Anti-murine IL-6 receptor antibody inhibits IL-6 effects in vivo

Thrombopoiesis, as well as antibody production, is one of the major events in which interleukin-6 (IL-6) has been reported to be involved. Polyclonal anti-murine IL-6 receptor antibody was prepared to examine the effect of the antibody on these events in IL-6-treated mice. Administration of the anti-mIL-6R antibody inhibited the IL-6-induced increase in the number of platelets. Enhancement of the serum level of DNP-specific antibody by intraperitoneal injection of IL-6 was inhibited completely with simultaneous administration of the anti-mIL-6R antibody. The level of DNP-specific antibody was decreased, even below the basal value, by the higher dose of anti-mIL-6R antibody, indicating its effect also on endogenous IL-6. This work provides evidence that anti-IL-6R antibody inhibits IL-6 function in vivo, and provides an animal model of the therapeutic use of anti-IL-6R antibody for IL-6-related disease.     

IL-6 induces hepatic inflammation and collagen synthesis in vivo.

IL-6 regulates the synthesis of a broad spectrum of acute phase proteins in the liver. Also, it is involved in the pathogenesis of many fibrogenic diseases. To study the inflammatory effects of IL-6 on the liver in vivo, human rIL-6, produced in Escherichia coli, was injected intraperitoneally into rats (25 micrograms/100 g body weight). The major fraction of injected IL-6 was accumulated in the liver within 40 min, and the number of platelets was increased during 72 h after injection. After 5 weeks of injection, the levels of serum glutamine pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) were not changed, but they were significantly elevated at 13 weeks of treatment. Meanwhile, serum albumin levels were slightly decreased compared with those of controls. The same phenomena were observed in carbon tetrachloride-treated rats. Collagen synthesis was increased in the liver tissues and in the culture supernatants of hepatic lipocytes isolated from the rats treated with IL-6 for 13 weeks. Histological analysis correlated well with biochemical analysis. At 5 weeks of treatment, only mild pathological changes were observed, but severe hepatocyte necrosis and the accumulation of fibres in necrotic area were developed in the liver of IL-6-treated rats after 13 weeks of treatment, confirming that hepatic inflammation and fibrosis were developed. IL-6 activities in the sera and in the culture supernatants of lipocytes from IL-6-treated rats were elevated compared with those in controls. These biochemical and pathological data indicate that IL-6 can induce hepatic inflammation, and it has important roles in the pathogenesis of fibrosis and diseases of the liver in vivo. In addition, these results will provide useful information for the clinical trials of IL-6.     

T lymphocytes need IL-7 but not IL-4 or IL-6 to survive in vivo

The role of IL-4, -6 and -7 in the survival of T lymphocytes was studied in vivo. The decay of polyclonal populations of CD4(+) and CD8(+) T cells was monitored in thymectomized anti-cytokine receptor mAb-treated and/or cytokine-deficient mice. The lack of IL-4 or -6 did not have any detectable effect on T cell survival, but IL-7 played an important role in the survival of the naive T cell compartment, especially of naive CD4(+) T cells.     

Ciprofloxacin treatment in vivo increases the ex vivo capacity of lipopolysaccharide-stimulated human monocytes to produce IL-1, IL-6 and tumour necrosis factor-alpha.

Because in vitro treatment with quinolones, at pharmacological concentrations, modifies lipopolysaccharide (LPS) induced production of cytokines by monocytes, we studied the effect of orally administered ciprofloxacin (25 mg/kg) on the capacity of peripheral blood monocytes of healthy volunteers to produce tumour necrosis factor-alpha (TNF-alpha), IL-1 activity, IL-1 alpha, IL-1 beta and IL-6 ex vivo in response to endotoxin stimulation. After 7 days of ciprofloxacin, the extracellular and cellular production of TNF-alpha, the cellular production of IL-1 activity, the extracellular and cellular production of IL-1 alpha, and the cellular production of IL-6 increased significantly. Seven days after the end of the treatment, values returned to basal levels or even lower. To our knowledge, this is the first demonstration that ciprofloxacin can modulate in vivo the capacity of human monocytes to react to an inflammatory stimulus such as endotoxin.     

