0041-0101/79/0101-0033102.0010

Toadcoe, Vol. 17,pp. 3~3-60. ® ~n Prow Ltà 1979. Printed in Great Britain.

CHEMOTACTIC ACTIVITY OF VENOM FROM THE BROWN RECLUSE SPIDER (LOXOSCELES RECLUSA) H, M. CrHBEL,* B. J. CAr~s>n,I,t and J. T. BARIesrr* "Department of Microbiology and tDepartment of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65201, U.S.A . (Acceptad

for publication 3 Apri11978)

H. M. a>3ren ., B. J. C`."uanur . and J. T. B,uexarr . Chemotactic activity of venom from the brown l+ecluse apida (Loxoacelea recluse). Taxlcon 17, 55-60, 1979r-The incubation of whok Loxosceles recluse venom with complement generated a chemotactic activity for gt»nea pig neutropht7s which had been harvested from peritoneal extldate teas . Venomguinea pig complement mixtures were 42 ~ more chemotactic than the complement alone and venom-human complement mixtures were 55 ~ more chemotadic than human complement alone. Vena~m plus dauent was no more chemotactic than datlent alone, although venom plea boat inactivated guinea pig serum was slightly chemotadic . PuriSed guinea pig or homes p or C5 incubated with recluse spider venom was not chemotactic nor did venom altar the aerological(y active quantity of p or CS in human serum. Both C4 deficient and properdin de9citnt sera were suitable substrates for the chemotaxigen in recluse spider venom. INTRODUCTION

Tt~ arsroP~Txol oolcAi, events which develop in rabbit skin following the injection of Loxosceles recluse venom (brown recluse or fiddle back spider) have been carefully analyzed (B~c~mt et al., 1973). Within 2 hr a marked elythema and edema surrounded the locus of the venom injection . By the 13th hr hemorrhage is the center of an expanded zone of elythema and edema was observed . During the interlude between the 8th and 16th post injection hr a marked influx of polymorphonuclear neutrophils into the tissues was noted and there was eatravasation of erythrocytes and fibrin diposition . Similar events are known to occur following natural recluse spider bites of the human (Az~is et al 1958), but these changes have not been analyzed histopathologically over the full time course of the reaction. SnaTx and Micgs (1970) reported that a depletion of the level of circulating polymorphonuclear neutrophile in rabbits by treatment with nitrogen mustard prior to an injection of recluse spider venom markedly reduced the severity of the lesion caused by this venom. These authors also noted an extensive infiltration of inflammatory cells into recluse spider lesions in both rabbits and guinea pigs within 6 hr after the injection of venom. In the case of guinea pigs which were depleted of serum complement by prior injections of zymosan or antigen-antibody aggregates, the typical necrotic lesion associated with rxluse venom failed to develop. Since complement activation is known to release chemotactic peptides from components C3 and CS (Bo>rlsx et al., 1969; Hu x, and WARn, 1969 ; WeRn and N$wMAN, 1969) these results suggest that the infiltration of polymorphonuclear leukocytes into the area surrounding recluse spider bites may be complement dependent. Support for the hypothesis that recluse spider venom may release chemotactic sub-

