Mycobacterial Ribonucleic Acid Preparations

Transcription

1 INFEcriON AND IMMUNITY, JUlY 1973, p Vol. 8, No. 1 Copyright Americn Society for Microbiology Printed in U.S.A. Reltionship Between Tuberculin Hypersensitivity nd Cellulr Immunity to Infection in Mice Vccinted with Vible Attenuted Mycobcteril Cells or with Mycobcteril Ribonucleic Acid Preprtions ROCHELLE G. NEIBURGER, GUY P. YOUMANS, AND ANNE S. YOUMANS Deprtment of Microbiology, Northwestern University Medicl School, Chicgo, Illinois Received for publiction 26 Februry 1973 The migrtion inhibition technique hs been used to study delyed hypersensitivity in vitro by using peritonel exudte cells nd splenic lymphocytes from mice vccinted with vible cells of the ttenuted H37R strin of Mycobcterium tuberculosis nd from mice vccinted with ribonucleic cid (myc RNA) preprtions obtined from vible mycobcteril cells of the sme strin. Inhibition of mcrophge migrtion ws noted when purified protein derivtive (PPD) or vible H37R cells were dded to peritonel exudte cells obtined from mice immunized with vible H37R cells nd not from mice immunized with myc RNA. Splenic lymphocyte cultures were exposed to the sme ntigens in vitro. Filtered superntnt fluids from these lymphocyte cultures, when dded to peritonel exudte cells obtined from nonimmunized mice, inhibited migrtion only when they were obtined from lymphocytes which cme from mice immunized with vible H37R cells. Injection of PPD intrvenously into vccinted mice resulted in inhibitory superntnt fluids from splenic lymphocyte cultures only when the lymphocytes cme from mice immunized with vible H37R cells. However, intrvenous injection of either vible H37R cells or of myc RNA preprtions into mice vccinted with myc RNA occsionlly produced inhibitory superntnt fluids when lymphocytes were obtined from these mice. On the other hnd, mice vccinted with myc RNA or vible H37R cell preprtions were consistently nd eqully protected ginst intrvenous chllenge with the virulent H37Rv strin. Thus, lthough some evidence ws obtined for delyed type hypersensitivity in mice vccinted with H37R cells or with myc RNA to ribosoml proteins or other proteins ssocited with the RNA preprtion, no evidence of tuberculin hypersensitivity could be detected in ny mice vccinted with the myc RNA. These results rgue ginst role for tuberculin hypersensitivity in immunity to tuberculous infection. The reltionship between tuberculin hypersensitivity nd cellulr immunity to infection hs not been fully resolved. The close ssocition of the two phenomen in nimls vccinted with vible ttenuted mycobcteril cells hs suggested to some tht tuberculin hypersensitivity my be responsible for the incresed resistnce to infection with virulent tubercle bcilli in vccinted nimls (5, 6). However, evidence is vilble which indictes tht the two phenomen cn be dissocited. For exmple, tuberculin hypersensitivity hs been 42 produced without concurrent increse in resistnce (9). Also, in immunized nimls reduction in tuberculin hypersensitivity by desensitiztion hs been chieved without loss of immunity to infection (10). Furthermore, mycobcteril ribonucleic cid (myc RNA) preprtion hs been isolted from vible cells of the ttenuted H37R strin which will immunize mice s effectively s the living H37R cells do (13, 15) without inducing tuberculin hypersensitivity in guine pigs or mice (18, 19).

