Inflammation 147

15.33 OXIDATIVE STRESS OCCURS IN SKIN IN THE PRE- IMMUNOLOGIC PHASE OF THE CELL-MEDIATED IMMUNE RESPONSE TO AIJ.,ERGENIC 2,4-DINITROHALOBENZENES Richard J. Schmidt and Lip Yong Chang Welsh School of Pharmacy, UWCC, PO Box 13, Cardiff CF1 3XF, U.K.

The 2,4-dinitrohalobenzenes (DN[Halo]Bs) are well known contact allergens. They are believed to be haptens which bind to skin protein by electrophilic attack at exposed uncleophilic centres such as -NH 2 and -SH groups. We have applied these allergens and related non- sensitizing analogues to mouse skin and later sacrificed the animals and examined changes in coloar, glutathione status, protein content, and glutathione S-transferase activity of microsomal and cytosolic fractions. Only DNFB caused a yellowing of skin and of skin fractions and a significant (P < 0.05) lowering of glutathione (GSH) levels to 39% of control that would be consistent with electrophilic attack; DNCB, DNBB, and DNIB caused a significant elevation of both GSH and glutathione disulphide (GSSG) levels to 135-161% and 131-157% of control respectively whilst non-sensitizing analogues failed to perturb glutathione status. Microsomal and cytosolic protein levels were significantly elevated by all four sensitizing DN[Halo]Bs; and, except in the case of DNFB which caused a decrease in cytosolic glutathione S-transferase (GST) activity to 55% of control, GST activities of both eytosolic and microsomal fractions were generally increased by DN[Halo]Bs to 138-166% of control in cytosolic fractions and to 185-325% of control in microsomal fractions. Our results clearly indicate that mouse skin exposed in vivo to allergenic DN[I-Ialo]Bs undergoes a classic oxidative stress response. They also implicate free radical species as the ultimate haptens in the case of DNCB, DNBB, and DNIB and suggest that the generation of oxidative stress may be an essential prerequisite to the expression of the cell-mediated immune response.

OXYGEN RADICALS MEDIATE IL-I-DEPENDENT CARTILA- GE MATRIX DEGRADATION THROUGH AUTOCRINE MECHAN- ISMS Masao Shingu, Masashi Nobunaga, Satoshi Shioka- wa, and Masahiro Yamamoto Medical I ns t i t u te of Bioregulat ion, Kyushu Uni- vers i ty 69, Beppu, 874 Japan

We have studied the mechanisms by which IL-I mediates car t i lage matrix degradation(CMD) and whether and how oxygen radicals are invo lv- ed in IL-l-dependent CMD, Chondrocytes obta- ined from the knee j o i n t of os teoar th r i t i s patients were cultured in 24-well dishes and label led with 35S, These cel ls were cultured in Hank's buffer with IL-I in the presence or absence of various scavengers and inh ib i to rs • CMD was expressed as % 35S release• I L - I - dependent 35S release by chondrocytes was signif icantly suppressed by the addition of catalase or methionine in advance. The addition of EDTA or tissue inhibitor of metalloproteina- ses(TIMP) signif icantly suppressed 35S release, but alpha-l-trypsin inhibitor did not. Collage- nase act iv i ty of the supernatants was s igni f ic- antly inhibited by catalase or EDTA. These findings were also obtained in the absence of exogenous IL-I. These results suggest that IL-l is involved in CMD through autocrine and paracrine mechanisms and that some oxygen radical species mediate IL-l-dependent CMD by autocrine mechanism.

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15.35 FAILURE OF SUPEROXIDE DISMUTASE (SOD) AND CATALASE (CT) TO PREVENT THE IMMUNOSUPPRESSION INDUCED BY UVB-IRRADIATED EPIDERMAL LANGERHANS CELLS (LC). J.C. Simon, P.R. Bergstresser, P.D. Cruz. Department of Dermatology, UT Southwestern Medical Center, Dallas, TX.

UVB irradiation causes profound morphologic and functional alterations in LC; in particular UVB converts the ab i l i t y of LC to induce Ag-specific T cell-mediated responses in vivo (J Invest Dermatol 92:160) and in v i t ro (J Invest Dermatol 94:5797 to suppression. UVB also generates free 02 radicals (02R 7 within epidermis and the %R quencher, SOD, prevents UVB-induced morpholoOic damage of LC (J Invest Dermatol 88:6997. To identi fy a role for 09R in UVB-irradiated LC- induced immunosuppres~ion, we f i r s t examined the effect of topical SOD on a standard assay for contact sensi t iv i ty (CS) in vivo. C3~/HeN mice were sham- or UVB-irradiated (800 J/m~), abdominal skin was sensitized with 0.5% DNFB (day O) and ears were challenged with 0.1% DNFB (day 6). Sham-irradiated mice mounted fu l l CS responses, whereas UVB-irradiated mice showed unresponsiveness; no difference was observed between untreated or SOD-pretreated mice. We next tested the effect of SOD and CT on the capacity of unirradiated or UVB-irradiated LC to ~timulate proliferation of antigen-specific CD4" T cel ls. Unirradiated LC, but not irradia- ted LC, stimulated T cell proli feration; pretreatment of LC with SOD or CT had no effect on this response. We conclude that SOD and CT do not prevent the immunosuppression induced by UVB-irradiated LC.

MODERATE EXERCISE POTENTIATES NEUTROPHIL OXIDATIVE AND MICROBICIDAL ACTIVITIES. John A Smith, Scott J McKenzie, Richard D Telford and Maurice J Weidemann. Department of Biochemistry, Faculty of Science, Australian National University, PO Box 4, Canberra ACT, 2600, Australia.

Neub-ophils constitute the "first-line-of-defence" against infectious agents but have also been implicated in tissue damage caused by inflammatory diseases. Moderate exercise of human subjects causes an increase in the plasma concentrations of some neutrophil-priming cytokines and hormones. Whilst regular moderate exercise may enhance resistance to common infections, intensive training appears to be detrimental. Neutrophil microbicidal activity was assessed in untrained and highly-trained human subjects (n = 9,11) before and after one hour of cycling at 60% of maximum aerobic capacity. Luminol-enhanced chemiluminescence was monitored to peak intensity following stimulation with opsonized zymosan (OZ) or phorbul myristate acetate (PMA) and, in untrained subjects only, their ability to kill Staphylococcus aureus was assessed by following the green 0iving) to red (dead) transition of acridine orange stained bacteria by fluorescence microscopy. Irrespective of training status, exercise caused significant priming of neutrophils to produce H202 and HOCI upon stimulation with OZ (P<0.01) but not with PMA. Killing eapaeity was also primed and this correlated with increased phagocytosis (P<0.01). Compared to untrained individuals, neutrophil oxidative activity of trained subjects was depressed about 50% at low (unit) OZ concentration (P<0.075) but not at saturation. The training treod has been.confirmed by a recent "longitudinal study" conducted over a.l~ month period. Regular moderate exercise may enhance resistance to infection by priming neutrophil phagocytic and killing capacities. In contrast, whilst intensive training may increase susceptibility to infection by diminishing neutrophil oxidative capacity below a critical threshold, it may also reduce inflammatory damage caused by intense exercise by limiting the extent of endogenous neutrophil stimulation.

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