Acute in vivo effects of IL-3 alone and in combination with IL-6 on the blood cells of the circulation and bone marrow.

Recombinant human IL-3 administered intravenously to rats as a single injection induced peripheral neutrophilia and monocytosis beginning at 4 to 6 hours after injection, peaking at 8 hours, and subsiding to normal by 12 to 24 hours. IL-3 did not induce an initial neutropenia such as accompanies endotoxin-, G-CSF-, and TNF-induced neutrophilia, or lymphopenia such as accompanies endotoxin-, IL-1-, and TNF-induced neutrophilia. The IL-3-induced peripheral neutrophilia was accompanied by a decrease in mature marrow neutrophils, indicating that the mechanism of neutrophilia was through marrow release rather than by demargination, which occurs after the administration of epinephrine or IL-6. The release of mature marrow neutrophils further suggests that IL-3 either has intrinsic neutrophil releasing activity or indirectly causes neutrophil release through the gene expression of a second cytokine. IL-3 induced a striking left-shifted myeloid hyperplasia in the bone marrow at 8 hours that morphologically was very similar to that observed after administration of endotoxin, a finding consistent with the hypothesis of previous investigators that endotoxin may in part act indirectly on hematopoietic cells by eliciting local marrow production of IL-3. Finally, IL-3 induced an increase in marrow pronormoblasts at 8 hours, consistent with the in vitro proliferative effect of IL-3 on erythroid stem cells. The combination of IL-3 and IL-6 induced a synergistic peripheral neutrophilia and monocytosis and a striking synergistic increase in marrow mast cells. The combination of IL-3 and IL-6 also induced an erythroid and left-shifted myeloid hyperplasia such as would be expected given the individual effects of these hematopoietic growth factors.     

In vivo expression of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumour necrosis factor-alpha and interferon-gamma in the fetal murine thymus.

Cytokines are known to play a role in T-cell lymphopoiesis as potent growth or differentiation factors, but many experiments focusing on their role in the thymus have been conducted only in vitro. We have thus used frozen sections obtained from fetal thymuses of normal C57BL 6 mice to investigate by immunohistochemistry the presence of interleukin-1 beta (I4-1 beta), IL-2. IL-4. IL-6. interferon-7 (IFN-7) and tumour necrosis facor-alpha (TNF-alpha). The results reveal that apart from IL-2, which was not detected, all these cytokines display a time-dependent expression pattern in the normal fetal thymus. First, production of IL-4, IL-6 and TNF-alpha is detected around days 13 14; this is followed by a second wave on days 16 17, with a production of IL-1 beta, IL-4 and IL-6, and finally, just before birth (day 19), by a third wave of IL-1 beta, IL-4, IL-6, IFN-7 and TNF-alpha production. This supports the hypothesis that cytokines play a rote in T-cell lymphopoiesis.     

Differential sensitivity of in vivo TNF and IL-6 production to modulation by anti-inflammatory drugs in mice.

The effect of dexamethasone and two non-steroidal anti-inflammatory agents ibuprofen and indomethacin on the production of serum interleukin 6(IL-6) and tumor necrosis factor (TNF) levels in mice treated with endotoxin (2.5 micrograms/mouse, i.p.) was investigated. Pretreatment of mice with dexamethasone (0.3-30.0 mg/kg, i.p., 30 min before endotoxin) completely blocked TNF production but did not affect that of IL-6. Conversely, pretreatment with indomethacin (5 mg/kg, i.p.) or ibuprofen (30 mg/kg, i.p.) potentiated the production of both IL-6 (+ 80% with INDO; + 100% with IBU) and TNF (+ 500% with INDO; + 50% with IBU). In the case of IL-6, the two anti-inflammatory drugs were able per se to induce significant levels of this cytokine even in the absence of LPS. These data indicate that IL-6 and TNF production are differently susceptible to glucocorticoids, and that prostaglandins can physiologically provide a negative feedback regulation of IL-6 and TNF synthesis.     

