Pyrogen and enzyme release from rabbit blood leukocytes promoted by endotoxin and polyinosinic polycytidylic acid

1IOCHEhlICAL

5, 227-236

MEDICINE

Pyrogen

and

Enzyme

( 1971)

Release

from

Rabbit

Leukocytes Promoted by Endotoxin Polyinosinic Polycytidylic Acid1 CAROL Department

of

G. COX

Biochemistry, Morgantown, Received

AND

GALE

and

W. RAFTER

West Virginia University West Virginia 26506 February

Blood

Medical

Center,

8, 1971

Materials released from granulocytic leukocytes play an important role in the inflammatory process in animals. Some of these materials are enzymes which attack extracellular substrates, and pyrogen a low-molecular protein (1, 2) which causes fever. Enzymes and pyrogen are released in response to stimuli such as bacterial lipopolysaccharide or endotoxin (3-5) and phagocytosis (6). Release of materials from the granulocyte in response to stimuli represents an important aspect of the host-parasite relationship which we know little about. In the case of pyrogen release the process is not a simple one. Because such agents as actinomycin D and puromycin inhibit its elaboration by blood cells protein synthesis appears to be involved (7, 8). I n our study enzyme inhibitors and a nonendotoxin promoter of pyrogen elaboration were used to investigate the process. MATERIALS

AND

METHODS

Materials. E. coli ATCC 11303 was obtained from the American Type Culture Collection, Rockville, Md. 14C-G1ucose was purchased from New England Nuclear Corp., Boston, Mass., l”C-Na acetate from Calatomic, from Tracerlab, Los Angeles, Cal., and I’C-algal protein hydrolysate Waltham, Mass. Endotoxin, E. coli 026:B6, was obtained from Difco Lab., Detroit, Mich. Puromycin HCl, actinomycin D, and RNA copolymer polyinosinic polycyticyclic acid [poly ( 1. C ) ] were purchased from riboNutritional Biochemicals Corp., Cleveland, Ohio. Cycloheximide, nuclease T,, and pancreatic ribonuclease, type A, were purchased from ‘This report was taken from a dissertation submitted by the senior West Virginia University in partial fulfillment for the Ph.D. degree. The supported by a research grant from the United States Public Health Service. of the work was reported at the 54th Annual Meeting of the Federation of Societies for Experimental Biology at Atlantic City, N. J. 227