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56

H. M. GEBEL, B. J. CAMPBELL and J. T. BARRETT

stances from complement stems from the reports(Flrrxe, 1974* ; KN1tcfiR et al., 1969) that this venom contains a complement inactivator. It has also bcen stated that serum complement depleted of hemolytic activity by recluse venom could be restored to its normal hemolytic potency by supplementation with C3 or CS (ICHII{SR and MORGAN, 1967). These experiments led us to investigate more directly by in vitro experiments, the potential chemotaxigenic activity of recluse spider venom and complement mixtures . MATERIALS AND METHODS Collection of venom The paired venom glands of L. recJnsa adults were surgically removed and the venom was expressed into saline or other dihrent by gentle pr+esatrre oa the glands (Wtuaxr et x1.,1973). The protein concentration of venom was measured by a micromodification (WRrakrr et al., 1973) of the method originally described by LowttY et al., (1951) . The venom solution was ndjtasted to a concentration of 250 Ng of protein per ml and frozen at -70°C in 02 ml aliquots until needed . Cells The peritoneal oxudate cells used in the chemotaxis assay were obtained from strain 13 guinea pigs (gifts of Dr. HarBrt Mt7uBrt, Departm~t of Medicine, Universityy of Missouri, Columbia). Each guinea pig was given an i.p . igjection of 50 ml of sterile 0"1 ~ shellfish glycogen (Sigma Chemical Company, St . Louis, Mo.) contained in physiological saline (Ar."r~x, 1975)t. The guinea pig was sacrificed 18 hr later and peritoneal exudate cells were harvested from the animal using heparinized saline (2U/ml) as the irrigating solution . The t+ecovorod cell suspension wes then centrifuged at a low speed at room temperature for S min. If the cell pellet was contaminated with erythrocytes, the cells were r+esuspended in 10 ml of cold ammonium chloride lysing buffex (Ar.~rr~,x, 1975)j' for 10 mix and reoentrifuged. The final cell pellet was nostrspeoded in medium 199 (Difco Laboratories, Detroit, Mn to aconcentration of 2"0 x 106 peritoneal extrdate cells/ml . The cell suspension consisted of >95% neutrophüs. Complement and complement activators Lyophilized, whole guinea pig complement, purified guinea pig C3 and C5, and purified human C3 and CS were purdtased from Cordia Laboratories, Miami, FL . The C3 and CS components of compliment contained 1000 CH 50 u/ml . (Ono CH 50 unit is the amount of the compliment component necessary to lyse 50~ of 3"2 x 10~ sheep erythrocytes in an excess of anti-sheep antibody). C4-deficient guinea pig serum wasa gift from Dr. Ulf Nilason(Dept. of Dental Pathology, University of Pennaylvaaia, Philadelphia, PA). Whole human serum was obtained by venipuncture of one of the authors (H .G.) Properdin-deficknt human serum was propared according to the method of PsxsrcY et al. (1968). Zymosaa (Sigma Chemical Company, St . Louis, MO) was aQiusted to a concentration of 0"5 % in physiological saline, autoclaved, and used as an activator of whole complement . Chemotaxis assays Chemotaxis assays wero performed according to a modification of the Boyden technique (BOYDEN, 1962) using filters with a porosity of 3 microns (Ntrckpore, Pkaaanton, CA and Neuroprobe, Bethesda, MD). The 81kd chambers were incubated in a 37C httmidiBed incubator for 90 min. The filters was rrmoved end stained according to a modification of the method of Wn.ratvaoN (1974) and the results were expressed as the total number of neutrophils per 10 high pow+or microscope fields . Each test was performed numerous times and each experiment incorporated pceitive and negative controls . The positive control consisted of a mixture of 0"5 ml of compkateat, 0"5 ml of zymosart (0 "5 %~ and 2"75 ml of medium 199. Tho negative control consisted of 0"5 ml of complement (or complement fracrion) plus 3"23 ml of medium 199. Experimental solutions were prepared by incubating 0"5 ml of complement or fraction tha+eof with 0"1 ml of venom (25 Pg protein) for 10 min at room temperature . This solution was then added to 3"15 ml of medium 199. In addition, medium 199 alone was also tested for chemotactic activity, ea was a mixtuue of 0"1 ml of venom (25 Ftg) plus 3"65 ml of medium 199. The serologic activity of C3 which remained in an equal mixture of whole human serum (150 mg % C3) and whole recluse apidea~ venom (7 irg protein) after a 60 min i~trbation at room temperature was determined by single radial immunodifftrsion (Msrrcn~a et al., 1965). In a parallel experiment the r+esid»el CS in whole human savor, originally at a concentration of 7"8 mg %, was determined after incubation with an equal volume of solution containing 60 Ng of venom protein. *FnaFe, J. H. (1974) Immunology of Loxosceles recluse venom. Ph.D . Dissertation, University of Missouri, Columbia. tAr.~rmwx, L. C. (1975) . Leucocyte chemotaxis, Procedure for the gtrantitation of leucocyte chemot a=ia titilizlng Ntu9aopore brand (R) polycarbonate filters. Nudeopore Corp. Pkasanton, CA .

Chemotaais and i3rown itxlvae Spider Venom

37

RPSULTS

The data in Table 1 indicate that both normal guinea pig and human complement when treated with spider venom become chemotactic for guinea pig peritoneal cells. The mean cell count of 709 for venom plus guinea pig complement represents a 42~ increase TAeFB

1.