Analysis of interleukin 6 (IL-6)/IL-6 receptor system using monoclonal anti-IL-6 antibodies.

To investigate the IL-6/IL-6 receptor system, we obtained five murine monoclonal antibodies against human IL-6, which neutralize its biological activity. We classified them into two groups (Type I mAb and Type II mAb) according to the epitopes they recognized. These two types of antibodies showed no difference in the manner in which they neutralized IL-6 activity, but they differed in the way they inhibited the binding of IL-6 to its receptor. While Type I mAb inhibited IL-6 binding to its receptor completely, Type II mAb inhibited only partially, even at a concn of Type II mAb sufficient to neutralize biological activity. Scatchard plot analysis revealed that in the case of the human myeloma cell line, the U266 cell, which showed two-phase binding of IL-6 to its receptor, the high affinity binding disappeared and the affinity of the low affinity binding decreased in the presence of Type II mAb. These results suggest that Type I mAb neutralizes IL-6 activity by the blocking of IL-6 binding to its receptor directly. In contrast, Type II mAb neutralizes IL-6 activity not by direct blocking of IL-6 binding to its receptor, but by modulating the binding affinity of IL-6 to its receptor, that is, inhibiting the formation of high affinity binding in IL-6 receptor system. This also indicates that the formation of high affinity may be necessary to transduce the IL-6 signal.     

Autostimulatory effects of IL-6 on excessive B cell differentiation in patients with systemic lupus erythematosus: analysis of IL-6 production and IL-6R expression.

Introducing avidin-biotin complex ELISA for anti-DNA antibody, the mechanism of in vitro production of anti-ssDNA antibody as well as of polyclonal immunoglobulin mediated by an IL-6-IL-6R loop was studied in patients with systemic lupus erythematosus (SLE). Regardless of the presence or absence of T cells, B cells from SLE patients could produce IgG anti-ssDNA antibody as well as total IgG without any stimulation. Low density B cells obtained by Percoll gradient density centrifugation responded to rIL-6 to produce IgG and IgG anti-ssDNA antibody. rIL-2 and rIL-4 had lesser effects on the differentiation of low density B cells. In fact, IL-6R was preferentially expressed on low density B cells from active SLE patients, as detected by anti-IL-6R MoAb, MT18, which did not inhibit IL-6 binding. SLE B cells, especially high density B cells, produced greater amounts of IL-6 in culture supernatants than did T cells, regardless of whether disease was active or inactive. Normal T cells and B cells did not produce significant amounts of IL-6. Thus, endogenous IL-6 produced by high density B cells bound to the IL-6R preferentially expressed on the low density B cells, and drove them into terminal differentiation, especially in active SLE patients. Further, addition of polyclonal anti-IL-6 or anti-IL-6R MoAb (PM1), which inhibited IL-6 binding, both inhibited IgG anti-ssDNA antibody as well as total IgG production by SLE B cells in a dose-dependent manner. These results suggest that interruption of the autocrine IL-6 loop would be of therapeutic value in SLE.     

Interleukin-6 in mouse hypersensitivity pneumonitis: changes in lung free cells following depletion of endogenous IL-6 or direct administration of IL-6.