author to work was A portion American

Sigma Chcl~lical Corp.. St. Louis. \lo. l)ric4 ,2~ic,~oc.oc.c,rts /ysorl&ktic:rls ~~~11swc’x obtaiued fronr th(b hlantl Rcscxrcll I ‘ah.. !%:1~ York. N. Y. General AIetlrot/.s. ,111 glassware, I\ ax 11rat1~.1~1 rog?;csll trcx~ t>\ ii~xti~q . tlits tirctltod ot Lowry et al. (9). Lysozynic activity n-as assayc,d using dried .\ficwcwcm lysotleikticus ~11s (10) and aldoluse b1; ‘1 tnodificatiorr of the* Sit&)and Lchningcr method ( 11 ) ( Sigma Chc~tnical . Tc~chnical Rullctin No. 750). Pyrogenicity of salnpl~s was iiicas1ir~~l I)!, injclctiilg tll(Wr ilIt the marginal ear vein of traincad New Zt~nland \vhit(l ~tralt, rabbits ( 1 1. Animals were usually made refractory to c~ndotoxin by injecting 2 ;Lg of K. cdi ondotoxin 23 hours bcforc using them. Thcx FI ,:(, illdcy \vas measured by the method of Keenc et ul. ( 12 1. Lc~ukocytic pyrogen injected into rabbits gave typically a monophasic fever curve: tvhich attained a maximum within 1 hour and rcturnrd to the starting tcarllptxlture within 2 hours. For pyrogen testing a group of about 12 rabbits selected l~ecxusc of their uniform rcspousc~ to tht* S~IW ;Lmormt ot Ieukocytic pyrogen was used. The amount of pyroq(‘n illjectcx(l illto ;L rabbit was adjusted so that the fcvcr produccad did not cxx~~l 1.2”. 1’0 achievca grcxater precision thr sa~nc rabbit ~~1s uscti on differf>ut tla>x to measure pyrogcn in diffcrc,nt samplrs fronr tlrcs s;m~(~ c*\;prxrinlctllt. Fc,\-cst data reported in Tables 1-5 in the Results section rq~rc‘s~nt results of ‘I single rcl”(‘s”ltati\,c, cqerimc~nt. Most cup(+mc~nts \\.(‘r(’ rc~prxtc~tl thrc>cx or more times. Prepur~ltion of Letik0cyte.s Und Leukocyte!-f?ich ~fist~lrcs. Rabbit ~)~OO(~ was obtained by cardiac puncture using hrparin as an anticoagulant. The blood was first centrifuged at 185g for 20 minutes and most of the supernatant plasma removed by aspiration. .4 pasteur pipette 1~;~s thcan carefully passed to the bottom of the tube and as ~nany red blood cells as possible were removed by aspiration without disturbing the layer of white blood cells. The remaining leukocyte-rich plasma was clilutctd \xTith 0.001 1\1 K phosphate buffered, 0.15 Hal saline to ;1 final concentration of 5 X 10” cells/ml. The pH of the suspension was 7.2 and it contained about 10% plasma. Purified leukocyte mixtures were prepared tram rabbit whole blood using a dextran method ( 13). Th esc ~11s incubated with endotoxin, released very little pyrogen compared to cells described in the previous paragraph. -4ddition of rabbit plasma did not impro\rc: tlwir pyr(jgeu \,icld.

LEUKOCYTIC

l’YROGEN

AND

ENZYMES

229

Leukocytes were counted by conventional methods after 1 in 20 dilution in a Levy counting chamber. All counts refer to the whole leukocyte population. Preparation and Handling of Reaction Mixtures. Usually 5 ml of leukocyte-rich mixture was used in each experiment. Additions were made to mixtures in a small volume. E. coli endotoxin or poly (I *C), 0.02 and 30 pg/ml, were added to mixtures as promoters of pyrogen elaboration unless otherwise indicated. Following incubation at 37” for 6 hours for endotoxin experiments and for 9 hours for poly (I * C) experiments, mixtures were centrifuged at 12,OOOg for 10 minutes and aliquots of the supernatant fluid removed for pyrogen and enzyme measurement. One milliliter of the supernatant fluid was routinely injected for pyrogen assay. If inhibitors were added to mixtures, the supernatant fluid was dialyzed against 0.15 M NaCl before pyrogen assay. Since poly ( I *C ) causes fever when injected into animals (14) and is not removed with the cells after centrifugation, it was necessary to remove it before measuring pyrogenicity. This was done by incubating each sample with 30 units of ribonuclease T,, and 20 ,ug of pancreatic ribonuclease/ml in 0.001 M EDTA at pH 7.0 for 2 hours at 37”. Control mixtures without leukocytes and containing poly (I .C) treated in this manner caused no fever when injected into rabbits. Preparation of Carbon-14-Labeled Endotoxin. E. coli was grown in a medium which contained in 1 liter of 0.1 M Tris-chloride buffer, pH 7.4, the following materials : 0.1 gm MgSO,*7H,O, 1 gm (NHa)$04, 0.5 gm Na citrate, 0.05 gm casein hydrolysate. 0.04 gm KH2P04, 2.5 mg glucose, and 0.3 mc l*C-uniformly labeled glucose (1 mc/mg). Cells were harvested when the culture reached the stationary phase of growth. Endotoxin was prepared from the cells as previously described (15). A typical preparation contained 27,000 cpm/ 100 pg. Incorporation of l”C-,Amino Acids into a Pyrogen Fraction. Forty milliliters of a leukocyte-rich mixture was divided into two 20-ml samples. To each sample was added 50 ,pc of l”C-algal protein hydrolysate (1.4 mc/mg) and they were incubated with shaking for 15 min at 37”. Then to one sample was added 0.4 pg of endotoxin and both samples incubated for 6 hours at 37”. The supernatant fluid was recovered by centrifugation and concentrated to about 4 ml by ultrafiltration. It was then passed through a Sephadex G-100 column (2 X 38 cm) with 0.1 M K phosphate, 0.15 M saline, pH 6.5’. Radioactivity, protein, and pyrogenicity were measured in each sample. RESULTS

