The Mechanism of Action of a Single Dose of

Methylprednisolone on Acute Inflammation In Vivo

STANLEYL. WIENEm, RoY WIENER, MORTONURIVETZKY, SHULAMrI SHAFER,HENRYD. ISENBERG, CALVIN JANOV, and EDWARDMEILMAN

From the Department of Medicine Research Laboratories, Long IslandJewish-Hillside Medical Center, NewHyde Park, NewYork 11040 (a clinicalcampus of Stony Brook Medical School, State University of NewYork)

A B S T R A C T A model system for the study of in-flammation in vivo has been developed using the 16-hpolyvinyl sponge implant in the rat. This system allowsfor simultaneous measurement of in vivo chemotaxis,volume of fluid influx, and fluid concentrations oflysosomal and lactic dehydrogenase (LDH) enzymes.In addition, the enzyme content of inflammatory fluidneutrophils may also be determined.

A parallel time course of neutrophil and lysosomalenzyme influx into sponge implants was observed. Thiswas characterized by an initial lag phase and a rapidincrease between 5 and 16 h.

The origin of supernatant LDH and lysosomal en-zymes was studied with anti-neutrophil serum to pro-duce agranulocytic rats. Inflammatory fluid in theserats was almost acellular and contained decreasedconcentrations of beta glucuronidase (-96%) andLDH (-74%).

In control rats all of the supernatant beta glucuroni-dase could be accounted for by cell death and lysis, asestimated from measurements of soluble DNA. Only 15-20% of the LDH activity could be accounted for on thebasis of cell lysis. The remainder was derived fromneutrophil-mediated injury to connective tissue cells.

Large intravascular doses of methylprednisolonemarkedly inhibited neutrophil influx into sponges andadjacent connective tissue. Secondary to decreasedneutrophil influx, fewer neutrophils were available forlysis, and lysosomal enzyme levels in inflammatory fluiddecreased.

No evidence for intracellular or extracellular stabili-zation of neutrophil lysosomal granules by methylpred-nisolone was found.

Received for publication 8 January 1975 and in revisedform 6 May 1975.

INTRODUCTIONCorticosteroids have been studied extensively in re-

spect to their effects on the acute inflammatory reaction.In various in vivo models of acute inflammation they

have been shown to inhibit neutrophil accumulation ininjured tissue. This has been demonstrated in the ratafter carrageenin injections into the paw (1) bypolyvinyl sponge implantation (2), and by subcutane-ous injection of air and carboxymethylcellulose (3).In man, a similar effect has been noted with Rebuckskin windows (4, 5) or Senn plastic skin chambers(6). Steroid-treated mice have shown decreased amountsof tissue neutrophils after subcutaneous injections ofcotton dust and Staphylococcus aureus (7).

Paramethasone (1 mg/kg orally) (1), methylpred-nisolone (30 mg/kg i.v.) (2), cortisol (2-3 mg/kgi.v.) (4), prednisone (0.5-1.5 mg/kg daily orally)(5, 6), and prednisolone (100 mg/kg i.p.) (7) were

among the drugs used in these in vivo studies. Tissuefluid levels of these steroids were not measured.

In vitro exposure of neutrophils to high concentra-tions of hydrocortisone succinate (100 Ag/ml) or

methylprednisolone (50 Ag/ml) inhibited rabbit neu-

trophil migration in Boyden chambers (8). However,Borel was unable to inhibit rabbit neutrophil chemo-taxis in vitro at hydrocortisone concentrations of 100Ag/ml (9). He has stated that higher in vitro concen-

trations were cytotoxic. Ketchel, Favour, and Sturgis,using human neutrophils, showed inhibition of migra-tion in capillary tubes after exposure of cells to 2-10Ag/ml of hydrocortisone (10). Perper, Sanda, Chinea,and Oronsky (1) have shown decreased chemotaxis inBoyden chambers of neutrophils isolated from steroid-treated rats.

The Journal of Clinical Investigation Volume 56 September 1975- 679-689 679

In vivo studies of lysosomal enzyme release from in-flammatory areas have involved the injection of car-rageenin or nystatin into the paws of rats. A perfu-sion technique was then used to collect inflammatoryfluid for enzyme assays (11). Dexamethasone (100Ag/kg, orally) and beta methasone (100 ytg/kg, orally)decreased lysosomal enzyme levels in perfusates. Pred-nisolone (1 mg/kg orally) had no effect. Lysosomalenzymes have also been extracted by homogenizationof rat paws afflicted with adjuvant arthritis. Hydro-cortisone (20 mg/kg, orally) decreased enzyme levelsin these extracts (12).

In vitro studies have failed to show steroid pro-tection of human neutrophil lysosomal membranes fromthermal or detergent injury (13).

Slight protection of guinea pig neutrophil lysosomesagainst hypotonic lysis has been demonstrated in vitroby Ignarro and Colombo (14). Goldstein has shownthat steroids inhibited release of lysosomal enzymesfrom cytochalasin B-treated human neutrophils in vitro(15).

The implantation of polyvinyl sponges for 16 h hasbeen shown to be useful for the study of in vivo chemo-taxis (16). We have extended the usefulness of thismodel by making simultaneous measurements of spongefluid neutrophil content, volume, protein, and solubleenzyme concentrations, and the intracellular enzymecontents of fluid neutrophils. Wehave used this systemto study the effects of antineutrophil serum (ANS)'and a single injection of methylprednisolone (1-60mg/kg) on these parameters.