In this study, we examined the role of interleukin-6 (IL-6) in the development of chronic lung inflammatory conditions, using a mouse model of hypersensitivity pneumonitis established by intranasal instillation of the thermophilic actinomycete Faeni rectivirgula. Challenged mice developed an early neutrophilic response at 24 h, followed by a macrophage/lymphocyte recruitment. The impact of IL-6 on the development of the inflammatory response was assessed by giving infusions of a monoclonal antibody against IL-6 so as to deplete endogenous levels of this cytokine or by giving exogenous IL-6 to challenged mice. Mice challenged intranasally with the actinomycete and given the anti-IL-6 antibody developed a strong, sustained neutrophilic response, with a significantly higher lung free cell number than control mice. Assessment of fibrosis by measuring lung hydroxyproline levels showed that challenged mice given anti-IL-6 developed more significant fibrosis than control mice. Conversely, infusions with IL-6 diminished F. rectivirgula-induced cell recruitments and the fibrotic response in the lungs. Moreover, alveolar macrophages from mice given 2 weeks of F. rectivirgula treatment released high levels of tumor necrosis factor alpha (TNF-alpha) bioactivity upon in vitro lipopolysaccharide challenge, compared to mice instilled with saline only. This TNF-alpha activity produced by macrophages was decreased by in vivo IL-6 treatment and enhanced by in vivo neutralization with anti-IL-6. These observations suggest that IL-6 may play a role in regulating the cellular recruitment in the lungs during an inflammatory response, with dramatic consequences for the cellular profile in the bronchoalveolar lavage and the subsequent fibrosis.     

IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo.

We have characterized a cytokine produced by Th2 cells, designated as IL-25. Infusion of mice with IL-25 induced IL-4, IL-5, and IL-13 gene expression. The induction of these cytokines resulted in Th2-like responses marked by increased serum IgE, IgG(1), and IgA levels, blood eosinophilia, and pathological changes in the lungs and digestive tract that included eosinophilic infiltrates, increased mucus production, and epithelial cell hyperplasia/hypertrophy. In addition, our studies show that IL-25 induces Th2-type cytokine production by accessory cells that are MHC class II(high), CD11c(dull), and lineage(-). These results suggest that IL-25, derived from Th2 T cells, is capable of amplifying allergic type inflammatory responses by its actions on other cell types.     

Polymorphisms in the IL-6 receptor (IL-6R) gene: strong evidence that serum levels of soluble IL-6R are genetically influenced

We have investigated the association of the recently identified IL6R polymorphisms with the serum levels of soluble IL-6 receptor (sIL-6R). sIL-6R is generated by shedding of the membrane-bound receptor (IL-6Ralpha) or alternative mRNA splicing. In total, 115 healthy volunteers were genotyped, with 70 of them analyzed for sIL-6R levels. Using the PCR/RFLP methods, two important polymorphic sites were selected for genotyping: the 48892A/C (D358A) in exon 9 and the -183G/A in the promoter region. In exon 9, C allele carriers had higher sIL-6R level (P<0.0001) showing that this sequence variation, which corresponds to the proteolytic cleavage site of IL-6Ralpha, strongly influences the serum sIL-6R levels. In the promoter region, G allele carriers had lower sIL-6R levels (P<0.0082) compared with the A allele carriers. This could be attributed to the linkage disequilibrium (D'=0.54, chi2=51.3, P<0.0001) between the -183G/A and the 48892A/C gene polymorphisms.     

FUT-175 inhibits the production of IL-6 and IL-8 in human monocytes.

We investigated the effect of FUT-175, a serine protease inhibitor, on the production of pro-inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8), by monocytes stimulated with lipopolysaccharide (LPS). Monocytes isolated from healthy volunteers were incubated with LPS and various concentrations of FUT-175 for 12 hours. The productions of both IL-6 and IL-8, measured by enzyme-linked immunosorbent assay, were significantly inhibited by FUT at the concentration of 1 to 100 microg/ml in a dose-dependent manner. Furthermore, the expressions of IL-6 and IL-8 mRNAs were also inhibited by FUT-175. These data suggest that FUT-175 may reduce the pathological inflammatory conditions associated with enhanced production of proinflammatory cytokines including IL-6 and IL-8.     

Blocking IL-1: interleukin 1 receptor antagonist in vivo and in vitro.

Clinical and experimental evidence suggests that shock, arthritis, osteoporosis, colitis, leukemia, diabetes, wasting and atherosclerosis are mediated, in part, by interleukin 1 (IL-1). Inhibition of this cytokine has been a strategy for studying disease and for new drug development. A naturally-occurring IL-1 inhibitor (IL-1 receptor antagonist, IL-1ra) that blocks binding of IL-1 to its receptors has been cloned and produced in recombinant organisms. IL-1ra reduces the severity of sepsis, colitis, arthritis and diabetes in animals and is presently being tested in humans with arthritis, shock and myelogenous leukemia.     

IL-6

In the late 1960s, the essential role of T-cells in antibody production was reported. This suggested to us that certain molecules should be released from T-cells for the stimulation of B-cells. We discovered activities in the culture supernatant of T-cells that induced proliferation and differentiation of B-cells. The factor that induced B-cells to produce immunoglobulins was named B-cell stimulatory factor (BSF)-2. The complementary DNA that encoded human BSF-2 was cloned in 1986. Simultaneously, interferon-β2 and 26-kDa protein in the fibroblasts were independently cloned by different groups and were found to be identical to BSF-2. Later, a hybridoma/plasmacytoma growth factor and a hepatocyte-stimulating factor also were proven to be the same molecule as BSF-2. Various names were used for this molecule because of its multiple biological activities, and thereafter, these names were unified as interleukin (IL)-6. Since the discovery of IL-6, rapid progress has been made in understanding its actions, the IL-6 receptor system, and the IL-6 signal transduction mechanism. More importantly, it was involved in numerous diseases, such as rheumatoid arthritis and Castleman’s disease. By accumulating the basic knowledge, a new therapeutic approach by blocking IL-6 actions appeared to be feasible for chronic inflammatory diseases.     

IL-6 secretion by ex vivo whole blood cultures upon allergen stimulation

Allergen induced IL-6 synthesis by whole blood cultures was compared with skin prick allergen test results for the same group of individuals. Whole blood cultures from both allergic and non-allergic individuals secrete IL-6 at high allergen concentrations. When whole blood cultures from controls were incubated with serial dilutions of allergens it was found that IL-6 induction was abolished at lower allergen dilutions (allergen threshold concentration or ATC). When whole blood cultures from patients with allergic rhinitis were stimulated with ATC it was found that some allergens induced IL-6 secretion. The allergens inducing IL-6 and the level of IL-6 secreted were dependent on the patient. The induction of IL-6 secretion by the cultures at ATC correlated very significantly with the patient's skin prick test results (r = 0.71 1; p = 0.0003).     

Role of IL-6 in cytokine-induced sickness behavior: a study with IL-6 deficient mice

Interleukin-6 (IL-6) is synthesized and released in response to the cytokine inducer lipopolysaccharide (LPS) and IL-1, and acts as an endogenous pyrogen. Systemic administration of LPS and IL-1 to mice induces signs of sickness, including reduction of social exploration, immobility and body weight loss. To assess the role of IL-6 in the induction of sickness behavior, male IL-6-deficient mice (IL-6 -/-, Balb/cAn genetic background) were used and compared to IL-6 +/+ littermates. The depressing effects of intraperitoneal LPS (2.5 microg/mouse) and IL-1beta (1.0 microg/mouse) on behavior and change in body weight were more marked in IL-6 +/+ than in IL-6 -/- mice. The same difference was observed when mice were injected with LPS (5 ng/mouse) and IL-1beta (1 ng/mouse) into the lateral ventricle of the brain (i.c.v.). These results show that IL-6 released at the periphery and /or in the central nervous system plays a role in the behavioral response to LPS and IL-1.     

IL-6 trans-signaling: the heat is on

The molecular consequence of the fever response has been illuminated by a recent study showing that a temperature shift to 40 degrees C resulted in increased leukocyte adhesion to tissue sections, which was mediated by L-selectin activation in lymphocytes. This L-selectin activation during heat responses was dependent on IL-6 trans-signaling via the soluble IL-6R.