~üIOTACI'IC IICIIVCTY OF VENOM AND 00lG'IFA~IPr OOI~INA'IIONS

Complment Zymosan plus Venom plus alone complement complemeat Normal guinea pig complement 499 f 40 (32)" 732 f 38 (32) 709 f 43 (32) Heat inactivatod guinea pig serum 434 f 34 (16) 338 f 42 (16) Normal human complement 240 f 3 (16) 333 f 17 (16) 373 f 12 (16) "Mean number of aeutrophl7ic leukocytes migrating/10 high paws microscope fields f standard error of the mean. The number in parenthesis is the number of trials . Fresh venom and Medium 199 alone or in combination gave < 30 cells migrating.

in cell counts above the 499 observed for complement alone. Also the addition of venom to normal human complement produces a 55% increase in the number of cells migrating in the Hoyden filters. The calculation for each experimental combination was calculated according to the formula : Chemotactic Quotient = Experimental cell count - control all count x 100 Control all count Chemotactic Quotient = 709 - 499 x 100 = 42~ -fguinea pig complement) 49g (Venom The all migrations in the experimental systems of venom plus guinea pig complement aced venom plus human complement were found to be statistically different than the controls which contained complement without venom (P , 0~00~. This calculation applied to heat inactivated guinea pig serum combined with venom yielded a value of 24~. Although this increment is less than that obtained when venom is mixed with unheated complement, the result indicates a significant increase. That these changes were not due to venom alone is supported by the observation that less than 50 cells/10 high power fields migrated in the venom plus medium 199 or the medium 199 control vessels. The expectation that purified guinea pig and human C3 and/or CS would be the source of venom induced chemotactic activity was not met. The data presented in Table 2 depict a constant chemotactic equality of these complement fractions alone when compared to their incubation with venom. That these data were not the result of faulty technique or dormant cells is corroborated by the high chemotactic activity recorded in the zymosan-whole complement controls conducted in each experimental set. Serological analysis of C3 and CS after exposure to venom in triplicate experiments revealed that there was absolutely no loss of C3 or CS due to the treatment. TAHIB 2 . FAILURE OF RECLUSE VENOM TO Avrnymç A CHEMOTAXIN FROM PURIFIED GUINEA PIG OR HUMAN p OR l;3

Zymosan plus Venom phla Complement complement whole compon~t alone complement component Guinea pig C3 38 f 13' 460 f 9 30 ~ 7 Guinea pig CS 33 f 2 314 f 26 36 f 4 Human C3 24 f 3 372 f 18 23 f 6 Human CS 190 f 8 730, 7801 199 f 1l 'Mean number of neutrophilic leukocytes migrating/10 high power microscope fields f standard error of the mean. The number of determination in each case was 16. tTwo determination only .

SE

H. M. CiEBEi,, H. J. csMpnf?r .r . and J, T. HARRETT

Guinea pig serum which was deficient in C4 was a source of a potent chemotaxin when exposed to recluse venom, reaching levels 73~ greater than the control. Likewise properdin deficient human serum treated with venom was nearly 53~ more stimulatory to peritoneal exudate cells than when not exposed to venom. These data are presented in Table 3. TABIB 3 . C,~~iOTAXI9 HY VEN01! AND A 88RA DSFII~sNT IN A ODMPI~IEAi1' COlIPON6N'I'

Serum alone 312 f 63 "

Zymosan plus serum 922 f 109

Veaom plus serum

C4 d~cieat guinea pig serum 540 f 86 Properdin deScJent human serum 398 f 32 390 f 30 608 f 64 " Mean number of neutrophilic leukocytes miglating/10 high power microscope ßdds f standard error of the mean . The number of determinations in each case was from 12 to 16 . DISCUSSION

