Altered interleukin-1 and tumor necrosis factor production and ...

Altered interleukin1 and tumor necrosis factor production and secretion during pyrogenic tolerance to LPS in rabbits GO WAKABAYASHI, JOSEPH G. CANNON, JEFFREY A. GELFAND, BURTON D. CLARK, KOICHI AIURA, JOHN F. BURKE, SHELDON M. WOLFF?, AND CHARLES A. DINARELLO Division of Geographic Medicine and Infectious Disease, Department of Medicine, Tufts University and New England Medical Center Hospital, Boston 02111; and Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114 Wakabayashi, Go, Joseph G. Cannon, Jeffrey A. Gelfand, Burton D. Clark, Koichi Aiura, John F. Burke, Sheldon M. Wolff, and Charles A. Dinarello. Altered interleukin-1 and tumor necrosis factor production and secretion during pyrogenic tolerance to LPS in rabbits. Am. J. Physiol. 267 (Regulatory Integrative Comp. Physiol. 36): R329R336, 1994.-Rabbits were injected intravenously with 10 &kg of endotoxin [lipopolysaccharide (LPS)] on days 0, 1, and 7, and rectal temperatures were monitored. The febrile responses were compared with circulating levels of interleukin-1P (IL-lp) and tumor necrosis factor (TNF) and in vitro synthesis of these cytokines by peripheral blood mononuclear cells (PBMC) isolated just before the injection of LPS. Fever after the first LPS injection was biphasic on day 0, attenuated and monophasic after the second LPS injection on day 1, and augmented after third injection of LPS on day 7. On day 1, circulating TNF and IL-lp levels were significantly (P < 0.05) decreased compared with those on days 0 and 7. Similarly, TNF and IL-ll3 synthesis by LPS-stimulated PBMC were significantly reduced on day 1. On day 7, cellular synthesis and secretion of IL- l(s were significantly increased compared with that on day 0. A significant positive correlation was observed between fever index and total in vitro IL-lp synthesis by LPS-stimulated PBMC (r = 0.866, P = 0.001). These data demonstrate that pyrogenic tolerance in the rabbit after a single LPS injection is associated with decreased circulating IL-lp and TNF levels as well as decreased production of these cytokines in vitro. In addition, the pyrogenic hyperresponsiveness to LPS after 7 days is associated with increased synthesis and secretion of IL- 1 l3 from PBMC in vitro. fever; cytokines; mononuclear cell; rabbit; protein synthesis; secretion

THE INITIAL EXPERIMENTS by Beeson (2) on tolerance to bacterial endotoxins [lipopolysaccharide (LPS)] were carried out using the febrile response in rabbits. Daily injections of LPS or bacterial suspensions rendered the animals hyporesponsive (tolerant) to the fever-inducing property of these agents. In the subsequent years, the pyrogenic response to LPS and other pyrogens has been attributed, in part, to changes in host-derived cytokines, namely interleukin-1 (IL-l), tumor necrosis factor (TNF), and/or IL-6 (reviewed in Ref. 20). In addition to fever, TNF and IL-l play a prominent role in the toxic and lethal effects of LPS. For example, passive immunization of mice with antisera against TNF increased survival following a lethal dose of LPS (3), and anti-TNF monoclonal antibody prevented septic shock and death induced by Escherichia coli bacteremia in primates (30).

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Similar data in mice, rabbits, and primates have been reported by blocking IL-l, using the IL-l receptor antagonist (reviewed in Ref. 11). Pretreatment with a small dose of LPS renders animals refractory to subsequent challenge by LPS or unrelated toxic insults. The same nonspecific resistance can be induced by administering small doses of IL-l or TNF 24 h before a lethal infection. Indeed, part of the mechanism by which LPS induces a state of tolerance is likely to be due, in part, to cytokines themselves (32). Although the precise mechanism of tolerance to LPS is not fully understood, studies suggest a prominent role for downregulation of macrophage function (reviewed in Ref. 23). Tissue macrophages from LPS-tolerant animals exhibit decreased production of “endogenous pyrogen” activity (10, 16). Production of granulocyte macrophage colony-stimulating factor (GM-CSF) by human adherent monocytes decreases after repeated exposure to LPS in vitro (29). Bone marrow cells from LPStolerant mice contain an increased number of macrophage progenitor cells, suggesting that LPS tolerance is related to a failure of these immature cell types to respond to LPS (22). In the present study, a rabbit model of LPS tolerance was used, in which a single injection of LPS induced pyrogenic hyporesponsiveness to a subsequent injection of LPS given 24 h later. Rabbits rechallenged after 7 days had more fever to the same dose of LPS than to the first injection. The febrile responses were compared with circulating levels of TNF and IL-lp, as well as to their total in vitro production from peripheral blood mononuclear cells (PBMC). In addition, the kinetics of synthesis and secretion of these cytokines in vitro during the hyporesponsive and hyperresponsive phases were studied. MATERIALS

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Fever. Female New Zealand-derived White rabbits weighing 3-4.0 kg were puchased from Pine Acres, Burlington, VT. Training and pyrogen testing took place in special restraining devices that allow free exchange of body heat as well as minimal neck restriction. The first two days of training were without rectal thermometers, whereas, 3 days before the experiments, rabbits were trained with flexible indwelling rectal thermistors (Yellow Springs Instruments, Yellow Springs, OH), which do not restrict defecation. Rectal temperatures were measured using these thermistors connected to a Scanning Tele-Thermometer (model 47, Yellow Springs Instruments) as described previously (9). Rabbits were injected with LPS (10 pg/kg, E. coli 0127:B8, Sigma, St. Louis, MO) via a marginal ear vein at 9:30 A.M. on days 0, 1, and 7.

o 1994 the American

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Clearance of 1251-labeled IL-l p from circulation. Rabbit IL-lp was labeled with sodium lz51 as previously described (8). After an intravenous bolus injection of 1 x lo6 counts/min (cpm) into anesthetized rabbits, blood samples were taken from an angiocatheter placed in the contralateral ear artery. Immediately after the bolus injection, a sample of blood was taken from the arterial catheter, and the cpm precipitated in this sample (t = 0) were assigned a value of 100%. After 15 and 30 s and 1, 1.5, 2, 3, 5, 7, 10, 20, 40, 60, 90, 120, and 180 min, additional blood samples were taken. Each sample was precipitated using 90% trichloroacetic acid (TCA), and the precipitates were separated by centrifugation (10,000 g x 2 min) and counted. After the 180-min sample, rabbits were killed, and organs were removed, weighed, and homogenized as described previously (8). Each tissue homogenate was precipitated with TCA, and the precipitates were separated by centrifugation (10,000 g x 2 min). Gel filtration over Sephadex G-75 (fine, 120 x 1.5 cm) was used to determine molecular weight of labeled products. The supernates were discarded, as they contained free iodine and/or small molecular mass fragments ( < 6,000 daltons) of 1251-labeled rabbit IL-l p; the precipitates were counted in a gamma counter and represent the unfragmented (17,000 dalton) labeled rabbit IL-lp. Preparation and incubation of PBMC. Rabbits intended for fever studies were bled at 9 A.M., just before the first (day O), second (day 1 ), and third (day 7) injection of LPS (10 kg/kg). Rabbit blood (10 ml) was taken from the central ear artery using a 19-gauge needle into a heparinized syringe (5 U/ml of blood final concn), and PBMC were isolated by density gradient centrifugation (400 g, 40 min) on Ficoll-Hypaque (Ficoll Type 400, Sigma; Hypaque-M, 90%, Winthrop Breon, New York, NY). Because rabbit leukocytes are denser than human leukocytes, the yield of leukocytes isolated with FicollHypaque is lower than that of humans (13). However, to reduce the number of polymorphonuclear cells (PMN) in PBMC preparations, Ficoll-Hypaque, with a density of 1.077 g/ml, was used instead of a higher density-gradient medium (Histopaque; density = 1.103 g/ml) (13). Whole blood was diluted 1:2 with pyrogen-free saline and underlayered with the Ficoll-Hypaque mixture. After centrifugation, the PBMC layer was aspirated, washed twice with saline, and resuspended in RPM1 1640 culture medium (Whittaker, Walkersville, MD), supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 &ml streptomycin, and 1 pg/ml indomethacin. RPMI had been subjected to ultrafiltration to remove microbial contaminants (26). Differential cell counts were assessed and cells were stained with following cytocentrifugation, cx-naphthyl acetate esterase and Mayer’s hematoxylin (Sigma). The numbers of esterase-positive cells were calculated in each PBMC preparation. The numbers of PMN were measured in each PBMC preparation. Because the number of contaminating PMN in PBMC preparations was different on day 1 (40.3 + 5.5%) from that on day 0 (16.4 + 4.4) and on day 7 (18.6 + 3.8), the number of mononuclear cells was adjusted accordingly to the differential cell count in each PBMC preparation to a concentration of 5 x 106/ml. The proportion of monocytes in PBMC preparations, which was determined by cx-naphthyl acetate esterase staining, did not differ between day 0 (17.0 + 1.6), day 1 (16.3 + 2.1), and day 7 (18.2 + 1.7%). Although PMN produce IL-l as well as IL-l inhibitors, which affect IL-l activity in bioassays, contamination by PMN does not interfere with the radioimmunoassay (RIA) of IL-l or TNF; futhermore, the amount of IL-l and TNF produced by PMN is negligible compared with monocytes (2 7). PBMC (5 x 106/ml) were incubated in triplicate 96-well flat-bottom microtiter wells at 37°C for 24 h with concanavalin A (Con A, Sigma, 10 &ml), LPS (5 rig/ml, E. coli 0127:B8,

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Sigma), or heat-killed Staphylococcus epidermidis (20 organisms/leukocyte) (34). One hundred microliters of cells were added to 100 ~1 of stimulant; the final concentration of stimulants in the PBMC culture is indicated. The final concentration of PBMC was 2.5 x 106/ml. To investigate the kinetics of synthesis and secretion of these cytokines, PBMC were prepared as described above but incubated for 1,3,6, or 24 h with LPS (5 rig/ml). At the end of each incubation period, the supernatants were aspirated, and then both the 96-well culture plate with the cells and the supernatants were stored at -70°C until assayed. The cell content of cytokines was determined after three freeze-thaw cycles, a method that has been validated using human PBMC cultures to determine cell-associated cytokines (12, 21, 28). This method has also been used for similar determinations in rabbit PBMC cultures (5, 8, 34). Supernatants and cellassociated lysates were pooled separately from duplicate wells before assay. Circulating TN% and IL-l@. To determine circulating TNF and IL-lp levels after LPS injections, a separate group of rabbits was injected intravenously with LPS (10 kg/kg) at 9 A.M. on days 0, 1, and 7, and blood (2 ml) was taken from the ear artery before and every 60 min after the LPS injection. Blood samples were collected in tubes containing EDTA and aprotinin (0.67 trypsin inhibitor unit/ml of blood) for plasma IL-ll3 measurement as previously described (6). Glass tubes with no additives were used for generating serum for TNF measurement. Plasma and serum samples were obtained after centrifugation (600 g for 20 min) and stored at -70°C until assayed. RIA for rabbit IL-1 p. Rabbit IL-lp was determined by a specific RIA (8) using goat antibody raised against recombinant rabbit IL-lp, as described previously (5). Rabbit IL-ll3 was iodinated by a modification of the chloramine T method and purified by using a Sephadex G-25 column (Bio-Rad, Richmond, CA). The sensitivity of the RIA was 90-120 pg/ml (95% binding) with 50% displacement at 1,500-2,000 pg/ml. The procedures for collection and extraction for detection of IL-lp in human plasma by RIA have been optimized (6), and the same procedures were used in the present studies for rabbit plasma. One milliliter of chloroform (Fisher Scientific, Fair Lawn, NJ) was added to 0.5 ml of plasma in 1.5-ml microfuge tubes. The tubes were agitated horizontally for 5 min at room temperature on a multitube vortexer (American Hospital Supply, Miami, FL) and spun for 5 min at 10,000 g at 4°C. The aqueous phase was recovered, extracted a second time with 1.0 ml of chloroform, and the aqueous phase was removed and assayed in the RIA. To investigate the effect of extraction, rabbits were injected with heat-killed S. epidermidis (2.0 x lOlo organisms), and blood was taken before and every 60 min for 4 h after the injection of the organisms. Plasma was obtained in EDTA with aprotinin. The plasma was split: one-half was assayed after the extraction as described above, and the other half was assayed directly in the RIA. The extraction of the rabbit plasma increased the IL- 1 p immunoreactivity (Fig. 1). In previous studies, there was a direct correlation (r = 0.84, P < 0.001) between the levels of IL-ll3 vs. IL-la in rabbit plasma after the injection of S. epidermidis, although the levels of IL-la were approximately one-third less than levels of IL-ll3 (34). Bioassay for TN%. TNF activity was measured by the cytotoxic effect on the L929 tumorigenic murine fibroblasts (ATCC CCL 1, American Type Culture Collection, Rockville, MD). Briefly, L929 cells were cultured in flat-bottom, 96-well microtiter plates (Nunc, Roskilde, Denmark) at 4.0 x lo4 cells/well in RPM1 1640 medium containing 5% fetal calf serum (Hyclone, Logan, UT), 2 mM L-glutamine, 100 U penicillin/ml, and 100 l&ml of streptomycin. After incuba-

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Fig. 1. Effect of extraction of detection of IL- l(s levels in rabbit plasma. Rabbits were injected with heat-killed S. epidermidis. Blood was taken before and after bacterial injection and then was divided and tested in radioimmunoassay (RIA) either unextracted or after chloroform extracted (see METHODS).

tion for 18 h, culture medium was removed, and recombinant human TNF (kindly provided by Genentech, South San Francisco, CA) as standards or test samples was added along with actinomycin D (1 pg/well). The cells were then cultured for an additional 20 h. The culture medium was removed, and the cells were stained with 0.1% crystal violet in 100% methanol for 40 min. Absorbance was measured at 600 nm by MR 600 Microelisa Auto Reader (Dynatech, Alexandria, VA). Cytotoxicity was calculated as follows: percent cytotoxicity = [l (absorbance of sample/absorbance of control)] x 100. Fifty percent cytotoxicity was established as 1 U/ml TNF. The lowest concentration of TNF which produced a statistically significant cytotoxic effect was 10 pg/ml of recombinant human TNF, whereas 1 rig/ml resulted in - 50% cytotoxicity (1 U/ml). Samples from PBMC were assayed either undiluted or diluted two- to fivefold with RPMI. Undiluted serum from healthy rabbits was cytotoxic; therefore, rabbit serum samples were diluted lo- to l,OOO-fold with RPM1 before assay on L929 cells. The units of serum TNF were calculated based on cytotoxicity at the 50% level of recombinant human TNF in the standard curve. The concentration of TNF in PBMC cultures was converted to nanograms per milliliter using the TNF standard curve for human TNF. Data analysis. Results were expressed as the means k SE. Statistical significance was determined by one- and two-factor analysis of variance (ANOVA) followed by Fisher’s leastsignificant difference test using StatView software (Abacus Concepts, Calabasas, CA) on a Macintosh SE computer (Apple Computer, Cupertino, CA). RESULTS

Effect of LPS injections on rectal temperature. The first injection (day 0) of 10 kg/kg LPS produced biphasic fevers, with the first fever peak occurring 90 min and the second peak occurring 140-250 min after the injection. As shown in Fig. 2, rectal temperature started to increase after 10 min; the increase in rectal temperature was statistically significant (P < 0.05) from 60 through 360 min after the first injection of LPS. After the second injection of LPS 24 h later, rectal temperature barely increased, although it became statistically significant from 40 to 240 min after the injection. The peak fever on .

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day 1 was dramatically reduced at 0.36 t 0.07”C at 60 min after the injection, and there was no second fever peak in these animals. In contrast to day 0 or day 1, greater fevers were observed after the third LPS injection on day 7. The initial increase in rectal temperature was faster compared with that on day 0 or day 1. The fever on day 7 was significantly higher during 30-50 min and 160-180 min than the elevation in body temperature (ABT) after the first LPS injection on day 0. To obtain a numerical expression of both the height and duration of the fevers, a fever index (36) was determined by the area under each fever curve (ABT) between time 0 and 360 min. Fever indexes were 656 t 156 units on day 0, 151 t 18 units on day 1, and 918 t 219 units on day 7. Comparison between febrile responses and in vitro PBMC IL-@ and TNF synthesis. PBMC isolated just before the first (day 0), second (day 1), or third (day 7) LPS injection were incubated with Con A, LPS, or heat-killed S. epidermidis. IL-lf3 and TNF synthesis in response to LPS were significantly reduced on day 1 compared with those on day 0 and day 7 (Fig. 3). A decrease in IL-lp synthesis in response to S. epidermidis was also observed on day 1. In contrast, IL-l(3 synthesis in response to LPS or S. epidermidis was increased significantly on day 7 compared with that on day 0. However, TNF synthesis in response to Con A or S. epidermidis did not differ significantly between day 0, day 1, and day 7. To evaluate the effect of the stress of removing 10 ml of blood and the intravenous injection procedure on subsequent production of cytokines by PBMC, 10 ml of blood were taken from the central ear arteries of three rabbits as described in METHODS, followed by an intravenous injection of pyrogen-free saline. After 24 h, a second sample of 10 ml of blood was removed. PBMC production of IL-lp and TNF was compared using LPS and S. epidermidis as stimulants as described in Fig. 3. There were no statistically significant differences in the production of IL-lp associated with the stress of bleed-

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MINUTES Fig. 2. Rectal temperatures after lipopolysaccharide (LPS) injections. Rectal temperature was monitored in awake rabbits (n = 5) after iv LPS (10 l&kg). There were statistically significant (P < 0.05) differences in body temperature elevation (ABT) between days studied as indicated by brackets labeled a, b, and c. ABT was significantly different day 0 vs. day 1 (a); day 0 vs. thy 7 (b), and du.y 1 vs. du.y 7 (4.

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Fig. 3. Total interleukin (IL)-ll3 synthesis and tumor necrosis factor (T&F) synthesis from peripheral -blood mononuclear cells (PBMC). PBMC from rabbits shown in Fig. 2 (n = 5) were obtained before LPS injection and stimulated with either concanavalin (Con A), LPS, or heat-killed S. epidermidis. Total IL-lp and TNF synthesis (secreted plus cell associated) was measured by RIA for rabbit IL- 1 l3 or by L929 assay for TNF. *P < 0.05, day 0 vs. 1 or day 0 vs. 7.

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IL-lp levels (Fig. 5B) were unchanged 60 min after LPS but rose after 120 min, and peak elevation occurred 180 min after the injection of LPS. This pattern is similar to that reported following injection of E. coli or S. epidermidis organisms into rabbits (33, 34). As shown in Fig. 5B, at each time point, plasma IL- 1 l.!!levels were lower on day 1 compared with day 0 or day 7 (P = 0.0027, Z-factor repeated-measures ANOVA), suggesting that total circulating IL-lp in response to LPS was diminished 24 h following the first exposure to LPS. Plasma IL-l l3 levels at 120 min on day 7 were significantly higher (P < 0.05) than those of day 1. To ascertain whether a previous cytokinemia-induced “tolerance” affected the clearance of cytokines following a second challenge, rabbits were injected with 1251-IL-lp, and blood clearance and organ distribution were determined. Control rabbits (n = 5) were given a bolus injection of saline; other rabbits (n = 4) were made “tolerant” with an intravenous injection of S. epidermidis (2.0 x lOlo organisms). This bacteremia induces more circulating TNF and IL-l than an injection of LPS (34). After 24 h, both groups received an intravenous bolus injection of 1 x lo6 cpm of rabbit 1251-IL-l(3. The mean cpm in the TCA precipitates of whole blood at t = 0 was used as the baseline (set at 100%). There was a rapid decrease in the mean cpm following the injection, and the lo-min samples contained - 10% of cpm at t = 0. There were no differences in percentage of TCA-precipiA 2000-

ing (for day 0 vs. 1, respectively: LPS = 3.097 t 1.607 vs. 2.662 t 0.644 rig/ml; S. epidermidis = 1.154 t 0.898 vs. 0.83 t 0.185 rig/ml). There were also no differences in TNF production in these experiments (for day 0 vs. 1: LPS = 13.675 t 5.842 vs. 12.962 t 6.557 rig/ml; S. epidermidis = 15.350 t 8.174 vs. 15.550 t 6.469 ng/mU

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As shown in Fig. 4, a significant positive correlation was observed between fever index and in vitro IL-1p production in response to LPS (r = 0.866, P = 0.0001). There was also correlation between fever index and in vitro TNF production in response to LPS (r = 0.669, P = 0.0064). However, the correlation of fever with TNF, although statistically significant, was less than that with IL-lp production in vitro. Circulating IL-l p and TNF after repeated LPS injection. In different rabbits from those used for the fever studies, circulating TNF and IL-lp levels were determined in the same experimental model. Serum TNF levels reached peak elevation at 60 min after the injection of LPS and then declined to levels below the detection limit by 180 min in all animals studied (Fig. 5A). The time course of these changes in rabbit serum TNF levels is consistent with those after LPS injection into humans. TNF levels at 60 and 120 min after the second LPS injection on day 1 were significantly decreased compared with levels after the first injection. There were no differences in TNF levels between day 0 and day 7.

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index and in vitro IL-lp and TNF correlation was observed between Fig. 2 and IL-l p synthesis from P = 0.0002). Correlation between less (r = 0.669, P = 0.006).

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tion were incubated with LPS for 1, 3, 6, and 24 h; the secreted portion of IL-lp and TNF was determined. Total IL-ll3 and TNF concentrations (secreted and cell associated) were also determined. The kinetics of in vitro IL-ll3 and TNF response to LPS varied on each of the days studied (Fig. 6). On day 0, total TNF synthesis (Fig. 6A) after 3 h of incubation reached 64.8 t 13.5% of the 24-h total synthesis, whereas total IL-ll3 synthesis (Fig. 6B) after 3 h of incubation was 22.6 t 3.9% of the 24 h total synthesis. After 24-h incubation on day 0, 94.7 2 2.5% of the total TNF synthesis was secreted, whereas 61.3 t 9.0% of the total IL-l(s synthesis was secreted. These data are consistent with the kinetics of

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Time [minutes] Fig. 5. Serum TNF and plasma IL-lp levels in rabbits after LPS injections. A: serum TNF levels from rabbits (n = 5) were determined by L929 cytotoxicity assay. *P < 0.05, significant differences were observed between day 0 and day 7. “fP < 0.05, significantly less on day 1 than day 0. B: plasma IL-l(3 levels from same rabbits were determined by RIA after chloroform extraction. *P < 0.05, significant differences were observed between day 1 and day 0 or day 7. $P < 0.05, significantly more on day 7 compared with day 1 (factorial ANOVA).

tated cpm from the blood between the two groups and any time period. After 180 min, the rabbits were killed, and liver, lung, kidney, spleen, heart, and urine (via a catheter) were removed. Total TCA precipitation in each organ was calculated using cpm per gram of tissue per weight of organ; the percent recovery in each organ was compared with the total cpm injected (1 x IO6 cpm). Radiolabeled IL-lp precipitated from the urine represented 1540% of the total cpm injected, the liver contained 10-15, the kidney 3-5, the spleen 0.5-1, and the heart