Experimental eosinophilia

Experimental eosinophilia VIII.

Cellular

responses

to altered

globulins

within

cutaneous

tissue

Infla~mmntory ctll r~~sponscs to cosinotactic agents were studicd in the gu.inca pig icndcr short-term experimental conditions designed: (1) to cxdthae possible associated rrntibody formation, and (2) to produce preliminary histaminic influences on chnllcngcd tkwe sites. Loeulizctl eosinopkil cdl infiltrations into dermis developctl si.2 hours after intracuta,neous ckallenge to: (1) molecular nggregates of rabbit so‘um gamma globulins, (SJ preformed soluble antigen-antibody com,plexes prcparcd itI vitro from bocinc gamma. globulin (BGG) and corresponding vnbbit antiseru,m, ctntl (3) BGG antigen-antibody systems through passiz;c sensitization techniques. Exogr910~s histamine neither caoked an cosinopltilia per se nor exerted synergistic 0) potcntia.ting influcnws on specifically challenged dermal sit& Pre-existing states of immctliate hypersensitivity to wnrelatcd antigen (iz., ovalbltmin or hemocyaniir t did not afleet cosinophil lcllkocytc responses to gamma globulin aggregates. The prompt cmergcncc of eosinophil cell-containing infiltrates within tissue devoid of tl~~modrntcd aatibody formCng elements suggests that the eosiaotactic in%lccnc>cs mny Anrc been related to the gamma. globulin complcxcs. This ,in turn may br it ,function of tlrdr stcctc of molecvlnr oggwgntion.

T

his study had the purpose of evaluating inflammatory ccl1 responses to eosinotactic agents1 under esperimental conditions designed : (1) to cxcludc possible associated antibody formation, and (2) to produce preliminary histaminic influences on challenged Gssuc sites. The basic esperimental method was quantitation of cosinophil granular leukocytes infiltrating the skin of guinea pigs after short-term treatment with immune and related reactants. Preliminary study indicated tlrat the height of dcrmal cosinophil ccl1 accumulations is generally rcachcd within sis hours after administration of thcl

From the Department of Biology, Wilkes College. This study was supported (in part) hy Research Grant Foundation. Wilkes College students, Catherine D. DeAngelis and wscareh studies t,hrou@l the National Science Foundation Program (Grant Ko. URP-1174). Received for pddiration SOY. 16, 196-t.

G-23913

from

the

National

Sri(~tlv~~

Howard G. Hughes, participated Undergraduntc~ Rcicnce Ehwti~n~

in

challenging materials. This histologic development wsc~nhled the t,imc scqurncc for other related models of tissue cosinophilia for this species, i.e., involvc‘ment of lymph nodes regional to foot pads treated with identiral test materials,‘-” and cellular infiltrations at dermal retest reaction sites of guinea pigs in dclayc~d hypersensitivity states rcportetl by Arnason and \\‘aksman. L MATERIALS

AND

METHODS

Our previous papers I- 3 hart described techniques for preparation of aggregated strum gamma globulins, antigen and corresponding rabbit antibody systems, and soluble antigen-antibody complrxcs for intracutaneous administration and the test procedures for ascertaining the absence of corresponding OI cross-reactive preformed antibody in the sera of experimental animals. Albino guinea pigs weighing 400 to 500 grams in series of eight each rcceivctl the materials described below. Xach was given into srparatc demarcated sitcls on the skin of t,he back or flank: E’ive milligrams of one of the following in 0.5 ml. 0.15M NaCl: a. Purified rabbit serum gamma globulin (RGG) .* 1~. Aggregated rabbit serum gamma globulins ( AgRGG) with components greater than 75 prepared according to the method of Christ.iam5 c. Soluble antigen-antibody complexes prepared in vitro at three times the antigen (excess. Dissolved precipitates were initially formed at, equivalence through the reaction of bovine gamma globulin (BGG) with corresponding antiserum containing 616 pg AbN per milliliter in a combining ratio of 14.5. Fresh normal rabbit serum in an amount containing 5 mg. total proteins. Antiserum to BUG (0.5 1n1. containing 310 pg AbN) followed in 30 minutes by 1.6 mg. BBC?; in 0.5 ml. 0.15?01 NaCl solution. These dosages were based on the predctcrmint~d Xn :Ah combining ratio of 1 4.5 at cqnivalencc fol this system. IIistamine as 10 pg of base solution. Series I consisted of previously unt,rcated animals. 111 Series II, each of the challenged skin sites of normal animals were pretruatcd with 10 ,ug histamine chalintracntancously 30 minutes prior to RGG, AgRG(:. or antigen-antibody lenge. Series III was composed of guinea pigs having serum content of precipitating ant.ibody ; four each to egg albumin (EA) f and to hemoc~~anin (TIP) $ produced by prior activcl immunization procedures by- intrapcritoneal md foot pad routes. Blood