When SbuTx and Mlcgs (1970) depleted the population of circulating polymorphonuclear neutrophil leukocytes in rabbits by treatment of these animals with nitrogen mustard, they observed a decrease in necrosis when they were injected with recluse spider venom. SMITH and Mlcgs (1970) sad FYN>~ (1974) " demonstrated a similar phenomenon when they caused a depletion of plasma complement in experimental animals by treatment with zymosan. These data constitute provisional evidence that a complement dependent chemotactic process was intimately involved in the development of L. redoes lesions in experimental animals. The in vitro chemotaxis assays demonstrated that the interaction of recluse spider venom with whole, guinea pig or human complement generated a chemotactic factors) for polymorphonuclear neutrophil leukocytes when compared to venomless controls. Venom alone was not chemotactic. Therefore, the venom from L. redoes is a cytotaxigen according to the usage advanced by W>t.x>rrsox (1974) . A chemotactic response also appeared upon incubation of heat inactivated guinea pig serumwith spider venom, although this did not reach the level of chemotaxis when whole fresh guinea pig sera was used. These results with heat inactivated serum-venom mixtures indicated that substances in sera other thaw the heat labile components may serve as a source of chemotaxins for recluse spider venom. The inability of purified C3 and CS from either human or guinea pig serum to serve as a source ofchemotaxinsfor reclusevenom was unexpected.. Both C3a and CSa have been repeatedly described as chemotactic split peptides from their parent complement com ponents. ICrr>~c and MORCiAN (1967) reported that recluse venom was anticomplementary by virtue of its ability to act upon guinea pig C3 and human C5. In retrospect this appears anomolous in view of the known homology of the complement system in guinea pigs and humans. The failure of purified C3 and CS to function as chemotaxin precursors cannot be accounted for on the basis of concentration since each was used at a level of 500 CH50 (see Materials and Methods Section) and their normal serum level is much less than this. Serologic experiments reported here have demonstrated that C3 and CS in whole serum remain at full serologic potency in radial immunodifiusion tests with their respective antisera after incubation with recluse spider venom. The proteinsee activity in whole recluse venom (WRICiHT et al., 1973) is apparently not active on these serum proteins . Human complement depleted of properdin interacted normally with spider venom to "P~rca, J. H. (1974) Immunology of Lox+nrce%a reeluaa venom. l?h.D. Disecrtation,University of Missouri, Columbia .

C~amotexis send Brown Recluse Spider Venom

59

generate a chemotactic factor. ProPerdin deßciart serum did not release a chemotaxin after exposure to zymosan, confirming that the alternate complement pathway was incomplete in the treated serum. C4-deficiart guinea pig scram was also ohemotactically active after exposure to recluse spider venom. A summation of these results yields the following conclusions. The alternate complomart pathway, if at all involved in the neutrophil chemotaxis examined here, can be so only below the level of properdin, since properdin deficient sera become chemotactic when exposed to varom. Since C4 deficient sera also liberated a chemotaxin, and C3 and CS treated factors were devoid of activity, a chemotactic source must reside in the complement system below the level of C5. This does not dispute the possibility of active molecules above CS in the complement system but these would not include properdin, C3 or C4, and these are apparently not acted upon nor do they generate the normal sequence of the complemart cascade. Dermacentor variabrHs, the dog tick, possesses a ohemotactic agent in its saliva and thiswas reported (BSRBNBERß et al., 1972) to act upon C5. Naja raja (hooded cobra) varom activates the alternate complement pathway (WILKIIdSON, 1974) and eventually liberates C3a. Venom of the ant Pseudomyrmex has recently been reported to contain two proteins which activate the complement system beginning with component Cl (ScfIULTZ and ARNOLn, 1977). L. recluse varom apparently acts in a different manner to liberate chemotaxin(s) from fresh serum. This mechanism may be differart than that responsible for the liberation of chemotactic activity from heat inactivated sera which was only half as chemotactic as fresh serum. dcbwwlaa~erunta--This research wes supported in part by Public Haslth Service reaeairh giant ES 00598 from the Division of Environmental Health 3ervla, and by Missouri Agiiculture Experiment Station, giant 7001-0150. REFERENCES J. A., Wn~roo, C. W., Soa®rsx, W. A. and Fixxrr, J. E. (1958) Necrotic aradmidism . Am. J. trop. Mad. Syg. 7,165. BBaCi~l, R. S., Aa~ar~x, E. Ii . and Arro~sox, P. C. (1973) Intravaswlar coagulation : the eauao of necrotic a:achnidisnn. J. im~t. Denrt. 61,142. B ,J. L., W~ta, P. A. and Sores, D. A. (1972) Tick bite igjury : mediation by a oomplementderived d~emotactic factor. J. Immraro1.109, 451. Bow, V. A., Mvu$a-Eden, H. J. and Coca~r~, C. O. (1969) Isolation of a fragrant (C3a) of the third oomponeat of human complement containing aaaphylatoadn and dxmotactic activity and descxiption of an anaphylatoxin iaactivator of human serum. J. axp. Mad.129, 1109. Homsrr, S. (1962) The d~tactic e8ect of mixtures of aat