2 VOL. 8, 1973 TUBERCULIN HYPERSENSITIVITY AND TB IMMUNITY 43 Youmns nd Youmns (17, 19) hve proposed multiple-response theory of immunity to tuberculosis. The work of Youmns nd Youmns (13, 19) nd Coppel nd Youmns (2, 3, 4) indictes tht the mjor immune response in this disese is specific nd tht the gent responsible cn be found in the RNA frction (13, 5). Secondly, immunity to tuberculosis depends, in prt, on reltively stble substnces (some of which cn induce tuberculin hypersensitivity) which will bring bout moderte mount of nonspecific immunity to infection becuse of stimultion of grnulomtous response or by ctivtion of mcrophges (17, 19). Mckness (6) sttes tht filure to demonstrte derml hypersensitivity fter vccintion with prticulr preprtion is not vlid rgument ginst the existence of hypersensitive stte, especilly becuse it frequently cn be demonstrted tht in nergic nimls rpid ppernce of derml hypersensitivity cn be noted fter second ntigen stimultion. According to this view, the so-clled "nonsensitizing" components merely produce very low nondetectble degrees of tuberculin hypersensitivity. This hypersensitivity would be rpidly boosted upon chllenge of the niml with virulent tubercle bcilli. Most of the work done with myc RNA vccine hs been performed in mice. Methods vilble for the detection of tuberculin hypersensitivity in mice re generlly poor, lthough derml sensitivity cn be noted fter injection of tuberculin into footpds of mice vccinted with H37R cells. However, recent evidence suggests tht the bility of sensitized lymphocytes to rect in vitro when exposed to ntigen is more sensitive mesure of hypersensitivity thn is derml rectivity (11). In guine pigs, the mcrophge migrtioninhibition technique is n in vitro correlte of delyed hypersensitivity. In previous investigtion we hve shown tht this technique cn be redily dpted for use with mouse lymphocytes (7). Under pproprite conditions the production of migrtion inhibitory fctor (MIF) by stimulted mouse lymphocytes lso cn be redily demonstrted (7). Therefore it is now possible, by using this more sensitive technique for the detection of tuberculin hypersensitivity, to more ccurtely compre the immune response of mice with their stte of tuberculin hypersensitivity fter vccintion with intct vible H37R cells or with myc RNA preprtions. MATERIALS AND METHODS Mice. Mle outbred CF-1 (Crworth Frms) nd inbred C57BI/6 (ARS/Sprgue-Dwley) mice, weighing 18 to 22 g, were used throughout. Mice were housed 10 per cge nd llowed unlimited food nd wter. Vccintion. Vccintion with vible H37R cells ws crried out s previously described (7) by giving intrperitonel or subcutneous injections of 1 mg, (moist weight) of H37R cells in 0.2 ml of 0.01 M phosphte buffer, ph 7.0. Immunogenic myc RNA, prepred from vible H37R cells, ws isolted by the methods described by Youmns nd Youmns (13, 15). Briefly, prticulte frction ws obtined by differentil centrifugtion of ruptured vible H37R cells. Ribosomes were obtined fter tretment of the prticulte frction with sodium dodecyl sulfte nd centrifugtion. RNA ws then prepred from the ribosoml frction by ethnol precipittion. Mice were vccinted intrperitonelly with 5.0 or 0.5 jig of the RNA preprtion in 0.4 ml of Freund incomplete djuvnt (14). In vivo chllenge nd mesurement of immunity. Immunized nd nonimmunized CF-1 mice were chllenged by intrvenous injection of 1 mg (moist weight) of fine suspension of the virulent H37Rv strin of Mycobcterium tuberculosis prepred s described for the H37R strin. The inbred C57B1/6 mice were chllenged with 0.25 mg of H37Rv becuse they were more susceptible to