Pyrogen Release. Endotoxin added to blood leukocyte mixtures causes the release of a leukocytic type pyrogen ( M). The effect of certain

230

(X)X

\NI)

I< \I‘I

I. I:

metabolic inhibi.tors on this process is shown iI1 Tabk 1. Dinitrophcnol, 60 phi, ditl not affect pyrogen release. As others hnw reported bcforca (7, 8) pyrogen appearance in the soluble phase of mixtures is inhibited by protein synthesis inhibitors. Both pyrogen elaboration and amino acid

I I 0 h 0 !I

.i( )

11

!I0

-I!)

100

a Each sample contained 5 ml of leukocyte-rich mixture prepared by t.he dextrail method (Materials and Methods), 10 PC of W-algal protSein hydrolysate (1.4 mr/mgt, and 0.1 rg of endot.oxin. Following incubation at 37” for 6 hours cells were separated from the supernatant fluid by rentrifugation at) 12,OOOg for 10 minutes. Samples were prepared for measuring radioactivity according to method of Baker et al. (231. A zero t.ime control which contained all the components wras performed for each sample. b Leukocytes not purified were used iii this experiment.

LEUKOCYTIC

PYROCEN

AND

231

ENZYMES

incorporation into protein showed a dose response relationship to the inhibitor (Table 2). Interestingly, actinomycin D at 10 pg/ml completely blocked pyrogen elaboration while only partially inhibiting amino acid incorporation into cellular protein and into protein released into the soluble phase during the incubation period. It should also be noted that the percentage inhibition obtained with the highest level of inhibitor was greatest in the pellet protein. To determine how rapidly the actinomycin D acted, it was added to cells at varying times after enclotoxin addition. As can be seen in Table 3 no effect of actinomycin D was obtained if its addition was delayed 1 hour after addition of enclotoxin. Because of the apparent requirement of protein synthesis for pyrogen production, incorporation of a mixture of carbon-14-labeled amino acids into a pyrogen fraction from leukocytes stimulated with endotoxin was sought. The pyrogen fraction was purified by passage of the supernatant fluid from incubated cells through Sephaclex G-100 as described in Materials and Methods. No increase in protein labeling was seen in the samples containing pyrogen activity. In some experiments about a 5% increase in amino acid incorporation into both cellular proteins and proteins released into the soluble phase was obtained in leukocyte mixtures incubated with endotoxin compared to mixtures with no enclotoxin. Cline et al. (16) f ouncl no enhancement of amino acid incorporation into leukocytes using much larger amounts of enclotoxin. To investigate the possibility that enclotoxin contributed a building block for the synthesis of pyrogen racliolabelecl endotoxin was incubated with leukocytes. The radioisotope and protein content of each sample after gel filtration of the incubated mixture are shown in Fig. 1. No inclication of increased radioactivity in the samples that contain pyrogen activity is seen. Poly ( 1. C ), a RNA copolymer, injected into rabits causes fever ( 14).

