136 / SECOND INTERNATIONAL WORKSHOP ON CYTOKINES

319

lTOWlEATION OF RAlELEf-CERIvEo @3lWlH FKTOR IN A CI~-(X~ MnEL OF THE ALVEOM bwLL. A.R. Body, J. Fangun. J. Everitt and J.C. Baurer. Nat’1 Inst. Bwirm. Hlth. 8ci. and Chart. Ind. Inst. Toxicol.. l&s. Tmole. Pk.. M: 27709

Ws are studying the biology and biochenfstry of ltng ma;xophqa&rived FULY (FKiEB J 2:2272, 1988; AIRY. J. Resp. Cell Fbl. Biol., In Press, 1989). A working hypothesis states that KX3F secreted by tract-ophqas an alveolar surfaces stirmlates a mitcgmic response in fibrcblasts of the lung intersti- tiun. ‘There currmtly are r0 data available m v&her cr not BGF will nave fms the alveolar air space to interstitial anpsrtnmt of the lung. To investigate this potentially sigificant pathobiolqical event, we have deve- loped a cell culture system in vhich a cmfluent mnolayer of rat alveolar Type II cells rests upm a nat of Type IV collagen coating bath sides of a Nuclepcre filtff (0.2 pn pore size). On the u&r-side of the filter, early passage rat lmg fitilasts lie upan the collagen n&r-ix and will replicate in log phase m retain @escmt dependirvg upm the culture caditims arployed. KIGF (25q) was ad&d to the uppar chrmbsr, and after 3 days the anxtnts of KGF in the uppsr and 1~ chmbers ware quintified by an enz)nre i-say. POGF readily diffused into the 1~ charber and equilibrium MS reached v&n the collagen tratrix alone hss present. l&en a cmflumt smc- layer of Type II cells vas pwent m the top or b&on of the filtq >90% of the A)GF was excluded frcn entering ths opposite charbe-. In carparism, vrhm Trpe II cell ncnolayers rmained sub-confluent or ware chemically or nechanically injured, PDGF diffused into the lacer charbar and an equilibiun was established. Fit&lasts ~aar m either side of the filter, with no Type II cells p-es&, utilized w degraded mre than BX of the KGF that was initially added to the system. These data suggest that pM;F is not readily translocated across an epithelial terrier-, except under pathological caditims in khich the barrio is injured a incarplete.

380

LOCALIZATION OF IL-15 IN TISSUE FOLLOWING TRAUMA. J.G. Canno& &I&g, M.A. FlatI. USDA Human Nutr. Res. Ctr. and Dept. of Medicine, Tufts Univ., Boston MA 02111

Eccentric exercise was used as a model to study non-infectious trauma in humans. In this damaging form of exercise, a muscle is forced to lengthen as it develops tension, resulting in local inflammation, leukocytosis, insulin insensitivity and proteolysis. Following eccentric exercise, plasma IL-15 levels are elevated for up to 9 hours, but systemic indicators of muscle damage remain elevated for 5 to 10 days. We tested the hypothesis that cytokines may persist in the damaged muscle during this period of time. Muscle samples were obtained by needle biopsy from 7 subjects before, 45 min after, and 5 days after a 45 min session of running downhill on an inclined treadmill. Transverse sections were stained immuno- histochemically for IL-la, IL-15, and TNFa. Samples obtained 45 minutes after, and 5 days after exercise exhibited increased staining for IL-15, compared to pre-exercise samples. The staining occurred only around the capillaries and along the cell membranes, not within the muscle cells themselves. Muscle tissue from 1 subject was homogenized in PBS containing protease inhibitors, and the extract tested for biological activity on DIO cells. Pre-exercise extract had no effect on DlO proliferation, compared to buffer control. The 45 minute- and 5 day- postexercise extracts increased proliferation 18 and 34%, respectively. Little or no staining was observed following exercise with anti-IL-la, anti-TNFa, non-immune rabbit serum, or with anti IL-15 which had been pre- absorbed with 50 kg/ml recombinant IL-15. These data indicate that IL-16 may remain localized in tissue for several days after plasma levels return to baseline. The rapid appearance and the pattern of IL-15 staining suggest that it may not be produced de nova within skeletal muscles themselves.

381

PLASMA IL-15 AND TNFa LEVELS IN HUMANS FOLLOWING THERMAL INJURY: ASSOCIATION WITH SEPTIC AND FEBRILE EPISODES. J.G.Cannon. J.A.Gelfand.. M.T. Heaartv. J.F. Bu&t. USDA Human Nutrition Research Center on Aging and Dept. of Medicine, Tufts University and Massachusetts General Hospital, Boston, MA.021 11.

Burned patients are at great risk of infection because the cutaneous barrier to microbial invasion is damaged and because factors from the burned tissue, and other inflammatory factors, contribute to a state of immunosuppression. In this investigation, 12 patients were studied for 24 f 5 days following injury, with plasma IL-15 and TNFa concentrations determined periodically (4 f 1 day intervals) by RIA. The prevailing cytokine concentrations were 140 f 22 pg/ml for IL-18 and 141 + 24 pglml for TNFa, which are higher than the concentrations usually found in healthy subjects (cl00 pglml). In addition, each of these patients exhibited at least one episode in which IL-15 or TNFa levels rose to more than 3 times the prevailing levels. Patient records were examined retrospectively to determine if these transient increases in IL-15 or TNFa were associated with other clinical parameters. In 7 of the 12 patients, the spikes in plasma cytokine levels coincided (+ 24 hours) with the observation of a positive blood culture. In 6 of the 12 patients, cytokine spikes preceded a worsening of clinical status as assessed by APACHE II scores. In 24 episodes of fever glO2”F), 6 coincided with elevated TNFa levels (>200 pglml), 6 coincided with elevated IL-18 levels, and 6 coincided with elevations in both cytokines. These data indicate that changes in circulating cytokine levels are related to changes in clinical status and suggest that routine monitoring of cytokine levels may, in some situations, prove to have prognostic value.

382

RADIOIMMUNOASSAY OF INTERLEUKIN-10 AND B AND THE CHARACTER- ISATION OF THEIR PLASMA IMMUNOREACTIVE FORMS. s J Capper, T H Mander and S Kalinka. ___ Amersham International plc, Life Sciences Business, Forest Farm, Whitchurch, Cardiff, CF4 7YT, UK.

We have developed specific and sensitive radioimmuno- assays for both Interleukin-la (IL-la) and Interleukin-16 (IL-16). The assay ranges are: IL-0 (0.5-lbfmol/tube, sensitivity 0.25fmolitube); IL-15 (0.3-4Ofmol/tube, sensitivity O.ZZfmol/tube). We have applied these assays to normal human plasma and found levels of immunoreactivity of 13-24fmol/ml for IL-131 and 18-32fmoliml for IL-15. To further characterise the nature of plasma IL-1 we subjected the plasma to G-75 and TSKZOOO column chromatography. The IL-1 immunoreactive material appeared in the void volume with G-75 and TSKZOOO. This suggested to us that plasma IL-1 is protein-bound or aggregated. To show whether endogenous IL-13 and IL-16 are found in plasma and that we are not measuring interference, we chromatographed plasma on TX2000 at acid pH(2.5). The immunoreactive peak moved to that corresponding to IL-l standard suggesting we had dissociated the IL-1 from a binding protein. It has been reported that a2-macroglobulin binds cytokines and has the molecular weight to correspond to our void volume immuno- reactive peak. Further studies will hopefully confirm whether a2-macroglobulin is the plasma binding protein.

383

PURIFICATION AND CHABACTERIZATION OF MONOCLONAL AND POLY- CLONAL ANTIBODIES TO NEUTROPBIL ACTIVATION PEPTIDE (NAP-l). THE DEVELOPMENT OF HIGHLY SENSITIVE ELISA- METHODS FOR THE DETEBMINATION OF NAP-l AND ANTI-NAP-l ANTIBODIES. Hiroslav Ceska, Friedrich Effenberger, Peter Peichl and Edith Pursch. Sandoz Forschungsinstitut A-1235 Vienna, Austria.

Monoclonal as well as polyclonal antibodies against NAP-l were purified by a combination of various biochemical purification techniques, including immunoaffinity chroma- tography. The purified antibodies were characterized and used in the development of ELISA-techniques. These tech- niques were with success employed to measure NAP-l con- centration at nanogram per milliliter levels in several biological samples. The double-ligand ELISA method for the determination of anti-NAP-l was developed and used to monitor the purification of anti-NAP-l antibodies from various species. The immunosorbent purified anti-NAP-l antibodies were also used to map the NAP-l determinants.

384

RELATIONSHIP BETWEEN INTERLEUKIN-1, INTERLEUKIN-6 AND PYREXIA IN BURNED CHILDREN, C B Ch' d Halt and S. J. Honkins North Western Injury and Rheumatic Diseases Research Centres, University of Manchester, U.K.

The aim of this study was to determine whether plasma and tissue levels of IL-1 or IL-6 were raised following burn injury in children with pyrexia. Plasma was collected from 10 healthy controls, aged 3-48 months (median 10.5). Blister fluid and serial plasma samples were collected from 6 patients, aged 9-84 months (median ZO), with 10-44X burns. Rectal temperature (Tr) was continuously monitored. Following removal of inhibitors, interleukin-1 (IL-l) was assayed using the DlO(N4)M cell line and interleukin-6 (IL- 6) was assayed using the B9 hybridoma cell line. Each assay was sensitive to plasma cytokine concentrations of <lO pg/ml. The mean plasma concentration of IL-6 in controls was 25.4 + 4.9 pg/ml (mean + SD). Plasma IL-6 was elevated in 4 patients on admission and rose in all patients to a median peak concentration of 194 pg/ml (range 73-4264) 12-24hr after the burn. Values of IL-6 were positively correlated with Tr in 5 patients from whom at least 4 samples were 5~:~nrr,'m"eth','~~~~t~f~~~~i~~~~ ;;g&f;,t '(y y;l;ne; << 0.001, 28 pairs). The concentration of IL-6 in blis'&r fluid collected on admission was only slightly elevated comnared to control plasma but was markedlv increased in later samples. IL-1 ;as not detectable in plasma but was present in blister fluid. Therefore, although IL-1 and IL-6 are present in wound exudate, only IL-6- is present in significant concentrations in plasma and is related to body temperature in burned children.