tuberculosis (20). A record ws kept of the time of deth of ech niml. The number of mice surviving 30 dys or longer in the immunized group ws compred with the number of mice surviving 30 dys or longer in the nonimmunized controls. The relibility nd vlidity of this technique hs been given by Youmns nd Youmns (16). Footpd tests. Tuberculin sensitivity to 2 jig of purified protein derivtive (PPD) injected into the footpds of mice ws mesured s previously described (7, 18). Migrtion inhibition test. The migrtion inhibition test using mouse cells ws performed s described in n erlier pper (7). In the direct test, oil-induced peritonel exudte cells were collected from nonimmunized nd immunized mice, nd the re of their migrtion ws determined by projection nd plnimetry in the presence or bsence of ntigen. For the indirect test, superntnt fluids from splenic lymphocyte cultures were prepred nd tested for the presence of MIF s previously described (7). Superntnt fluids lso were dilyzed ginst physiologicl sline nd then ginst distilled wter. The dilystes were then lyophilized nd stored t -70 C for lter use. Smples to be tested were reconstituted in the complete tissue culture medium nd sterilized by filtrtion. Results will be reported s percent migrtion in the presence of ntigen compred with percent migrtion in the bsence of ntigen. RESULTS Effect of PPD on mcrophge migrtion. CF-1 mice which hd been vccinted with vible cells of the H37R strin of M. tuberculosis or with myc RNA preprtions were highly

3 44 NEIBURGER, YOUMANS, AND YOUMANS INFECT. IMMUNITY protected, s shown by concurrent studies (Tble 1). Similrly immunized mice were tested for footpd sensitivity to tuberculin. Only the mice immunized with vible H37R cells showed significnt degree of footpd swelling t 24 h s previously reported (7). Peritonel exudte cells were collected from nonimmunized, H37R-immunized, nd myc nd tested for migrtion in the presence of PPD (Tble 2). None of the PPD concentrtions used hd ny effect on the migrtion of cells from nonimmunized or myc. Migrtion of cells from H37R-immunized mice ws significntly inhibited in the presence of PPD. Similr results were found using peritonel exudte cells from C57B1/6 strin mice. Effect of vible H37R cells. The ddition of vible H37R cells to the migrtion chmbers gve similr results (Tble 3). Peritonel exudte cells from nonimmunized or myc RNAimmunized CF-1 or C57B1/6 mice migrted in t'he presence of the bcilli. Sttisticlly significnt inhibition of migrtion ws detected only when vible H37R orgnisms were dded to chmbers contining peritonel exudte cells from H37R-immunized mice. Effect of myc RNA. myc RNA ws dded to the medium in the chmbers contining peritonel exudte cells from nonimmunized, H37R- TABLE 1. Immunogenicity of myc RNA nd vible ttenuted cells of the H37R strin immunized, nd myc. The ddition of myc RNA hd no inhibitory effect in ny of the chmbers (Tble 4). Fifty microgrms of myc RNA is the equivlent of 1 mg (moist weight) of the H37R microorgn- Per- Immunizing Experi- Amount No. of No. of centge preprtions preprtonsment injected mc mice S-30 of S-30 no. (Mg) mice mice myc RNA H37R cells Controls myc RNA H37R cells Controls myc RNA H37R cells Controls myc RNA H37R cells Controls Number of mice which survived 30 dys or longer. TABLE 2. Effect of purified protein derivtive (PPD) on the migrtion of peritonel exudte cells from nonimmunized, H37R-immunized nd myc Source of peritonel PPD Migr- Probbilexudte (ug/ml) tion (%) ity NS NS H37R-immunized mice < <0.005 mice NS NS NS, Not significnt = P > TABLE 3. Effect of vible H37R cells on the migrtion of peritonel exudte cells from nonimmunized, H37R-immunized, nd myc Source of peritonel c37nl Migr- Probbilexudte (mg/ml) tion (%) ity NS NS H37R-immunized mice < <0.005 mice NS NS NS, Not significnt = P > isms. One milligrm of H37R cells mrkedly inhibited the migrtion of peritonel exudte cells from H37R-immunized mice. Addition of 50 to 400.ug of myc RNA hd no inhibitory effect on ny cells. Effect of MIF on migrtion. Superntnt fluids were prepred from 72-h cultures of splenic lymphocytes obtined from nonimmunized, H37R-immunized, nd myc RNAimmunized C57B1/6 mice. The lymphocytes were incubted for this period with nd without ntigen. The ntigens used were 50 to 75 gg of PPD per ml, 0.3 to 3.0 mg of vible H37R microorgnisms per ml, or 200 to 333,sg of myc RNA per ml. Superntnt fluids were tested for inhibitory ctivity on peritonel exudte cells obtined from nonimmunized mice. MIF ctivity ws detected in superntnt

4 VOL. 8, 1973 TUBERCULIN HYPERSENSITIVITY AND TB IMMUNITY TABLE 4. Effect of myc RNA on the migrtion of peritonel exudte cells from nonimmunized, H37R-immunized, nd myc Source of peritonel RNA Migr- Probbilexudte (pg/ml) tion (%) ity NS NS NS H37R-immunized mice NS NS NS mice NS NS NS NS, Not significnt = P > fluids from H37R-immunized mice incubted with PPD or incubted with vible H37R cells (Tble 5). Incubtion of lymphocytes from nonimmunized or myc with ny of the ntigens nd incubtion of lymphocytes from H37R-immunized mice with myc RNA gve no inhibitory superntnt fluids. Concentrtion of superntnt fluids. Superntnt fluids prepred nd tested for MIF ctivity lso were dilyzed, lyophilized, nd reconstituted to obtin more concentrted preprtions. These reconstituted fluids were tested to determine whether ctivity could be detected in concentrted superntnt fluids which ws not discemible in the unconcentrted preprtions. Inhibition obtined with lyophilized inhibitory superntnt fluids reconstituted to five times the originl concentrtion ws virtully identicl to the inhibition seen when the superntnt fluids were fresh; the superntnt fluids which did not inhibit in their originl form were still not inhibitory t five times their originl concentrtion. Intrvenous stimultion. Antigen ws injected intrvenously into nonimmunized nd immunized mice to determine the effect of in vivo dministrtion of ntigen on subsequent MIF production by spleen cells in vitro (7). MIF production ws found only with lymphocytes from H37R-immunized mice fter intrvenous injection of 100 to 150 Ag of PPD (Tble 6). This fmding is consistent with our previously published results (7). Injection of between 0.25 nd 1.0 mg of vible H37R microorgnisms resulted in in vitro MIF production by lymphocytes from H37R-immunized mice nd by lymphocytes from myc (Tble 7). Positive footpd test rections to tuberculin were found only with H37R-immunized mice, either before or fter the intrvenous injection of ntigen. No positive footpd rections to tuberculin were seen in nonimmunized or myc either before or fter the injection of the H37R orgnisms. A smple (50 ug) of myc RNA ws given intrvenously to nonimmunized, H37R-immu- TABLE 5. Effect of superntnt fluids from lymphocytes from nonimmunized, H37Rimmunized, nd myc RNA-immunized mice cultured in the presence of purified protein derivtive, vible H37R cells, or myc RNA on the migrtion of mouse peritonel mcrophges Source of lympho- Concn of Migr- Probbilcytes ntigen per tion () ity ml 75 Ag PPD NS 3 mg H37R 95.9 NS 200 ug RNA 98.4 NS H37R-immunized mice 75 Ag PPD 56.3 < mg H37R 63.8 < ug RNA 88.5 NS mice 75 Ag PPD NS 3 mg H37R 87.8 NS 200pug RNA 99.8 NS NS, Not significnt = P > TABLE 6. Effect of intrvenous dministrtion of purified protein derivtive (PPD) on the relese of migrtion inhibitory fctor by lymphocytes, obtined from nonimmunized, H37R-immunized, nd myc Source of lymphocytes PPD Migr- Probbil- (Mg) tion (%) ity NS H37R-immunized mice < <0.01 mice NS NS NS, Not significnt = P >