These studies made possible the evaluation of therelative quantitative importance of methylprednisoloneeffects on neutrophil chemotaxis, lysosomal granulestabilization, and lysosomal enzyme secretion in themediation of its anti-inflammatory effect.

METHODSMaterials

Drugs. Methylprednisolone sodium succinate in mix-o-vials with diluent was a gift of the Upjohn Company(Kalamazoo, Mich.). Nembutal sodium was puchased fromAbbott Laboratories (North Chicago, Ill.).

Chemicals. Phenolphthalein monoglucuronic acid, sodiumpyruvate, and NADHwere products of the Sigma Chemi-cal Co. (St. Louis, Mo.). [U-"C]Tyrosine (404 mCi/mmol) and [carboxyl-'4C] dextran (1.28 mCi/g) were pur-chased from New England Nuclear (Boston, Mass.). Ben-zidine and trypan blue were products of Matheson Coleman& Bell (East Rutherford, N. J.). Triton-X-100 was pur-chased from the Rohm and Haas Co. (Philadelphia, Pa.).Yeast transfer RNAwas a product of Schwarz/Mann Div.,

'Abbreviations used in this paper: ANS, antineutrophilserum; IF, sponge inflammatory fluid; LDH, lactic aciddehydrogenase; NS, normal serum; PBS, phosphate-buf-fered saline; WCEE, white cell equivalent of enzymes.

Becton, Dickinson & Co., (Orangeburg, N. Y.). Calf thy-mus DNA was a product of General Biochemicals (Div.,North American Mogul Products Co., Chagrin Falls,Ohio). Electrophoretically pure deoxyribonuclease I waspurchased from Worthington Biochemical Corp. (Freehold,N. J.) and pronase B (nuclease-free) was a product ofCalbiochem (San Diego, Calif.). All other chemicals usedwere of reagent grade.

Animtals. Female Wistar rats (wt range 180-210 g)were used in all experiments. They were purchased fromthe Charles River Breeding Laboratories, Inc. (Wilming-ton, Mass.).

Sponges. Polyvinyl sheets, dry- or formaldehyde-packed,were obtained from the Unipoint Co., High Point, N. C.Dry sponges were cut to a uniform 1.3 X 1.3 X 0.3-cm size(dry wt 30-32 mg) and washed in tap water by 35-50 soak-ings and squeezings. The sponges were then washed in fil-tered deionized water in the same manner, before sterilizationby boiling in deionized water for 30 min before transferto 0.15 M NaCl. Before implantation, wet sponges weredried by compression between two sterile cloth towels. Aresidual of 40-50 ,ul of 0.15 M saline was present in eachof these sponges at the time of implantation. This wasdetermined by weighing 20 sponges dry and after compres-sion, as described. Air-dried, ultraviolet light-sterilizedsponges were also used in some experiments.

ANS and control serum. Preparation and assay. RabbitANS was prepared by a modification of a method describedby Simpson and Ross (17). Rat neutrophils were preparedfrom peritoneal exudates produced by injection of 30 mlof 3%o proteose peptone. The cells were collected in anequal volume of heparinized (10 U/ml) 0.15 M NaCl andthen centrifuged at 700 g for 10 min at 25°C. Red cellswere removed by one or two cycles of hypotonic lysis, andthe neutrophil pellet was washed three times with 9 mlof phosphate-buffered saline (PBS) (0.14 M NaCl, 0.01M phosphate buffer, pH 7.3). Two New Zealand rabbits(wt 3 kg) were each immunized with 1 X 108 cells mixedwith 2 ml of complete Freund's adjuvant. Each dose wasgiven at four subcutaneous sites. Weekly boosters of 1 X 108cells in PBS were given for 5 consecutive wk. In one rabbitthe ANS agglutination titer at 5 wk was 1: 640 and itscytotoxic titer 1: 1,280. This animal was used as a sourceof ANS. Agglutination titers were measured as describedby Simpson and Ross (17) and cytotoxic assays as de-scribed by Simpson and Ross (18) and modified by Millerand Wilson (19). ANS and normal rabbit serum (NS)were absorbed as described by Simpson (17), Millipore-filtered (Millipore Corp., Bedford, Mass.), and frozen at- 20'C in 2.5-ml aliquots before use.

Neutrophil lysosomal granule sonicates. Rat neutrophilswere obtained from 18-h proteose peptone peritoneal exu-dates. From 2 X 108 neutrophils, lysosomal granules wereprepared according to Chodirker, Bock, and Vaughan (20).These granules were suspended in 0.15 M NaCl and lysedby ultrasound (Branson Sonifier, Heat Systems-Ultrasonics,Inc., Plainview, N. Y.) at a setting of speed 3 for 10 s.The Millipore-filtered sonicate was assayed for beta glu-curonidase (21), LDH (22), acidribonuclease (23), cathep-sin D (24), and protein (25).

Sponge model system techniquesSponge implantation was accomplished as previously de-

scribed (16), except that three sponges were implanted oneither side of the midline in the lower thoracic and lumbarregions.

680 Wiener, Wiener, Urivetzky, Shafer, Isenberg, Janov, and Meilman

Sponge removal. Sponges were removed through an ex-tension of the original incision, with Nembutal (AbbottLaboratories) for anesthesia (16). They were placed inpreweighed beakers and reweighed to determine fluid con-tent. Cultures were obtained from all sponge sites. Datafrom infected rats were discarded. Sponge capsular con-nective tissue was biopsied, fixed in 10% formaldehyde, em-bedded in paraffin, sectioned, and stained with hematoxylinand eosin. Sponges were squeezed manually with glovedfingers to obtain fluid free of skin enzymes. The fluid fromthree sponges was pooled, giving two samples for analysisfrom each rat.

Experimental schedules

Time studies. The rats were divided into four groups ofsix to eight animals. Sponges were removed from the firstgroup at 5 h and from the others at 11, 16, and 21 h, re-spectively.

Methylprednisolone studies. Rats were divided intogroups of six to eight animals. Methylprednisolone was in-jected as an intracardiac bolus 5 min before sponge im-plantation. Sponges were removed at 16 h. Group dosageswere 0, 1, 10, 20, 30, and 60 mg/kg.

ANS-treated rats. Agranulocytosis was produced in sixrats given 0.5 ml of ANS i.p. every 4 h for five doses. Allrats had neutrophil counts of less than 100/mm3 beforesponge implantation. A control group was treated with anidentical schedule of injections of NS. Sponges were re-moved in both groups 16 h after implantation. All fluidcultures on agranulocytic and control rats were negative.

Lysosomal enzyme-containing sponges were implantedinto four agranulocytic rats rendered neutropenic as de-scribed above. Two enzyme-containing sponges were im-planted in the left lumbar area and two 0.15 M NaCl-containing sponges in the right lumbar area of each rat.Separate incisions were made on each side. Sponges wereremoved at 16 h. Each sponge contained 300-400 ul oflysosomal enzyme solution. The enzyme solutions used con-tained no LDH activity. The lysosomal enzyme concen-tration of this solution was beta glucuronidase, 3 U/ml(4 X 106/ml); Cathepsin D, 0.1 U/ml (5 X 106/ml); andacid ribonuclease, 14 U/ml (3.5 X 106/ml). The numbers inparentheses indicate the cell numbers required to supplythese enzyme concentrations, as determined from assays ofintact peritoneal neutrophils.

Assays of sponge fluidAliquots were taken from each pool for total and differ-

ential cell counts (Wright-Giemsa stained smears). Totalcounts were done by hemocytometer and checked by Coulterautomatic counting (Coulter Electronics Inc., Hialeah,Fla.). Squeezed sponges were fixed in 10% formaldehydeand processed for histologic study as described. Fluid con-tent per sponge was determined by weighing.

Centrifugation and dilution of sponge fluid. An aliquotof each pool (100-400 ,ul) was centrifuged at 700 g for 20min at 4°C after addition of 10 ,IA of [4C] dextran (30,000cpm). Supernates were removed and diluted 1: 10 withPBS. Pellets were lysed in PBS and 0.1%o Triton-X-100in a volume of 1-1.5 ml.

Assays of inflammatory fluid and blood

Aliquots of diluted supernates and cell lysates were as-sayed for protein (25), hemoglobin (26), and for betaglucuronidase (21, 27), LDH (22), and acid ribonuclease

23). Triton-X-100 was added to supernates to a concen-tration of 0.1% to measure the enzyme content of lysosomalgranules present in this fraction. Aliquots of undilutedsupernate were combined with an equal volume of 10%trichloroacetic acid (TCA) at 4°C. DNAinternal standardswere added to duplicate aliquots. The precipitates werewashed three times with cold 5% TCA with centrifugationat 20,000 g for 5 min after each wash. The precipitateswere then extracted with 5%o TCA for 1 h at 70°C andDNAwas determined (28).

A blood sample was obtained from each rat immediatelyafter sponge removal. Aliquots were used to measurehematocrits, hemoglobins, white counts, and differentialcounts. Heparinized plasma was obtained, as well as a1:10 hemolysate of whole blood in distilled water for de-termination of enzymes, hemoglobin, and protein, as de-scribed above. The hemolysate was used after centrifuga-tion at 1,500 rpm for 15 min.

The data obtained from inflammatory fluid, plasma, andhemolysate assays were inserted into a computer programbased on the equations presented below.

EquationsCalculation of inflammatory fluid (IF) supernate enzyme

concentrations.Eso = EsT- (E8p + ERR) (1)

EXc, corrected IF supernatant enzyme concentration; EsRT,total IF supernatant enzyme concentration; E8p, plasma:derived supernatant enzyme concentration; ERR, hemolysedred cell-derived supernatant enzyme concentration, all inunits per milliliter. Eq. 1 corrects the total enzyme activityof supernatant fluid for contributions due to enzyme activityderived from plasma and hemolysed red cells. The Eqs.2-5 are used to calculate the terms E8P and ERR used inEq. 1. R, and C are calculated by dividing the hemoglobinconcentration of plasma and of inflammatory fluid super-nate, respectively, by the hemoglobin content of a micro-liter of red cells.

EPO= EPT- (RJ X ER) (2)EP, corrected plasma enzyme concentration in units permilliliter; EPT, total plasma enzyme concentration in unitsper milliliter; R, microliters of red cells per milliliter ofplasma; ER, enzyme concentration of red cells in units permicroliter of red cells.

Esp = Epc X(IF protein, mg/ml)

(serum protein, mg/ml) (3)

Gel electrophoresis of plasma and inflammatory fluid pro-teins (29) gave identical patterns. This indicated that therewas no differential diffusion of plasma proteins into thesponge fluid. This provided the rationale for the use ofthis simple ratio to calculate the plasma contribution (EBP)to total supernatant enzyme activity.

EHT - EPT X 103 X (1 - HCT/100)ER = HCT/100 (4)

EHT, total enzyme concentration of hemolysate of wholeblood in units per microliter; HCT, hematocrit in percent.

EsR = C X ER (5)C, microliters of hemolysed red cells per milliliter of in-flammatory fluid supernate.

Mechanism of Action of Methylprednisolone on Acute Inflammation in Vivo 681

Calculation of intracellular enzyme concentrations of in-flammatory fluid neutrophils. An aliquot of each inflam-matory fluid pool was centrifuged for 20 min at 700 g inan International Centrifuge (Damon/IEC Div., DamonCorp., Needham Heights, Mass.) at 4VC after addition of10 IAl of ["C] dextran (30,000 cpm). The supernate wasremoved and the neutrophil pellet was lysed in 1.5 ml ofPBS and 0.1%o Triton-X-100. ["C]dextran counts of neu-trophil lysate and supernate were used to calculate thevolume of supernatant present in the lysate fraction (Eq. 7).Lysate hemoglobin concentration was used to calculate thevolume of red cells present in this fraction (Eq. 8). Eq. 6corrects the cell lysate activity for enzyme content derivedfrom supernatant and intact red cells. Eqs. 7 and 8 calcu-late the terms on the right side of Eq. 6.

ENo = ENT - (ENS + ENR) (6)

ENo, corrected IF neutrophil enzyme concentration of theneutrophil lysate fraction; ENT, total enzyme concentrationof the neutrophil lysate fraction; ENs, supernate-derived en-zyme concentration in the neutrophil lysate fraction; ENR,intact red cell-derived enzyme concentration in the neutro-phil lysate fraction; all in units per milliliter.

jAl supernateENS = mlnutro ate X (EST X 10-3) (7)ml neutrophil lysate

jdl red cellsEivi = .Are el

X ER (8)ml neutrophil lysate

ENCX VNL (9)ENI C = TTy

VNL, volume of neutrophil lysate; TN, total number of cellsin the lysate in millions, based on cell counts done on theinflammatory fluid before centrifugation and lysis; ENIC,intracellular enzyme content of neutrophils in units per 106cells.

Estimation of supernatant enzyme activity dependent onthe presence of neutrophils in the inflammatory fluid.

END= Es - ESCA (10)

END, enzyme concentration in the IF supernate dependenton the presence of neutrophils, in units per milliliter; ESCA,mean enzyme concentration in the IF supernate of agranulo-cytic rats. These fluids are almost acellular and the ac-

S tivity probably is derived from local connective tissuesources.

WCEE -END X VN (11)ENIC

VN, volume of fluid for sponge pool N. WCEE, the whitecell equivalent of the enzyme content of supernatant fluid(number of cells) is a derived parameter. It is a theoreticalestimate of the number of white cells whose death andlysis would be required to provide the excess enzymefound in supernatant fluids of control rats when comparedto agranulocytic rats. This excess activity is dependent onthe presence of neutrophils but is not necessarily dueentirely to neutrophil lysis. An alternative source is neutro-phil-induced injury to local tissue cells with enzyme re-lease. WCEEas calculated attributes the total amount ofexcess enzyme to neutrophil lysis.

Additional studies

Studies of the effect of sponge compression on the me-chanical breakage of cells with possible enzyme releasewere done. Sponge fluid was obtained from 16-h implantsby compression, and duplicate aliquots were taken for cellcounts and enzyme assays. The fluid was then absorbed intofresh dry sponges and resqueezed. All assays were repeated.Decreased cell concentration and increased supernatantLDH were used as indicators of cell lysis.

["C] Serum protein fluxes in sponges in inflammatoryareas were studied. Five rats were given 5 ,uCi of ["C]-tyrosine i.p. 4 days and 1 day before the experiment. The["C]tyrosine labeled the serum proteins and was not circu-lating as free ["C]tyrosine at the time of the study.

10 sponges were implanted in each of four rats withradioactive serum proteins. 10 sponges were also implantedin three unlabeled rats. At 16 h supernates were obtainedby centrifugation from each group.

Dry sterile sponges were used to absorb "C-labeled in-flammatory fluid proteins (300-400 ,ul). These spongeswere placed in the inflamed subcutaneous tissue space ofthe unlabeled rats. Sponges filled with a similar volume ofunlabeled inflammatory fluid were placed in the labeled rats.Sponges were removed from each rat at 1-h intervals upto 4 h. Fluid from all sponges were assayed for ["C]pro-tein content, specific activity (cpm/mg protein) and forenzyme content. Sponge capacity is about 600 ,ul of 0.15 MNaCI.

Enzyme studies. DNase I, 200 U in 1 ml of 0.05 MTris acetate buffer, pH 7, 0.05 M magnesium sulfate wasadded to 1 ml of inflammatory fluid supernate. Incubationwas carried out at 37'C for 2 h. DNA content at zerotime and after incubation was determined as describedabove. Pronase (2 mg/ml in Tris acetate buffer 0.02 M,0.005 M CaCl2) in a volume of 0.05 ml was added to 1 mlof supernate and incubation was carried out for 2 h at45'C. Then pronase was inactivated by heating to 80'Cfor 3 min. After it cooled, DNase was added as above.DNA was determined on aliquots after treatment withDNase alone, with pronase alone, or with pronase fol-lowed by DNase.

RESULTS

The time-course of neutrophil and enzyme influx intosponges implanted in normal rats. Neutrophils beganto appear in sponge inflammatory fluid after 2 h ofimplantation. The relatively slow influx observed dur-ing the first 5 h was accelerated during the intervalbetween 5 and 16 h (Fig. 1A). Results have been ex-

pressed for both neutrophils and enzymes as content persponge (i.e., sponge volume X concentration of neutro-phils or enzymes). Changes in sponge contents of betaglucuronidase (Fig. 1B) and acid ribonuclease (Fig.1C) showed a similar time course to neutrophil influx.Initially, there was a slow accumulation of these en-zymes with a more rapid increase in enzyme contentbetween 5 and 16 h. LDH activity increased linearlyfrom zero time until 16 h and did not show an initiallag period (Fig. 1D).

No significant change in neutrophil or enzyme con-tent occurred between 16 and 21 h. 50% or more of

682 Wiener, Wiener, Urivetzky, Shafer, Isenberg, Janov, and Meilman

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FIGURE 1 Changes in total neutrophil (A), beta glucuroni-dase (B), acid ribonuclease (C), and LDH (D) contentof sponges in control rats over a 21-h period after spongeimplantation. Vertical bars represent ± 1 SEM. Hatchedareas indicate the portion of enzyme activity present inintact cells.

the total sponge enzyme content at all times studied wasfound in the supernatant fraction.

Sponge fluid volume changed most rapidly between0 and 5 h with a more gradual but significant risethereafter (Fig. 2A). Protein concentrations in super-natant fluid were at maximum levels by 5 h and didnot change over the next 16 h (Fig. 2B).

Fig. 3 displays the changes in peripheral blood neu-trophil and total leukocyte counts in these rats as afunction of time after implantation. During the first 5h a marked fall in the total leukocyte concentration oc-curred. During this period the rate of fall of circulatinglymphocytes was greater than that of neutrophils. Afteran initial fall, neutrophil levels rose to zero time valuesat 16 h. During the 16-21-h interval when spongeneutrophil and enzyme contents showed no change,peripheral neutrophil concentrations fell to their lowestlevels. The initial fall in neutrophil levels was mostlikely due to influx into the inflammatory zone. In theexperiments described below sponges were removed at16 h because neutrophil and enzyme contents hadreached a plateau at this time.

Supernatant enzymes and their origins. 16-h inflam-matory fluid supernate was found to contain concentra-tions of LDH, beta glucuronidase, and acid ribonucleasethat were 17, 13, and 6 times higher, respectively, thanthe levels of these enzymes in plasma. Supernatant fluidprotein concentration was only 0.63 of that of plasma.Since all supernatant enzyme concentrations had al-

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Hours

FIGuRE 2 Changes in fluid volume (A) and protein con-centration (B) in sponges from control rats removed overa 21-h period. Vertical bars represent ±-1 SEM.

ready been corrected for plasma and red cell origins byEqs. 1-5, the source of these high enzyme levels ob-served was most likely the neutrophil and the adjacentconnective tissue. Sponges were capable of rapidly ab-sorbing enzymes from the surrounding inflammatoryarea.

The ability of sponge implants to retain ["C] pro-teins is demonstrated in Fig. 4A and to absorb inflam-matory fluid proteins rapidly is shown in Figs. 4Band 4C. When ["C]proteins were implanted in spongesin the inflamed subcutaneous connective tissue of un-labeled rats, no loss of radioactive proteins occurredduring the first 3 h (Fig. 4A). The specific activity ofthe ["C]proteins in these sponges fell rapidly becauseof the rapid sponge uptake of unlabeled proteins (Fig.4B). Fig. 4C shows a rapid uptake of ["C]protein andLDH when sponges were implanted in labeled rats.

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Mechanism of Action of Methylprednisolone on Acute Inflammation in Vivo

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FIGURE 4 (A) The percent change in totalcontent of sponges implanted with labeled inflamproteins in the inflammatory connective tissuerats for 4 h. The initial number of ['C] prcimplanted with each sponge varied in a rang4,382 cpm. (B) Decrease in specific activity csponges shown in A due to influx of unlabeled irfluid proteins present in the area of implantationperiod. The initial specific activity of the implhinflammatory fluid protein was 410 cpm/mg protof [14C]protein specific activity in sponges corlabeled inflammatory fluid implanted in the itconnective tissue of [14C]protein-labeled rats forcontent (lower line) of these sponges also rsorption of locally present enzyme. Vertical b±1 SEM.

ANS-treated rats were used to estimatebution of neutrophils to the supernatant enzypresent at 16 h. Table Ia shows that ANS a

pletely inhibited all neutrophil influx. Without neutro-phils, the supernatant LDH fell 74%, beta glucuroni-dase 96%, and acid ribonuclease 72,o when comparedto control animal fluids. Although enzyme concentrationsshowed neutrophil dependence, no effect on fluid volumeor protein concentration was found in the agranulocyticrats. All residual enzyme activity in these rats musthave come from the adjacent connective tissue cells,since there were no neutrophils.

The neutrophil itself or its interaction with localconnective tissue cells were the possible sources ofneutrophil-dependent supernatant enzyme activity innormal rats. The latter possibility was explored by im-plantation of sponges containing soluble lysosomal en-zymes or saline into agranulocytic rats. An increase insponge fluid LDH levels above the values found on thesaline-implanted side was sought as evidence of localtissue injury. No difference in LDH levels between thelysosomal enzyme and saline sides was found (TableIb), indicating a lack of effect of soluble lysosomalenzymes.

Implantation of lysosomal enzymes is not equivalentto the tissue and fluid influx of neutrophils that occursin normal rats. The failure of lysosomal enzyme im-plants to cause LDH release from the adjacent con-nective tissue of agranulocytic rats should be inter-preted with caution.

0, An attempt was made to estimate the role of neutro-200 > phil death and lysis as a source of supernatant enzymes.

*. The neutrophil-dependent portion of supernatant en-zyme activity was estimated by subtracting the mean

100 n concentration of each enzyme in agranulocytic ratse) from the corresponding concentrations of each enzymec, in fluid samples from control rats. From the sponge' volume, the amount of neutrophil dependent enzyme per1-100 sponge could be estimated for each of the three en-

zymes (Table II). This amount of enzyme was then di-vided by the intracellular enzyme content of 106 neutro-

[mC] protein phils (Eq. 11).Ofunlabeled The resulting parameter, WCEE, was a theoreticalotein counts estimate of the number of neutrophils that would have

,e of 2,445- had to die and release their enzymes into the supernatantf the same fluid to account for the amount of neutrophil-dependent

nflaomatory enzyme present.

intve [14C]- If the supernatant LDH resulted only from neutro-tein. C. Rise phil lysis, then 17.5 X 10. neutrophils/sponge would beitaining un- required to provide the amount of LDH found pernflanmmaDH sp6nge. On the other hand only 3 X 10' neutrophilsises by ab- would be required to account for the amount of betatars indicate glucuronidase observed. Measurement of the quantity

of soluble DNA present in supernatant fluid showedthe contri- concentrations of 60-70 ug/ml or 20-25 oLg/sponge.'me activity This was equivalent to the remnants of 3 - 3.5 X 10'Imost com- nucleated cells/sponge and agreed with the neutrophil

684 Wiener, Wiener, Urivetzky, Shafer, Isenberg, Janov, and Meilman

TABLE I

Inflammatoryfluid

Neutrophils Neutrophils Fluid protein LDH Beta glucuronidase Acid ribonuclease

Rats n Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Change Mean SEM Change Mean SEMChange

1O'/mm' 106/sponge gd/sponge mg/ml U/ml % U/ml % U/ml %

a. Inflammatory Fluid Cell and Enzyme Concentrations in Agranulocytic and Control Rats

Normal (NS) 4 11.1 1.8 4 0.7 359 14 33 0.9 10,895 468 16.1 1.4 99 8Agranulocytic 6 0.03 0.01 0.01 0.01 316 16 31 0.7 2,808 852 -74 0.58 0.28 -96 28 6 -72

rats (ANS)

b. Effect of Neutrophil Lysosomal Enzymes on Sponge Fluid Enzyme Levels in Agranulocytic RatslRight lumbar implants Control, saline sponges 31.5 0.6 2,521 482 1.3 0.4 74.3 11Left lumbar implants Lysosomal enzyme sponges 29.4 1 2,254 470 -11 1.6 0.3 +23 78 16 +5

All data were from fluid obtained at 16 h. Abbreviations: NS, normal serum-injected rats; ANS, antineutrophil serum-injected rats.

lysis estimate based on beta glucuronidase. This datasuggests that supernatant beta glucuronidase may havearisen entirely by neutrophil lysis, while less than 20%of supernatant LDH originated from this source. Theremainder was probably released by injured connectivetissue in the inflammatory area. Fluid DNAwas presentin the form of DNase-susceptible DNA (62%) andnucleoprotein (38%). No endogenous DNase activityin sponge fluid was demonstrated.

The effect of methylprednisolone on sponge neutro-phil and enzyme content. When rats received dosesof 10 mg/kg or more of methylprednisolone, a significantdecrease of neutrophil influx into the sponges occurred.At a dose of 30 mg/kg neutrophil chemotaxis was re-duced by 82% (Fig. 5A). Fluid accumulation was notaffected until doses in excess of 30 mg/kg were given.The effect on fluid influx amounted to less than a 20%decrease (Fig. 5B). Sponge content of soluble betaglucuronidase (Fig. 5C) and acid ribonuclease (Fig.5D) decreased at doses of 20 mg/kg and higher andparalleled the decrease in sponge neutrophils.

LDH levels did not fall after steroid administration(Fig. 5E), in contrast to the significant decrease seenin agranulocytic rats (Table Ia). At 10 mg/kg a sig-

nificant fall of LDH was noted but this may repre-sent only experimental variation.

The effect of methylprednisolone on neutrophil ly-sosomal stabilization. The intracellular contents of ly-sosomal enzymes were not elevated in inflammatoryfluid neutrophils isolated from methylprednisolone-treatedrats (Table III). If this drug had an effect on neutro-phils to decrease secretion, or "regurgitation" losses,then higher intracellular contents of lysosomal enzymesshould have occurred.

No significant difference in lysosomal enzyme con-tent occurred between control neutrophils and steroidneutrophils at a dose of 1 mg/kg. At this dosage, betaglucuronidase levels in neutrophils were decreased.This may have been due to stimulation of secretion orintracellular protein degradation. The elevated intra-cellular level of LDH seen at a dose of 30 mg/kg couldbe due to increased synthesis in precursor cells or todecreased intracellular degradation.

Treatment of supernate from steroid-injected andcontrol rats with 0.1% Triton-X-100 released nearlyequal amounts of granule-associated beta glucuronidasefrom each group. Control rats showed latent enzymeequal to + 12% while fluids from rats given 30 mg/kg

TABLE I ICalculated Neutrophil-Dependent Sponge Soluble Enzyme Content* and WCEEt

No methylprednisolone 30 mg/kg methylprednisolone

Neutrophil-dependent enzyme WCEE Neutrophil dependent enzyme WCEE

Mean SEM Mean SEM Mean SEM Mean SEM

U/sponge X10-6 U/sponge X10JO

LDH 2,413 336 17.6 2.4 2,725 573 11.4 2.4Beta glucuronidase 3 0.4 3 0.4 1.3 0.1 1.4 0.1Acid ribonuclease 28 5 8 1.4 13 0.2 2.8 0.05

Eq. 10, Methods section.* Eq. 11, Methods section.

Mechanism of Action of Methylprednisolone on Acute Inflammation in Vivo 685

A

0

0

m22-0.

0.0.

z

B

300

0titr- 200

00

0 10 20 30 60Methylprednisolone, mg/kg

7 C E3 Supernate 0

26 O~~0Pellet 00.0o44 -

32 o

0 0 20 3060Methylprednisolone, mg/kg

H H0 10 20 30 60

Methylprednisolone, mg/kg

E

0 10 20 30 60 0 10 20 30 60Methylprednisolone, mg/kg Methylprednisolone, mg/kg

FIGuRE 5 (A) Total number of neutrophils per sponge, (B) total volume of fluid persponge, (C) total amount of beta glucuronidase per sponge, (D) total amount of acid ribo-nuclease per sponge, (E) total amount of LDH per sponge in 16-h implants from rats givendifferent dosages of methylprednisolone. Vertical bars are ±1 SEM. Enzymes are designatedas supernatant or cell-associated in C, D, and E.

of methylprednisolone showed a 13% increase in ac-tivity. Failure to find more free granule activity influids from steroid-treated rats indicated a lack ofeffect of methylprednisolone on the "stabilization" ofextracellular lysosomal granules.

Fig. 6 provides some evidence that the fluid cellcounts reflect events occurring in the adjacent con-nective tissue. Neutrophil counts of histologic sectionsof connective tissue capsules in direct contact with thesurface of specific sponges was strongly correlated withthe neutrophil concentrations in fluids of these sponges

(r = 0.803). Rats receiving more than 20 mg/kgmethylprednisolone all had sponge fluid neutrophilc3unts below 4 X 103/mm' and constitute all the pointsbelow this value in Fig. 6.

Peripheral blood neutrophil counts obtained at 16 hwere not decreased in steroid-treated or control rats.Decrease in the circulating neutrophil pool was not thebasis of the decreased in vivo chemotaxis of neutrophilsinto sponge implants in methylprednisolone-treated rats.

Studies for possible system artifacts. Mechanicalbreakage of neutrophils with enzyme release did not

TABLE IIIEffect of Methylprednisolone on Sponge Fluid Neutrophil Intracellular Enzyme Content*

LDH Beta glucuronidase Acid ribonuclease

Dose M SEM P+ M SEM Pa M SEM Pt

mg/kg U/l06 cells

0 (control) 137 13 1.1 0.13 3.5 0.51 125 17 NS 0.63 0.04 0.001 1.5 0.5 0.001

10 152 17 NS 1.2 0.1 NS 3.6 1 NS20 202 67 NS 1.7 0.3 NS 3.9 1.5 NS30 239 53 0.05 0.9 0.1 NS 4.7 0.6 NS

* Observations made on cells removed at 16 h after implantation.t Probability as determined by Student t test.

686 Wiener, Wiener, Urivetzky, Shafer, Isenberg, Janov, and Meilman

occur after fresh inflammatory fluid was absorbed intodry sponges and then recollected by sponge compres-sion. Single neutrophils or clumps were not adherentto the sponge matrix when histologic sections ofsqueezed sponges were examined. Such sections fromcontrol rats showed 7-10 neutrophils/10 oil fields and1-2 neutrophils/10 oil field from rats treated withdoses of methylprednisolone in excess of 20 mg/kg.

Correction of supernatant and cell lysate enzymeactivities. It was most important to correct super-natant enzyme concentrations for contaminating plasmaenzyme activity. The magnitude of the correction variedwith the enzyme. For LDH the plasma correction was- 3.3%, while for ribonuclease it amounted to - 22%.Corrections of supernate for enzyme activity releasedfrom hemolyzed red cells were less than - 1% for allenzymes. Supernate contamination of the cell lysatewas most important, amounting to 47% for LDH. Redcell enzymes accounted for less than 1% of the lysoso-mal enzyme activity in the neutrophil lysate.

DISCUSSIONThis model system has provided data that have helpedin understanding the origin of inflammatory fluid en-zymes and the mechanism of action of high doses ofmethylprednisolone.

Neutrophil influx and the appearance of soluble su-pernatant enzymes in sponge fluid followed a similartime course in normal rats. This was because super-natant enzyme activity resulted in large part from eitherneutrophil lysis or neutrophil-mediated tissue injury.Some supernatant enzyme activity still was presentin agranulocytic rats, and injured tissue cells were themost likely enzyme source in these animals.

The calculation of WCEEwas an attempt to esti-mate the magnitude of white cell lysis required to pro-duce the neutrophil-dependent enzyme activity foundin sponges. Soluble DNA present in the sponge su-pernatant fluid gave positive evidence of neutrophillysis and death. Estimates of neutrophil lysis basedon beta glucuronidase and DNA content of spongeswere in good agreement. A large amount of LDH couldnot be accounted for on the basis of DNA-estimatedcell lysis. It is likely that the source of this excess LDHwas enzyme leakage from injured connective tissuecells. An alternative hypothesis for the different esti-mates of neutrophil lysis required to produce theamounts of LDH, beta glucuronidase, and DNA foundin supernatant fluid would depend on different rates oflocal destruction or removal of these molecules. Sincethere was no detectable DNase activity, DNA wouldhave had to diffuse from the sponges six times fasterthan LDH afLer cell death. This is unlikely because ofthe large size of DNA relative to LDH.

0 200 *

0

z 100 *

2 4 6 8 10 12 14 16 18 20 22

Neutrophils x 103/mm3, sponge fluid

FIGURE 6 Neutrophil counts of hematoxylin and eosin-stained sections of connective tissue capsules of 16-h spongeimplants were plotted against the neutrophil concentrationof the fluid contained in the corresponding sponges. Doublecircles indicate counts from control rats. All of the pointswith low neutrophil concentrations (less that 4,000/mm3)are from rats treated with methylprednisolone in doses of20-60 mg/kg. The higher counts are from control rats orrats given 1 mg/kg.

The high LDH concentrations in supernatant fluidsfrom steroid-treated rats relative to fluid from agranu-locytic rats was of interest. In the agranulocytic ratsno neutrophil-mediated tissue injury or lysis could oc-cur. In the methylprednisolone-treated rats, neutrophilinflux was decreased by over 80% at doses of 30 mg/kgor higher. However, the neutrophils that did enter thesponges and tissue were still capable of cell lysis andof producing connective tissue cell injury. The higherLDH content of neutrophils isolated from steroid-treated rats may also have contributed to the higherlevels of supernatant LDH activity found in theseanimals.

Doses of methylprednisolone of 20 mg/kg or highergiven immediately before sponge implantation had amarked inhibitory effect on neutrophil influx intosponge implants. Decreased lysosomal enzyme contentsof sponge fluid paralleled the decreased influx of neu-trophils. Lower levels of these enzymes were probablysecondary to the decreased numbers of neutrophilsavailable for lysis and enzyme release. The individualneutrophil can be thought of as a lysosomal enzyme-carrying unit. With fewer of such units entering theinflammatory area, decreased amounts of lysosomal en-zymes are carried in. The primary effect of methyl-prednisolone was to decrease neutrophil influx into theinflammatory zone.

Inhibition of neutrophil chemotaxis in vivo by cor-ticosteroids has been described in skin pouches byIshikawa, Mory, and Tsurufuji (3), in the [51Cr]neu-trophil rat paw assay (1) and by use of Rebuck skin

Mechanivn of Action of Methylprednisolone on Acute Inflammation in Vivo 687

windows in humans (4. 5). In vitro studies of neutro-phil migration in Boyden-type chambers have givenconflicting results. Ward reported inhibition of chemo-taxis by hydrocortisone (8), while Borel reported no

effect (9). Inflammatory area perfusates from rat pawsinj ected with nystatin have shown increased levels oflysosomal enzymes that are markedly reduced by neu-

trophil-depleting methotrexate pretreatment or steroids(11). No simultaneous quantitative studies of in vivochemotaxis were made in this model.

The mechanism of steroid inhibition of neutrophilchemotaxis in vivo is unknown. Some qualitative ex-

planations, such as decreased stickiness to capillary walls,preservation of endothelial cells, and direct effects onthe neutrophil, have been suggested (8, 30).

No "stabilizing" effect of methylprednisolone on in-tracellular or extracellular lysosomal granules was ob-served. Failure to find higher intracellular lysosomalenzyme levels in steroid-treated rats was evidenceagainst an in vivo effect of steroids on neutrophilgranule secretion or granule losses during phagocy-tosis.

Lack of increased amounts of granule-associated betaglucuronidase in supernatant fluids from steroid-treatedrats was evidence against a stabilizing effect of methyl-prednisolone on extracellular lysosomal granules. Inagreement with our in vivo studies, Persillin and Kuhave recently shown failure of steroids to stabilize humanlysosomes in vitro (13). The in vitro effects of steroidson the stabilization of liver lysosomes have been ques-tioned (31), and probably will vary with steroid con-

centration, species, method of preparation, and methodof injuring the membranes (32). Goldstein has shown

less release of lysosomal enzymes from cytochalasinB-treated neutrophils after steroid treatment in vitro(15). In this system lysis did not occur.

This in vivo model system has permitted multiplesimultaneous measurements of neutrophil chemotaxis,supernatant and cellular enzymes, and other parameters.In this system the anti-inflammatory action of methyl-prednisolone was mediated by a marked inhibition ofchemotaxis. Decreased supernatant levels of lysosomalenzymes were secondary to the fewer neutrophils en-

tering the inflammatory area. No effect on lysosomalstabilization was observed after steroid treatment.

ACKNOWLEDGMENTSWe wish to thank Dr. Peter Ward of the University ofConnecticut School of Medicine for several helpful dis-cussions and Dr. Ira Goldstein of New York UniversityCollege of Medicine for making available a preprint of hispaper. We wish to thank Sarai Lendvai, Rasma Upmanis,Victoria Kranz, and Rosemary Darmstadt for excellenttechnical assistance, and Gail Bessel for expert secretarialassistance.

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