TIME Time of addition of actinomycin D (min) No actinomycin 0 1.5 30 00 90 120

D

COURSE

TABLE 3 OF ACTINOMYCIN

Maximum (“C) 1 0 0 0.S.5 0 .i.i I 05 1 .05 I 0

D INHIRITIOX

fever I&C 8.0 0 1.3 4.4 S” 7 9 s 1

232 ,110 i I00 IS-

j90

16-

180

7

14-

470

>

ii?-

360

E

g

IO-

E

8-

250 -40

6 v

6

130

42-

1'"

E"

10 0

oI

3

5

7

9

fsEsil II 13 15 17 FRACTION

19 21 23 25 27

29

NUMBER

FIG. 1. (kl filtration profilt’ of supernatant flllid ohtainetl tro!ll leukocytes irrcuhated with “C-labeled endotoxin. Sixty milliliters of leukocyte-rich miutnre and endotoxin were incubated with shaking for 12 hours at 59,000 cpm of “C-labeled 37’. The supernatant fluid was recovered hy centrifugation and concentrated to about -4 ml. This fluid was then passed through a Sephade.1 (:-IO0 cohmm I 2 ‘~1 3X em ) with 0.1 hf I( phosphate, 0.15 hl KaCl, pH 6.5. Carbon-1.4 and protein cwntent a~ttl measured. Carbon-l-l, a--0-0; protein, pyrogenicity of each sample were O--O---D: and pyrogenicity by the cross-hatched area.

When added to blood leukocyte mixturc~s it promoted the rt+asc of a leukocytic type pyrogen (Tables 3 and 5 ). Maximum pyrogcn release was obtained with 30 ,&ml of the copolymer. Like ondotoxin-pro(noted pyrogen r&we both metabolic and protein synthesis inhibitors inhibited the poly (I .C)-p romoted pyrogen releast~ ( Tables 4 and 5 1. Enzyme Release. Along with pyrogcn, cndotoxin promoted c~nzymc~ release from leukocytes. Aldolase, an cnzymc~ foulid in the cytosol, and lysozymc, an enzyme found in the gram& or lysosonws, wcr~~ sclwtc~cl

LEUKOCYTIC

PYROGEN

AND

233

ENZYMES

TABLE 5 @hFBCT OF PH~TKIN ~YNTHYYIH INHIISITOHS ON PYIWWN R~LIUSIC FROM LEUKOCYTES TKKATBD WITH POI~YINOSINIC POLYCYTIDYLIC ACID hlaximum (“C,

Additions Actinomycin Cycloheximide Puromycin

fevel

0.7 0 0 0

D (10 @g/ml) (10 pg/ml) (50 pg/ml)

4.0 0 0 0

to examine the effect of inhibitors on the release process. Compared to pyrogen release enzyme release was rapid. Maximum lysozyme release was obtained in about 3 hours contrasted to maximum pyrogen release in 12 hours. The effect of metabolic inhibitors on enzyme release is shown in Table 6. It should be noted that protamine sulfate which did not inhibit pyrogen release inhibited enzyme release. Unlike aldolase, lysozyme release without added endotoxin was inhibited by all the reagents. The protein synthesis inhibitors (Table 2) did not affect release of either enzyme nor did poly (I-C) promote their release from leukocytes. It was reported earlier (15) that leukocytes separated from peritoneal exudates degraded endotoxin and that the process was inhibited by protamine sulfate. For this reason, degradation of endotoxin was looked for in the blood leukocyte system. As can be seen in Table 7 endotoxin products soluble in lipid solvents were obtained and their appearance, in part, was blocked by protamine sulfate.

EFFECT

OF ~~ETABOLIC LIGJKOCYTE:S

TABLE INHIBITORS TREATED

6 ON ENZYME RELEASE WITH ENDOTOXIN

Aldolase S-L units/ml Addition -

Protamine SO4 (80 pg/ml) EDTA (10 mM) X-Ethylmaleimide (1 mM) a Minus endotoxin. 6 Percentage inhibition

+E

3 2

11.0 5 .j 4.6 -

4.6 4.6 -

is calculated

from

Lysozyme 7% Inhibition

-Ea

FROM

81 103

endotoxin-stimulated

a/ml ~ -E

+E

1.7 0.72 0.63 0.50

3.43 1.92 1.18 1 23

release

alone.

% Inhibition* 32 66 57

DISCUSSION

The mechanism by which certain materials cause leukocytes to relt~asc~ pyrogen appears not to depend on their rlniquc chemical composition. Previous work ( 17) indicated that the lipid portion oF clndotosin was essential to the procc~. As found in this study poly ( I.(: 1) which contains no lipid promotes pyrogrn release. Furthermore, as judged b! the effect of metabolic and protein synthesis inhibitors on th(l process the manner by which it causes pyrogrn relcasc is the same as c,ndotoxin. That poly (1.C) itself and not an endotosin impurity in the copolymck1 promotes pyrogcn release is indicated by t\vo findings: poly (1.C ) treated with nucleascs is not pyrogc,n‘c, \vl1c11 injected into rabbits and poly (I.C) unlike cndotoxin does not promote lysozymc~ and adolasc~ r(tleast from leukocytes. The notion that endotosin contributes m cwt’tlti:il lipid building block to the synthesis of pyrogcn ( 18) also appears unlikely in light of the result obtained with poly ( I .C). Failure to obtain transfer of radioactivity from radiolabeled cndotoxin to a pyrogcn fraction supports this idea. Long chain fatty acids previously found in pyrogen fractions ( 1)) therefore. must come from the leukocyte. Activation of leukocytes to elaborate a pyrogen comprises at least three steps: attachment of promoter to the cell, protein synthesis, in response to this interaction, and release of pyrogen from the cell. Previous work indicated that EDTA prevents the first step (15). In thv second step from our results with actinomycin D it is concluded that

LEUKOCYTIC

PYROGEN

AKD

ENZYMES

23.5

messenger RNA is rapidly transcribed and is absent in unactivated cells. What protein is synthesized is conjectural. Working with exudate leukocytes the third step can be separated from the second step and in this system it has been shown that sulfhydryl reagents prevent pyrogen release (19). In the blood leukocyte system used in these studies the inhibition with N-ethylmaleimide probably also represents interference with ATP synthesis required for protein synthesis. Previously, based on studies with sodiunl fluoride, a requirement for energy at this stage of pyrogen elaboration has been noted (17). The site of inhibition with NaCN is not clear; however, the lack of inhibition with dinitrophenol suggests that it is not aerobic energy metabolism. While endotoxin and poly (1.C) are chemically dissimilar they both have physical properties of large size and of many anionic groups widely distributed over the molecule. These properties suggest that the essential event of promoter activity occurs at the plasma membrane of the leukocyte. One possible result of the interaction is a damaged or altered membrane (20). The finding that certain steroids promote pyrogen elaboration in human but not in rabbit leukocytes (21) emphasizes the important role of the plasma membrane in this interaction. The requirement of protein synthesis for pyrogen elaboration is then explained by the requirement for repair or replacement of the protein portion of damaged membrane. Data are not available to decide whether or not pyrogen is synthesized de rlovo at this time, but the protein synthesis which does occur at this time must be coupled to ultimate pyrogen release. In the simplest model pyrogen is a membrane component or is made from a membrane component. The final or release step still entails further reactions on the protein which is to become pyrogen because active pyrogen is not found in cells after the protein synthesis step. Lysosomal or granular enzyme release from leukocytes promoted by endotoxin has been described (16). The stimulus for release was apparently phagocytosis of an endotoxin-antibody complex. The endotoxinpromoted enzyme release described here includes both lysosomal and soluble enzymes and requires much smaller amounts of endotoxin. In contrast to pyrogen release it seems to depend on the unique chemical composition of endotoxin and to require endotoxin degradation. As shown by the EDTA results both processes require endotoxin binding to leukocytes, but after this step they diverge as shown by the different responses to protaminc sulfate and protein synthesis inhibitors. A plausible hypothesis is that leukocyte enzymes leak nonspecifically through a damaged plasma membrane produced by an endotoxin breakdown product, possibly unesterified fatty acid. Protamine sulfate which inhibits enzyme release also inhibits lipase activity (22).

236

(:0X

ANI)

ILAl.‘I‘l~:ll