNF alpha) in the treatment of patients with rheumatoid arthritis (RA). METHODS: Twenty patients with active RA were treated with 20 mg/kg of anti-TNF alpha in an open phase I/II trial lasting 8 weeks. RESULTS: The treatment was well tolerated, with no serious adverse events. Significant improvements were seen in the Ritchie Articular Index, which fell from a median of 28 at study entry to a median of 6 by week 6 (P < 0.001), the swollen joint count, which fell from 18 to 5 (P < 0.001) over the same period, and in the other major clinical assessments. Serum C-reactive protein levels fell from a median of 39.5 mg/liter at study entry to 8 mg/liter at week 6 (P < 0.001), and significant decreases were also seen in serum amyloid A and interleukin-6 levels. CONCLUSION: Treatment with anti-TNF alpha was safe and well tolerated and resulted in significant clinical and laboratory improvements. These preliminary results support the hypothesis that TNF alpha is an important regulator in RA, and suggest that it may be a useful new therapeutic target in this disease.

High-level expression and characterization of a mouse-human chimeric CD4 antibody with therapeutic potential.

The use of murine anti-CD4 monoclonal antibodies (MAbs) has shown considerable promise for the treatment of allograft rejection and rheumatoid arthritis. We have constructed mouse-human anti-CD4 antibodies with the goal of increasing their clinical potential by decreasing immunogenicity and improving effector functions. The chimeric antibodies were constructed by cloning the heavy and light chain variable regions of M-T412, a murine antibody raised against the human CD4 antigen, and joining them to the human G1, G4, or kappa constant regions in mammalian expression vectors. After transfection into mouse myeloma cells, stable cell lines were isolated that secrete up to 140 micrograms/ml chimeric antibody in static culture. The chimeric antibodies were equivalent to the murine antibody in their binding characteristics and relative affinities. However, the chimeric M-T412 MAbs have enhanced activity when compared to the murine G2a MAb in mediating antibody-dependent cell-mediated cytotoxicity using human CD4+ target and effector cells.

Stable expression of cloned human antibody genes in murine myeloma cells.

Human monoclonal antibodies (MAbs) offer potential advantages over murine MAbs for therapy because they are not likely to elicit immune responses and are expected to interact more efficiently with the human immune system to activate therapeutically useful functions. Traditional methods for obtaining human MAbs (i.e., immortalization of B cells by cell fusion or transformation) can result in low and unstable antibody secretion. Recently, methods have been devised for direct cloning of human variable region genes via polymerase chain reaction and phage combinatorial libraries. Both types of human MAb production can benefit from expression systems that support the stable, high-level antibody secretion required for therapeutic use. Using an existing human-derived hybridoma that secretes a human IgM antibody as a convenient source of antibody genes, we have demonstrated that cloned human antibody genes can be efficiently expressed in murine myeloma cells and that cell lines with properties suitable for large-scale economical production can be obtained. We were unable to detect any differences between the antibodies produced by the original hybridoma and the engineered cell line. In addition, we were able to express an IgG form of the antibody, showing that expression of a recombinant human antibody need not be limited to the original antibody class.

Pharmacokinetics of a mouse/human chimeric monoclonal antibody (C-17-1A) in metastatic adenocarcinoma patients.

The pharmacokinetic characteristics of a mouse/human chimeric monoclonal antibody (C-17-1A) were determined in 10 patients with metastatic adenocarcinoma following the administration of either 10-mg or 40-mg infusions as a single or multiple dose. The administration of single 10-mg (n = 5) and 40-mg (n = 5) doses infused over 1 hr resulted in mean apparent steady-state distribution volumes of 4.13 +/- 0.97 and 5.16 +/- 1.92 liters, respectively, indicating that C-17-1A appears to distribute throughout the vascular compartment and into limited extracellular fluid volume. The disposition of C-17-1A was adequately characterized using a two-compartment open model with mean distribution half-lives of 15.8 and 18.5 hr and mean elimination half-lives of 90.0 and 97.6 hr for the 10- and 40-mg groups, respectively. A linear relationship was observed between AUC and dose (micrograms/kg). The clearance of C-17-1A was correlated linearly with total Ig, IgG, and tumor size. Multiple administration of either 10-mg (n = 3) or 40-mg (n = 3) doses of C-17-1A infused over 1 hr every 14 days for a total of three doses resulted in superimposable mean serum concentration versus time data and consistent mean pharmacokinetic characteristics. These data indicate that C-17-1A exhibits linear, nonsaturable distribution and elimination characteristics in man over the dose range studied (i.e., 130 to 880 micrograms/kg). The multiple-dose pharmacokinetics of C-17-1A were predictable, indicating a lack of an antibody response to C-17-1A over a period of 42 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of B-cell stimulatory activity of HIV-1 to the carboxyl terminus of gp41.

Patients with AIDS are known to have B-cell hyperactivity. We have previously demonstrated that an extract of HIV-1 could induce differentiation of peripheral blood B lymphocytes of healthy volunteers into immunoglobulin-secreting cells. In an attempt to delineate the B-cell stimulatory subregion in HIV-1, we have investigated the influences of native glycoproteins and recombinant proteins of the envelope. The complete envelope glycoprotein, gp160 and a recombinant protein in the carboxyl terminal region of gp41 termed PE-8 were effective in inducing terminal differentiation of normal peripheral blood B lymphocytes and did so in a T-lymphocyte-dependent manner. The activity was not present in the native exterior envelope glycoprotein, gp120 and several other recombinant proteins, viz PE-2 an PE-3, which are in the amino terminal region of gp120 or in env-9, a protein in the junctional region of gp120 and gp41. Polyclonal and monoclonal antibodies directed to diverse regions of the envelope abrogated the influence of gp160. The PE-8-induced B-cell differentiation was abrogated by goat anti-gp160 antibody but not by goat anti-gp120 antibody or monoclonal antibody to the amino terminal of gp41. These studies suggest that a putative polyclonal B-cell stimulatory epitope of HIV-1 is located in the carboxyl end of the envelope glycoprotein.

Post-translational modifications of human interleukin-6.

We have previously reported that interleukin (IL)-6 secreted by human fibroblasts induced with either IL-1 or tumor necrosis factor (TNF) consists of at least six differentially modified phosphoglycoproteins of molecular mass 23-30 kDa: a triplet in the mass range from 23 to 25 kDa and another triplet in the range from 28 to 30 kDa. We now report that a combination of metabolic labeling, glycosidase digestion, and lectin chromatography experiments demonstrates that the 23- to 25-kDa species are O-glycosylated and that the 28- to 30-kDa species are both O- and N-glycosylated. Pulse-chase experiments reveal that newly synthesized IL-6 polypeptides rapidly enter two separate protein modification pathways: one leads to O-glycosylation and the other to both N- and O-glycosylation; polypeptides in both pathways are further modified (phosphorylation) prior to secretion. Although both pathways appear to be equally utilized in IL-1- or TNF-induced fibroblasts, the relative proportion of polypeptides proceeding through one or the other pathway can be experimentally modified. In the presence of tunicamycin, IL-6 is secreted exclusively in the O-glycosylated form, whereas in the presence of cycloheximide the pathway leading to both N- and O-glycosylation is dominant. The inclusion of monensin (1 microM) does not inhibit IL-6 secretion from fibroblasts even though it inhibits glycosylation. Combined immunoprecipitation, immunoblotting, and immunoaffinity chromatography experiments reveal additional IL-6 species with mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions corresponding to molecular masses 17-19 kDa and 45 kDa, suggesting that this cytokine undergoes further alterations. These observations highlight an aspect of IL-6 biosynthesis that appears to represent an excellent model system for studying the mechanisms regulating post-translational protein modifications in human cells and also suggest a basis for reconciling conflicting descriptions of IL-6 structure.

IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular sources.

We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays. IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line. IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting. The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera. In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay. Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay. Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml). No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6. The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6. Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied. Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation. Similarly, there was no constitutive production of IL-1 by these cells. However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS. The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells. These findings suggest that IL-6 is involved in inflammatory joint disease. However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate.