Jon E. Silver

Strategies of Anti-Cytokine Monoclonal Antibody Development: Immunoassay of IL-10 and IL-5 in Clinical Samples

The identification of cytokines, a class of intermediate molecular weight, intercellular regulatory proteins, and an understanding of their function has been facilitated tremendously by the use of recombinant DNA technology. Following closely upon these developments has come the ability to detect and quantify cytokines in clinical and biological samples, in large part made possible by the use of specific and sensitive immunoassays using anti-cytokine antibodies. Cytokine detection with antibody-based techniques is particularly useful because so many of these factors have overlapping, parallel, or counter-regulatory bioactivities and it is extremely difficult to distinguish and measure them in complex mixtures on the basis of biological activity alone, Therefore, both neutralizing antibodies in bioassay systems and cytokine immunoassays of complex samples are required (Mosmann & Fong 1989). In the first part of this review, we outline the general strategy we have followed for preparing a large panel of anti-mouse and human cytokine monoclonal antibodies (mAb) and setting up cytokine immunoassays. While there is a general consensus that many of the frequently produced cytokines such as IL-1, TNF, IFN-y, IL-6, and IL-8 may be general inflammatory mediators, see for example (Dunn 1991), definitive links between the detection of specific cytokine patterns and the pathogenesis of particular diseases are in the early stages of being understood. The eventual elucidation of such cause-and-

Interleukin-6 Production by Schwann Cells and Induction in Sciatic Nerve Injury

Abstract: Interleukin‐6 (IL‐6) was produced by the spontaneously immortal Schwann cell clone, iSC, when cocultured with PC12 cells. The iSC cell‐derived IL‐6 in coculture conditioned media caused the neuronal differentiation of naive PC12 cells and this bioactivity was neutralized by preincubation of conditioned media with antisera to IL‐6. Cocultured iSC transcribe IL‐6 message as confirmed by northern analysis. Stimuli that induce IL‐6 production in the hematopoietic lineage induced transcription and production of IL‐6 by iSC cells. Lipopolysaccharide, tumor necrosis factor‐α, IL‐1α, IL‐6, and serum withdrawal induced iSC cell IL‐6 mRNA. The kinetics of IL‐6 production was confirmed in the mouse IL‐6‐dependent B9 bioassay and that activity could be neutralized with antisera to IL‐6. Expression of both the IL‐6 receptor and the gp130 signal transduction component by iSC as determined by northern analysis suggests an autocrine regulatory mechanism. The observed iSC production of IL‐6 in vitro led to an investigation of the sciatic nerve crush model of Schwann cell activation in vivo. In the initial 12 h after crush injury, IL‐6 message is induced. IL‐6 mRNA expression was highest distal to the crush injury. Our in vitro data demonstrate that iSC cells produce IL‐6 in response to PC12 cell coculture and to stimuli that induce IL‐6 production in the hematopoietic lineage. The induction of IL‐6 message distal to a crush injury suggests another mechanism by which Schwann cells facilitate peripheral nerve regeneration.

Interleukin-5 and the posttreatment eosinophilia in patients with onchocerciasis.

To understand the role of the eosinophilopoietic cytokine IL-5 in humans, the posttreatment eosinophilic response in a group of microfilaria (mf)-positive patients with onchocerciasis (n = 10) was examined before and after treatment with diethylcarbamazine (6 mg/kg for 7 d). Sequential blood samples were assessed at 24 and 1 h before treatment (baseline values), then at frequent intervals over the next 14 d. Symptom scores, skin microfilariae (mf), and peripheral blood eosinophil counts were recorded as a function of time after treatment, and serum levels of IL-5 were quantitated by a highly sensitive (sensitivity greater than or equal to 20 pg/ml) monoclonal-based ELISA. Pretreatment eosinophil counts ranged from 240 to 1,186 eosinophils/microliter (geometric mean, 675), and the mf counts from 10 to 218 per mg skin (geometric mean, 79). After an initial decline in the peripheral eosinophil count to 28 +/- 8% of pretreatment levels at 8 h after beginning treatment, the eosinophil counts steadily increased over the next 2 wk, reaching a maximum at 14 d (257 +/- 38% of pretreatment levels). Serum levels of IL-5 rose sharply from pretreatment levels to a peak of 70.5 +/- 11 pg/ml by 24 h after treatment. Serum IL-5 remained elevated over the next 2-3 d and declined toward baseline by approximately 6 d after treatment, at which time the eosinophil levels were steadily increasing. IL-3 and granulocyte macrophage colony-stimulating factor, two other cytokines implicated in eosinophilopoeisis, were not detectable in the serum at any time before or after treatment. The rise in serum IL-5 before the posttreatment eosinophilia seen in this group of patients with onchocerciasis demonstrates a temporal relationship between IL-5 and the subsequent development of eosinophilia and implicates IL-5 as an important mediator of eosinophilia in humans.