counts

Prior to initial intracntancous administration of any test material and again prior to biopsy of challenged sites, samples of peripheral blood were taken for: (a) total white blood cell chamber counts, (1)) differential leukocyte cell study of Wright-Giemsa stained slide smears, and (c) total eosinophil chamber counts.” “Pentex Corporation, liankakee, Ill. t5x crystallized, Nutritional Biochemicals, : Prepared by fractionation of blood wrum

Cleveland, Ohio. from Romrcru.s o ~1, ricw~~s.

Skin biopsies

Six hours after the intracutaneous administration of the above listed test materials, challenged sites were excised and sections prepared for histologic study. Biopsies identically prepared were t.aken from untreated sites for control study and from other skin areas nonspecifically traumatized, i.e., by pinching with a surgical clamp and by injections with 0.5 ml. of physiological saline. One portion of each specimen was fixed in formalin and stained with hematoxylin and cosin. A second portion was fixed i-n Icad subacctatc, acetic acid, and alcohol, and stained with an alcoholic solution of thionine for selective

Fig.

1

Skin biopsy taken Prom a guinea pig six hours after intracutancous administration of aggregated rabbit gamma globulin. Interstitial etlrms and a group of bilobed nuclear eosinophilie granular leukocytes nrc seen within the corium. (Hr~mxtosylin and twin. A. x500; B and C’, r970.)

and mast cell idcntifivation ;Iccordirrg to the tcchniyue of Nota and co-workers.’ Quantitation of leukocyte ceil typvs was attempted bj- microscopic* studj- with averages of their rcspcrtivc nnmber*s taken from ten r’cgr(‘s(‘rrt;lti~,(! fields of involved dermis and \-isualizcd at x970 magnific~ation.

hasophil

RESULTS

Cellular infiltrates consisting of eosinophil and pal?-rrrorplronuclear granular leukocytes were found in varying degrees within the dermis and occasionally penetrating into adjacent subcutaneous tissue of guinea pigs treated with RGG both in native and in altered (AgRG(: ) states and with related antigen-antibody systems and soluble complexes (Fig. 1) . Eosinophils were often found in groups but neither in particular relationship to blood vessels nor associated with pathognomonic findings of early Arthus rccation. Quantitation of cells within the given parameters of studr for prcriousl,v normal animals and for those pr(‘1. Injlammatoq cell responses in treated* cutw~eous sites of guinea pigs:+ OtherxGse normal (I), p-e-txposed to histamine (II), 01’ dimly scnsitizcrl to 012 unrelated adigen (III) Table

(‘(11 X‘oSiMIphil Trratmoat

xema

AgRGG RGG :R Ab complrs HGG + R anti-HGG

1: 1 (i 20

scrirs

at skiu

JInximwn

Mran

site: RUSOphil

___.

1 Xazirnwn

AIIcan

/ Xaxim

um

I x

99

35 :10 .50

89 63 210

1iX 156 111 L’j3

2:

4

84

ISi


4 ti 0

6 13 14

191 1% 163

‘25 193 “30

17

"g

"91

43s

4 II L’ 2 15 0.4

12 ”2 30 20 2

208 1x4 259 ‘29 X8

250 350 410 410 1 io

HGG

S(‘rics II Histamine$ Histamineg plus : RGG AgRGG RGG:R Ab complex HGG + R anti-BGG SC rirs

irbfiltration

Polymorphontccl~ni

1.5

1 I

.I 3 3 4 1

.,

i

.?

!L? 2


4

111

Nonrelated active %nsitization plus: Ij RGG AgRGG RGG : R Ab complcs HOG + K anti-BGG Histaminr$

=;

; 0

:‘1

0

.) i

*Treatment-local administration of 5 mg. dosages : RGG, Rabbit serum gamma glohulin. AgRGG, Aggregated RGG. RGG:R Ab romple.u, Antigeu (An) bovine gamnra globulin (BGG) :dh (corwsponding rabbit species Rutiserum) soluhilized in 3x antigcu c~cess. BGG + R anti BGG, An :Ah system hascd upon An :Al) ratios at equivalence and given by local passive sensitization prowdurcs. t Iudicatcd figures rcpwwnt mean tlc~tcrminations for ~~11 group of identically treated animals. $Cells per ten represeutatiw firlds at x970. $Histaminc base 10 pg per skin site. /IEgg albumin or lwnocyanin.

treated with histamine or activel>T sensitized to unrelatrd antigen is rqortcd in Table I. Eosinophils were not found in biopsies of untreated or of saline-injected sites regardless of local histamine treatment at other skin arcas (Series 1 I ) or of an associated state of nonrelated systemic sensitization (Series 111). Alt skin sites subjected to nonspecific trauma by pinch-clamp in both urrtrcatcd and stlnsitixed animals, components of cell infiltrates per 100 x970 mitryoscopic fields averagetl less than 1 cosinophil, 39 polSnro~phorruclear leukocytes, and 1. to 3 basophils. For those injcctcd with normal rabbit. strum, polymorphonuclear leukocytes were evident (ES avcragc) but fewer cosinophils (I.-l avcrngc) wcrc dcmonstrxted wit,h little variation or relationship to the associalt~tl treatment. Quantitation of mean values for eosinophil ct~ll counts within affcctcd cutancous tissue specimens was subjected to analysis of variants for n two-way classification. The F test was made to evaluate ccl1 rcsponscs as a function of: ( 1 ) charact.er of the locally administered materials, and (I)) effect of other treatment given prior to local challenge. l’rclirninary histamine influence and an associat,etl stattl of active systemic sensitization to unrclatrd antigen wcrc not. found significant. The effect of AgBG(:, of soluble antigen-antibody complcscs, and of in \-ivo antigen-antibod,v union was to produce a \ariablc increase in eosinophils significant at. the 5 per cent 1~~1 (11‘ = 52 with 3 and 6 degrees ol’ frt+ dam) . 1Icmatologic changes gcncrally showed rises of 800 to 2,000 cells in counts of total circulating leukocytes six hours after intracutaneous injections of COYresponding immune reactants, soluble antigen-antibody complcscs, and AgBG( ;. This was generally reflected in increases in pol~rrrorphonuclear granular Icukocytes and proportionate dcrreascs in lymphocytes on differential counts of stained fiscd preparations. However, a uniform pattern for rncmbers of each treated series could not bc demonstrated. Fluctuations in total circulating eosinophil cell counts of pcriphrral blood samples wt~r~~ncithcr consistent nor considtred significant. DISCUSSION

Our earlier reports’-” s-‘Jhave agreed with those of other investigators”‘-I’ that the eosinotactic influence of irnmunc reactions is related to circumstant~rs of antigen-antibody union. IEowever, similar findings in studies of the guinea pig lymph node regional to injection sit,es have been given divergent, basic interpreta.tions. Litt,” suggested that the prompt cosinophilic ccl1 infiltrations were a response to the specific reaction between residual administered antigen and corresponding antibody newly synthesized within the lymphoid tissue. On the other hand, our findings’ suggested t,hat the eosinotactic inflncnccs of antigcn-antibody union may reside in the inflammatory response to structurally altered states of the reactants, i.c., molecular aggregates formed by antibody globulins with intcrpositioned corresponding antigen. Esperirnentnl conditions may therefore be considered relevant. This is especially true where cosinophil leukocyte infiltrations can be drmonstratrd within tissue lacking both histologic

structure and physiological function foi antibody formation and without dependence upon circumstances of immune challenge. An assumption that t,he skin is devoid of antibody-forming clcmcnts can be open to just question. llowever, failure to find plasma cells and/or other pyi*oninopliilic cells at sllch c.utancously affected sites in the rcspectivc models described by Xrnason and \Vaksman4 and reported in this stud>- may be cAonsideret1 signific*ant,. Thus, inflammatory cell responses at. the dermal site of introduction of foreign protein within 0nlS 4 to 6 hours after initial exposure in a nonsensitirc animal does not favor a hypothesis invoking the presence of dc nova formed specific antibody’1 to explain the cosinotactic inflncnce. In the guinea pig, relative (~osinophil leukocyte responses t.o molcc*ular aggregates of serum gamma globulins, to soluble antigen-antibody compleses, and to their in vi\-0 union within cutaneous tissue resembled those dentonstratcd within lymphoid tissue’ althongh milder in degree. In both situations, it is significant that valnc~s t’or ~11 counts rcprescnting cosinotactic influcanvcs of nonspecifically aggregated serum gamma globulins closely approac*hetl those arising from the effects of’ in \ivo reaction between antigen and precipitating antibody and hotit soluble products of their in vitro union. On the assumption that a e011l1non otiologic~al tlcnominator functioned at, both sites, anatomical t’actars favorable for tlrainag(~ and retention of the reactants and for ~ICCWS to circulating formed clemcnts of the blood would in turn be cspected to favor acc*umuIiltiOllS of larger nunil)c~i~sof c+culating eosinophils within regional lymph nodes. The apparent lack of consistent ~ol~rrlntion hetwecn local and circulating eosinophi1 cell quantitation is in agrecmc~nt with other reports”’ -I2 illustrating technical difficulties of random sampling t’or dcmonstratin, u time and extent of bloodborne cosinophil leukoc?-tcs in tl*ansI~ol*t from hone 111itrI’o\v stows to affected tissue sites. In the present experiments, a significant diffcrcnce (p <0.05) was noted in the local eosinophilia associated, on the one hand, with tissue exposure to gamma globulin molecular aggregates produced through tither nonspecific bonding or immune mechanisms and, on the other hand, resulting from the native form with the same ant,igenic character. &act reasons for the finding of even few eosinophils after the administration of KGG are unknown. That similar findings were not produced by fresh rabbit serum containing an equivalent amount of total serum proteins suggests that this reactivity may ho related to the reported tendency of isolated gamma globulins to undergo spont.aneous aggregat,ionl” and/or the presence of altol*ed forms resulting from chemiral fractionation of whole serum.‘; Eosinophils were absent both after injections of saline and after the mechanical trauma of skin pinching. Thus, it is unlikely that they are nonspecific components of cellular inflammatory infiltrates. WC have therefore taken note of the work of Stougllton’8 reporting that breakdown products of human epiderof his cxpcrimental mis are capable of act,ing as eosinotoxins. Consideration technique is of special interest, It, is not. unlikely that subjecting skin samples to trypsin-treatment or thermal damage, subsequent incubation at 3’7’ C, and estraetion proredures could result in a denat,uring effect on the tissue pmtcins.

It is thus possible t,hat the association of such end products with eosinophil cellular inflammat.ion is a function of their physicochemically altered state. Within the limitations of this study, we have been unable to demonstrate participation of the basophil or mast cell in the reactivity of guinea pig skin to immune complexes or altered gamma globulins. In this connection, we hare also examined special histochemical preparat,ions of formed elements of peripheral blood, histologic sections of intracut.aneous rhallengr, and the cxudatc taken from denuded surface contact at ear sites,‘” according t.o the skin window technique of Rebuck.“” The reported attraction of cosinophils to sites of cell damage as a consequcncc of immune reaction?’ and their phagocytic function related to resnlbant cornpIcxe~“-‘~ need not necessarily preclude a chcmotactic effect for cosinophlis by associated histamine release. Though the histamine dosages per SC were not found to exert, eosinotact,ic influence in the guinea pig, it. is possible that histnmine chemically potentiated the cell response to antigen-antibody union and the inflammatory stimuli produced by soluble complexes and molecular aggregates of gamma globulins. Not, only did this not prove to be the case, but specifically challenged sites, each pretreated with histamine in dosages of 10 to 20 pg! wcrc characterized by the lowest. observed levels of eosinophil ccl1 infiltrations. Such localized tissue findings are in contrast to reported peripheral blood changes in guinea pigs, both normal?* and sensitized,““, “‘; which were injected with histamine by both intraperitoneal and intracardial routes. Possible differences might be arcounted for by systemic histamine cffcc~ts. lt is therefore pertinent that cumulative influences of histamine absorbed from each cutaneous site in dosages totaling up to 120 ,IL~ frequently caused symptoms of delayed histamine shock before completion of the six hour experimental period. The enhancement, of eosinotactic mechanisms and/or bone marrow precursor cell stimulation was sought through induced states of associated hppersensitivity involving an unrelated antigen-antibody system of demonstrated cosinotactic potential.“, ” Sue11 pretreatment, however, was also found to be without effect in animals suhjectcd to subsequent local challenge. Species differences may indeed be important in interpreting pathophysiological mechanisms of eosinophilia. The lack of histamine influence on eosinophil cell attraction in guinea pig skin at six hour intervals agrees with data rcportctl by Litt” but is in contrast to a reported positive effect in the horse?; and in man.28 With the utilization of peroxidase reaction techniques”” for eosinophil cell dctect.ion in rabbit skin, we were able to demonstrate localized cell responses to antigen-antibody union, soluble complexes, and aggregated gamma globulins similar to those effected in the guinea pig. However, in the rabbit skin histamineinduced inflammatory changes at six hours were characterized by interstitial edema but with a paucity of cells even of the polymorphonuclear lcukoc,vtc series. Although this stud; suggests that eosinotactic influences of antigen-antibod,v union in the guinea pig may be related to resultant immune complexes prcsenting as molecular aggregates, it is apparent that a. common denominator for pathophysiologic mechanisms of eosinophilia remains to be defined.

I 2.

6. i.

8. 9. . 10. 17. 12. 13.

14. 15. 16.

(!ohen, 8. G., Happ, T. ill., Rizzo, A. P., and Kostage, S. T. : E;xperimental Eosinophilin. VII. Lymph Node Responses to Altered Gamma Globulins, J. ALLERGY 35: 346. 1964. Cohen,-R. b., Sapp, 1~. &M., and Gallia, A. R.: Expcriment~l Eosinophilix. V. Specificity of Regional Lymph Node Responses to AntigcwAntibody Systems, Proe. Sot. Exper. Riol. & Med. 113: 29, 1963. Cohen, 8. G., and Sapp, T. M.: Polysaccharide Effects Simulating Hypersensitivit! Cellular Responses in the Rabbit, Am. J. Physiol. 207: 3X9, 1964. Arnason, B. G., and \Vaksman, H. H.: The Retest Rravtion in Delayed Sensitivity, La11. Inrcst I. 12. .c, 77i 1963 . Christian, C. I,: : Characterization of the ‘ i Reactant * ) (Gamma Globulin Factor) in the F 11 Precipitin Rl,action and the F II Tanned Sheep Cell Apglut,ination Test, J. Espw. Mod. 108: 139, 195x. Pilot, M. L. : T:se of Base in Fluids for Counting Eosinophils; d M&hod for Staining Eosinonhils. Am. J. Chin. Path. 20: 870. 1950. Mota, ic., &raldo, IV. T., and Junquerira, L. C. U.: l’rotamine-like Property of Conipounds 48/X0 and Stilbamidine and Their Action on Mast Cells, Prov. Sot. Espcr. Rioi. & Med. 83: 455. 1953. Cohen, R. G., and Sapp, T. M.: Plasma Protein Antigens in Histopathologic Vascwlar Responses to Expwimental Sensitization, J. Lab. & Clin. Med. 58: 644, 1961. Cohen, Y. G., Kantor, M., and Gatto, L.: Experimental Eosinophilia. III. Regional Lyml)ll Node Responses to Reactions of Tissue Sensitization, J. I\LLEKGY 32: 214, 1961. Samter, M., Kofoecl, M. A., and Pieper, XV.: A Factor of Anaphylactically Shocketl Guintw Pigs Which Can Induw Eosinophilia in Normal i\nimals, Blood 8: 1078, 1953. I,itt, M.: Studies in Experimental Eosinophilia. IV. l)c+erminants of Eosinophil Locnlizntion. J. ALLEKOY 33: 5.12. 196%. Litt, M.: Studies in E&rimental Eosinophilia. V. Eosinophils in Lymph Nodes of Guinea Pigs Following Primary Antigcwic Stimulation, Am. J. Path. 42: 529, 1963. Speirs, R. S., and Droisl)ach, M. E. : Quantitative Studies of tho Cellular Rrsponscs to Antigen Tnjcctions in Normal Mice. Technique for Determining Cells in Pwitmwal Fluitl. Rlood 11: 44, 1956. I,itt, M.: Rtudics in Expwimental Eosinophilia. I. Repeated Quantitation of Pwitoncwl Eosinophilia in Guinea Pigs by a Method of Peritoneal Lavage, Blood 16: 1315, 1960. Gross, R.: The Eosinophils, in Braunsteiner, H., and Zucker-Franklin, I)., editors: The Phvslology and Pathology of Leukocytes, New York, 1962, Grune & Stratton, Iw., p. 30. Tsliizakn, K., and Ishizaka, T. : Biologic Activity of Aggregated-Globulin. IT. ;i Stud) of the Various Methods for Aggregation and Species Differences, J. Immunol. 85: 163,

14fill _ .,~,. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26.

27.

Milnrom. Z’.. and Witelwkv. E. : Studies on the Rheumatoid and Related Serum ~actorr. 1. Auto&m&ization of Rahl,its With Gamma Globulin, J. A. M. A. 174: 56, 1960. Atoughton. R. E. : Eosinophil Attracting Substances (Eosinotzoxins) Extrwtctl From IIunmn ‘$pid&mis, J. TnvestDrrmat. 31: b9, 1958. ohsrrvations. Cohen, 9. G., and DeAngelis, C. I). : Unpublished Rebuck, J. W., and Crowley, 3. H.: A Method of Study of Leukocq-tic Functions in Viw, Ann. New York &ad. Hc. 59: 757, 1955. Speirs, B. S., and Hpeirs, E. E.: Cellular Localization of R,adioactire Antigen in Immunized and Nonimmunized Miw, J. Immunol. 90: 561, 1963. %besin, S. M.: A Function of t,hr Eosinophil: Phagocytosis of Antigen-Antibody (‘omplexrs, Proc. Hoc*. Espw. Biol. 6; Med. 112: 670, 1963. Archer, G. T., and TTirsch, J. G.: Motion Picture Studies on Degranulation of Horse F:osinophils During Phagorytosis, J. Exper. Med. 118: 287, 1963. Vaughan, J.: The Function of the Eosinophile Leukocyte, Blood 8: 1, 1953. (‘amnhrll. D. II.: Relationshin of the Eosinoahil Rrsnonse to Factors Involvrd in Ananhv-L d I lax& .T. infect. Dix. 72: 42, 3943. Hamtw, M.: The Rcsponsc of Eosinophils in the Guinea Pig to Sensitization, <1naphylasis and Various Drugs, Blood 4: 21i, 1949. Archer, R. K. : The Eosinophil Leukocytes, Oxford, 1963, Blackwell Scientific Publications, pp.

2x. 29.

42-60.

Kline, allergic

B. S., Cohen, M. R., and Rudolph, .T. A. : Histologic Changes in Allwgic ant1 NonWhcals, J. AI.IJCRCTY 3: 531, 1932. Staining Combined With AntoPopp, R. -4., Gudr, W. I)., and Popp, I). M.: Peroxidase Stain ‘I’whnol. 37: 243, 1962. radiography for Studying Eosinophilic Granules,