Interleukin-6 (IL-6) is overproduced in the joints of patients with rheumatoid arthritis (RA) and, based on its multiple stimulatory effects on cells of the immune system and on vascular endothelia, osteoclasts, and synovial fibroblasts, is believed to participate in the development and clinical manifestations of this disease. In this study we have analysed the effect of ablating cytokine production in two mouse models of arthritis: collagen-induced arthritis (CIA) in DBA/1J mice and the inflammatory polyarthritis of tumor necrosis factor α (TNF-α) transgenic mice. IL-6 was ablated by intercrossing an IL-6 null mutation into both arthritis-susceptible genetic backgrounds and disease development was monitored by measuring clinical, histological, and biochemical parameters. Two opposite responses were observed; while arthritis in TNF-α transgenic mice was not affected by inactivation of the IL-6 gene, DBA/1J, IL-6−/− mice were completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints. These results are discussed in the light of the present knowledge of cytokine networks in chronic inflammatory disorders and suggest that IL-6 receptor antagonists might be beneficial for the treatment of RA.

Stunted growth is a major complication of chronic inflammation and recurrent infections in children. Systemic juvenile rheumatoid arthritis is a chronic inflammatory disorder characterized by markedly elevated circulating levels of IL-6 and stunted growth. In this study we found that NSE/hIL-6 transgenic mouse lines expressing high levels of circulating IL-6 since early after birth presented a reduced growth rate that led to mice 50-70% the size of nontransgenic littermates. Administration of a monoclonal antibody to the murine IL-6 receptor partially reverted the growth defect. In NSE/hIL-6 transgenic mice, circulating IGF-I levels were significantly lower than those of nontransgenic littermates; on the contrary, the distribution of growth hormone pituitary cells, as well as circulating growth hormone levels, were normal. Treatment of nontransgenic mice of the same strain with IL-6 resulted in a significant decrease in IGF-I levels. Moreover, in patients with systemic juvenile rheumatoid arthritis, circulating IL-6 levels were negatively correlated with IGF-I levels. Our findings suggest that IL-6-mediated decrease in IGF-I production represents a major mechanism by which chronic inflammation affects growth. ( J. Clin. Invest. 1997. 99:643-650.) Key words: interleukin 6 • insulinlike growth factor-I • growth disorders • juvenile rheumatoid arthritis

SummarySystemic and localized inflammation elicit a number of host responses which include fever, cachexia, hypoglycemia, and major changes in the concentration of liver plasma proteins. Interlenkin 6 (IL-6) is considered an important mediator of the inflammatory response, together with IL-1 and tumor necrosis factor c~ (TNF-c 0. The purpose of this study was to unequivocally determine the role of IL-6 in these phenomena making use of IL-6-deficient mice that we have recently generated by gene targeting. We report here that in the absence of IL-6, mice are unable to mount a normal inflammatory response to localized tissue damage generated by turpentine injection. The induction of acute phase proteins is dramatically reduced, mice do not lose body weight and only suffer from mild anorexia and hypoglycemia. In contrast, when systemic inflammation is elicited through the injection of bacterial lipopolysaccharide (LPS), these parameters are altered to the same extent both in wild-type and IL-6-deficient mice, demonstrating that under these conditions IL-6 function is dispensable. Moreover, we show that LPS-treated IL-6-deficient mice produce three times more TNF-c~ than wild-type controls, suggesting that increased TNF-c~ production might be one of the compensatory mechanisms through which a normal response to LPS is achieved in the absence of IL-6. We also show that corticosterone is normally induced in IL-6-deficient mice, demonstrating that IL-6 is not required for the activation of the hypothahmicpituitary-adrenal axis. Our results reinforce the idea that different patterns of cytokines are involved in systemic and localized tissue damage, and identify IL-6 as an essential mediator of the inflammatory response to localized inflammation.

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 g of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.Genes can be transferred into skeletal muscle cells of rodents and primates by intramuscular injection of plasmid DNA, and the resulting gene expression has been reported to last as long as several months (1, 2). Similarly, various viral vectors such as adenoviral, retroviral, and AAV-based vectors (3), have been used to transduce myofibers in vivo. The i.m. injection of plasmid DNA, however, has several advantages over viral vectors. First, plasmid DNA vectors are easier to construct and can be prepared as pharmaceutical-grade solutions (4) without the risk of contamination with wild-type infectious particles. Second, previous infection by wild-type adenovirus or AAV may induce a neutralizing antibody response that could preclude administration of the recombinant virus. In contrast, anti-DNA antibodies have never been detected in experiments of muscle DNA injection (2), therefore it is possible to readminister plasmid DNA by i.m. injection if repeated therapy or escalation is required.Despite the promise of i.m. injection of plasmid vectors for treating serum protein deficiencies, several important issues remain to be addressed before this approach becomes feasible for human gene therapy. The potential clinical usefulness of direct gene transfer to muscle of plasmid DNA is in fact limited by the low and highly variable level of gene expression (1, 2, 5, 6). Therefore, although DNA injection is potentially very powerful as a vaccination method because a low level of gene expression is sufficient to trigger immunoresponses, it is necessary to increase the efficiency of DNA uptake after i.m. injection of plasmid vectors before using this technique as a standard gene correction procedure.One of the most efficient methods implemented to achieve gene transfer and expression in mammalian cells is based on electric pulses (7). Electroporation has been used to introduce foreign DNA in different cell types (7), but it has also recently met with some success in in vivo applications. Gene transfer by electrical permeabilization has been obtained in skin (8, 9), corneal endothelium (10), melanoma (11), brain (12), liver, (13) and muscle (14) of experimental animals.We have shown previously that electropermeabilization can increase severalfold the uptake by rat muscle of a plasmid encoding the Escherichia coli lacZ gene (15). In this study...

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob͞ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db͞db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.

Tuberculosis rarely developed among contacts, and preventive chemotherapy effectively reduced the tuberculosis risk among IGRA-positive contacts. Although the negative predictive value of IGRAs is high, the risk for the development of tuberculosis is poorly predicted by these assays.

The modified EPO gene yields higher levels of circulating transgene product and a more significant biological effect than the wild-type gene in all the species tested, thus showing great potential in clinically developable gene therapy approaches for EPO delivery.

Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations. A viable alternative is to induce a neutralizing antibody response. Using transgenic mice with high circulating levels of human interleukin-6 (hIL-6), we show that injection of the hIL-6 receptor antagonist Sant1 (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hIL-6 antibody response. The elicited antibodies bind circulating hIL-6 with very high affinity, totally masking it, and neutralize hIL-6 bioactivity both in vitro and in vivo.