5 46 NEIBURGER, YOUMANS, AND YOUMANS INFECT. IMMUNrrY TABLE 7. Effect of intrvenous dministrtion of vible H37R cells on the relese of migrtion inhibitory fctor by lymphocytes from nonimmunized, H37R-immunized, nd myc H37R Mg rbbl (mg) % t Source of lymphocytes cells Migr Pr bbil NS NS H37R-immunized mice < < <0.001 mice NS < <0.001 NS, Not significnt = P > 0.1. TABLE 8. Effect of intrvenous dministrtion of myc RNA on the relese of migrtion inhibitory fctor by lymphocytes from nonimmunized, H37Rimmunized, nd myc Source of lymphocytes RNA Migr- Probbil- (Mg) tion (%) ity NS H37R-immunized mice NS mice NS NS, Not significnt = P > nized, nd myc, nd spleen cells were hrvested s before. Pooled dt from five such experiments re shown in Tble 8. There ws no sttisticlly significnt inhibition of migrtion. However, in two of five individul experiments there ws sttisticlly significnt inhibition with superntnt fluids from lymphocytes from H37R-immunized mice injected intrvenously with myc RNA, nd in one experinlent there ws sttisticlly significnt inhibition by superntnt fluids from lymphocytes tken from myc RNA-immunized mice (Tble 9). This is in contrst to the results of ll experiments in which lymphocytes were stimulted with myc RNA in vitro. In ll such experiments no MIF could be detected. DISCUSSION The dt in this pper show tht hypersensitivity to PPD s mesured in vitro by using the migrtion inhibition technique cn be found by using lymphocytes from mice immunized with vible cells of the ttenuted H37R strin of M. tuberculosis nd not with lymphocytes of nonimmunized nimls or myc RNA-immunized nimls. Equivlent protection ginst chllenge cn be chieved by vccintion with vible H37R cells or with the myc RNA preprtions. Thus tuberculin sensitivity, s mesured in vitro, is not ssocited with the bility of immunized nimls to withstnd chllenge with virulent microorgnisms. The detection of MIF ctivity fter the injection of vible H37R cells into myc RNA-immunized mice nd the occsionl detection of MIF ctivity fter the intrvenous injection of myc RNA in H37R-immunized nd myc RNAimmunized mice my be due to hypersensitivity to the protein ssocited with the RNA. Ortiz-Ortiz et l. (8) nd Bker et l. (1) hve demonstrted delyed hypersensitivity to ribosoml protein prepred from BCG cells. Animls vccinted with BCG hd rections of delyed hypersensitivity to PPD or ribosoml protein; nimls sensitized with ribosoml protein gve delyed rections when tested with either the ribosoml protein or PPD (8). Efforts by Youmns nd Youmns (18) to detect hypersensitivity to ribosoml nd myc RNA frctions in mice nd guine pigs were unsuccessful. The hypersensitivity to the protein in the myc RNA preprtions cn ply no role in the immune response becuse the protein cn be removed in number of wys without ffecting the immunogenicity (12, 19, nd unpublished dt). In this respect it is importnt to point out tht hypersensitivity to RNA protein cn be de- TABLE 9. Effect of intrvenous dministrtion of myc RNA on the relese of migrtion inhibitory fctor by lymphocytes from H37R-immunized nd myc Source of lymphocytes RNA Migr- Probbil- (4g) tion (%) ity H37R-immunized mice <0.001 k mice k <0.005 Pooled dt from the two positive experiments out of five performed. b Dt from the one positive experiment out of five performed.

6 VOL. 8, 1973 TUBERCULIN HYPERSENSITIVITY AND TB IMMUNITY 47 tected only occsionlly, but the RNA-immunized mice re protected ginst chllenge to the sme degree whether or not this hypersensitivity cn be detected. Evidence obtined in this study by using the murine migrtion inhibition technique supports the seprtion of delyed hypersensitivity to tuberculin nd cellulr immunity to infection becuse vccintion of mice with myc RNA does not result in production of tuberculin hypersensitivity. These observtions strongly suggest tht tuberculin hypersensitivity is not responsible for the cellulr immunity to tuberculous infection. ACKNOWLEDGMENT This investigtion ws supported by Public Helth Service reserch grnt Al from the Ntionl Institute of Allergy nd Infectious Diseses. LITERATURE CITED 1. Bker, R. E., W. E. Hill, nd C. L. Lrson Delyed hypersensitivity rections provoked by ribosomes from cid-fst bcilli. I. Ribosoml isoltion, chrcteriztion, delyed hypersensitivity, nd specificity. Infect. Immunity 6: Coppel, S., nd G. P. Youmns Specificity of cquired resistnce produced by immuniztion with Listeri monocytogenes nd listeri frctions. J. Bcteriol. 97: Coppel, S., nd G. P. Youmns Specificity of cquired resistnce produced by immuniztion with mycobcteril cells nd mycobcteril frctions. J. Bcteriol. 9X: Coppel, S., nd G. P. Youmns Specificity of the nmnestic response produced by Listeri monocytogenes or Mycobcterium tuberculosis to chllenge with Listeri monocytogenes. J. Bcteriol. 97: Mckness, G. B The immunologicl bsis of cquired cellulr resistnce. J. Exp. Med. 120: Mckness, G. B The reltionship of delyed hypersensitivity to cquired cellulr resistnce. Brit. Med. Bull. 23: Neiburger, R. G., nd G. P. Youmns Inhibition of migrtion of mouse mcrophges by tuberculin-sensitive mouse lymphocytes nd by mouse migrtion inhibitory fctor. Infect. Immunity 7: Ortiz-Ortiz, L., E. B. Solrolo, nd L. F. Bojlil Delyed hypersensitivity to ribosoml protein from BCG. J. Immunol. 107: Rffel, S The components of the tubercle bcillus responsible for the delyed type of "infectious" llergy. J. Infect. Dis. 82: Rich, A. R The pthogenesis of tuberculosis. Chrles C Thoms. Springfield, Ill. 11. Schlossmn, S. F., H. A. Levin, R. E. Rocklin, nd J. R. Dvid The comprtmentliztion of ntigenrective lymphocytes in desensitized guine pigs. J. Exp. Med. 134: Youmns, A. S., nd G. P. Youmns Effect of trypsin nd ribonuclese on the immunogenic ctivity of ribosomes nd ribonucleic cid isolted from Mycobcterium tuberculosis. J. Bcteriol. 91: Youmns, A. S., nd G. P. Youmns Preprtion of highly immunogenic ribosoml frctions of Mycobcterium tuberculosis by the use of sodium dodecyl sulfte. J. Bcteriol. 91: Youmns, A. S., nd G. P. Youmns Preprtion nd effect of different djuvnts on the immunogenic ctivity of mycobcteril ribosoml frction. J. Bcteriol. 94: Youmns, A. S., nd G. P. Youmns Fctors ffecting immunogenic ctivity of mycobcteril ribosoml nd ribonucleic cid preprtions. J. Bcteriol. 99: Youmns, G. P., nd A. S. Youmns The mesurement of the response of immunized mice to infection with Mycobcterium tuberculosis vr. hominis. J. Immunol. 78: Youmns, G. P., nd A. S. Youmns Nonspecific fctors in resistnce of mice to experimentl tuberculosis. J. Bcteriol. 90: Youmns, G. P., nd A. S. Youmns Allergenicity of mycobcteril ribosoml nd ribonucleic cid preprtions in mice nd guine pigs. J. Bcteriol. 97: Youmns, G. P., nd A. S. Youmns Recent studies on cquired immunity in tuberculosis, p In J. A. Brody et l. (ed.), Current topics in microbiology nd immunology, vol. 48. Springer-Verlg, New York. 20. Youmns, G. P., nd A. S. Youmns Response of vccinted nd nonvccinted syngeneic C57Bl/6 mice to infection with Mycobcterium tuberculosis. Infect. Immunity 6: