Specific and Nonspecific Antibody Responses in Different Segments

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1 INFECTION AND IMMUNITY, OCt. 1991, p /91/ $2./ Vol. 59, No. 1 Specific nd Nonspecific Antibody Responses in Different Segments of the Respirtory Trct in Rts Infected with Mycoplsm pulmonis JERRY W. SIMECKA,1* PADMA PATEL,' JERRY K. DAVIS,"2 SUZANNE E. ROSS,' PHYLIS OTWELL,1 AND GAIL H. CASSELL' Deprtment of Microbiology1 nd Deprtment of Comprtive Medicine,2 University of Albm t Birminghm, University Sttion VH 57A, Birminghm, Albm Received 2 Mrch 1991/Accepted 13 July 1991 Murine respirtory mycoplsmosis resulting from Mycoplsm pulmonis infection in rts provides useful model for the study of immunologicl nd inflmmtory mechnisms opertive in the respirtory trct. We hve previously shown tht LEW rts develop more severe disese thn do F344 rts. To further study the production of ntibody responses in chronic respirtory disese due to M. pulmonis infection, we exmined the distribution nd development ofm. pulmonis-specific ntibody-forming cells (AFC) in different segments of the respirtory trcts of infected LEW nd F344 rts. In these studies, the upper respirtory nodes were the initil site of ntibody production fter infection nd remined the mjor site for recovery of AFC. Since infected LEW rts hd equl or higher numbers of AFC thn did infected F344 rts, these results suggest tht the level of locl ntibody production lone is not responsible for the decresed susceptibility of F344 rts to murine respirtory mycoplsmosis. The differences in totl ntibody responses pper to be due to the greter numbers of cells recovered from the tissues of infected LEW rts compred with those recovered from F344 rts, suggesting tht LEW rts my hve greter production of chemotctic fctors. Also, we demonstrte tht nonspecific ctivtion nd/or recruitment of B cells occurs in the respirtory trcts of both LEW nd F344 rts fter infection with M. pulmonis. Murine respirtory mycoplsmosis (MRM) resulting from Mycoplsm pulmonis infection in rts provides useful model for the study of immunologicl nd inflmmtory mechnisms opertive in the respirtory trct (6). Nturl or experimentl infection with M. pulmonis results in the development of otitis, rhinitis, trcheitis, nd bronchopneumoni (16). Like mny other infectious respirtory diseses, MRM begins in the nsl pssges nd progressively spreds downwrd into the lungs (26). The mjor feture of the lesions of MRM is the lrge mononucler cell ccumultion present in ll the mucosl regions of the respirtory trct, including hyperplsi of the bronchilly ssocited lymphoid tissue in the lung (8, 16). Eventully, disese of the lung prenchym cn lso develop. We previously demonstrted differences between MRM lesion severity nd disese progression in two strins of rts. After infection, LEW rts develop more severe upper nd lower respirtory trct lesions thn do F344 rts (8). Also, severe lesions in the lung prenchym often develop in LEW rts, wheres the lung disese in F344 rts is predomintely ssocited with the mucosl surfces nd irwys. The lung lesions in F344 rts begin to resolve in 28 dys fter infection, wheres the lesions in LEW rts continue to worsen (1). In LEW rts, there is n increse in ll clsses of lymphocytes in the lungs throughout the course of disese. In contrst, the numbers of lung lymphocytes in F344 rts increse only in the first 28 dys fter infection, nd not ll lymphocyte clsses increse during this time (1). Specific immune responses lso seem crucil to the pthogenesis of MRM. The respirtory lesions of MRM, including lymphocyte infiltrtion, cn be prevented or reduced by intrvenous vccintion with killed orgnisms; however, intrnsl immuniztion with killed orgnisms reduces the * Corresponding uthor severity of rhinitis but does not ffect lesions in ny other region of the respirtory trct (7). Thus, this disese provides unique opportunity to study interctions between the systemic nd mucosl immune systems, including interctions between the upper nd lower respirtory trcts, in nturlly occurring infection. In previous studies, we exmined the bility of LEW nd F344 rts to produce ntibody to M. pulmonis. First, we evluted specific immune responses to nonreplicting M. pulmonis ntigens in both rt strins fter prenterl immuniztion (29). Immunized F344 rts produce higher levels of M. pulmonis-specific serum immunoglobulin G (IgG) ntibody thn do LEW rts, wheres IgM serum ntibody responses do not differ. In more recent studies, we exmined the temporl sequence of specific serum ntibody production in LEW nd F344 rts fter M. pulmonis infection (31). In contrst to the results in the immuniztion studies, infected LEW rts produce t lest s much of ech of clss of specific ntibody to M. pulmonis s do infected F344 rts. Although these studies suggest tht serum ntibody is not responsible for differences in the disese in LEW nd F344 rts, ntibody production long the respirtory trct my still ply role in disese, becuse locl immunity hs not been exmined. Additionl studies on the distribution of ntibody-forming cells (AFC) re needed not only to understnd the role of ntibody in disese progression but to further understnd the effect of chronic inflmmtory responses on immunity. In recent study (33), lymphoid tissues of the upper respirtory trct in infected F344 rts were shown to be the mjor sites of M. pulmonis-specific ntibody production, wheres the contribution of the lung s site of ntibody production ws miniml. In ddition to ctivtion of specific immune mechnisms, nonspecific ctivtion of B cells my lso occur during disese pthogenesis. We (1, 3, 37) nd others (18, 19) hve suggested tht nonspecific ctivtion of lymphoid

2 3716 SIMECKA ET AL. cells my contribute to the development of disese; this is supported by our studies (9) showing tht the LEW lymphocyte response to mitogens in vitro is greter thn tht of F344 lymphocytes. To further study the production of ntibody responses in chronic respirtory disese due to M. pulmonis infection, we exmined the distribution nd development of M. pulmonisspecific AFC in different segments of the respirtory trcts of infected LEW nd F344 rts. In these studies, the upper respirtory lymph nodes (URN) were the initil site of ntibody production fter infection nd remined the mjor site for the recovery of AFC. Also, we demonstrte tht nonspecific ctivtion of B cells occurs in the respirtory trcts of both LEW nd F344 rts fter infection with M. pulmonis. MATERIALS AND METHODS Animls. Pthogen-free F344 nd LEW rts 8 to 12 weeks old were rered nd mintined in Trexler-type plstic film isoltors (22). The pthogen-free sttus of niml colonies ws monitored by using serologicl (including n enzymelinked immunosorbent ssy [ELISA] for serum ntimycoplsml ntibodies) nd/or culturl techniques for mycoplsml, virl (Sendi virus, pneumoni virus, silodcryodenitis virus, Kilhm rt virus, H-1 virus, reovirus type 3, GD-VII virus, lymphocytic choriomeningitis virus, mouse denovirus [serologicl ssys by Chrles River Biotechnicl Services, Wilmington, Mss.]), fungl, nd other bcteril pthogens. The rt colony ws consistently negtive for ll pthogens, nd the nimls were negtive for IgG nd IgM ntimycoplsm ntibodies. Before ny experimentl mnipultion or collection of tissues, rts were nesthetized with combintion of ketmine hydrochloride (Bristol Lbortories, Syrcuse, N.Y.) nd xylzine (Rhompun, 5 mg/ml; Hver-Lockhrt, Shwnee, Kns.). For experimentl infection, nimls were intrnslly inoculted with 5 RId of M. pulmonis contining 5 x 16 CFU, unless otherwise indicted. In selected experiments, rts were intrperitonelly immunized with 125 jig of keyhole limpet hemocynin (KLH). Mycoplsm nd immunizing ntigens. M. pulmonis UAB 651 ws originlly isolted from the lungs of rt with nturl MRM. The isolte ws cloned three times with the medium of Hyflick (13), nd cultures were identified s pure M. pulmonis by using immunofluorescence (11). M. pulmonis ws grown in modified Hyflick medium (13, 29) nd stored in 1-ml liquots t -7 C. The number of M. pulmonis CFU ws determined by inocultion of gr pltes with seril dilutions of the stock in Hyflick broth medium. KLH ws purchsed from Clbiochem-Behring (L Joll, Clif.). Protein concentrtion ws determined by the micromethod of Bio-Rd Lbortories (Richmond, Clif.) (3). Before immuniztion, ntigens were diluted to 5 jig/ml in phosphte-buffered sline (PBS). Isoltion of lymphocytes. Lung cells were isolted by using Ficoll-Hypque (LymphPque; Phrmci) density grdients fter mechnicl disggregtion of tissue (9, 1); nerly ll of the lung lymphocytes re recovered by this method, since very few cells re retined in the remining tissue frgments (1). Spleen lymphocytes were isolted s previously described (9). URN nd lower respirtory lymph node (LRN) cells were isolted by mechnicl disggregtion. URN contined the deep cervicl lymph nodes nd retrophryngel lymph nodes, wheres LRN contined the trcheobronchil lymph nodes. Nsl pssge (NP) cells were INFECT. IMMUN. collected fter longitudinl section of the hed nd removl of tissue from the nsl cvity, nsl sept, ethnoid sinuses, nd mxillry sinuses by scrping; the tissue ws further disggregted nd pushed through 25-,um-pore-size nylon mesh to remove ny remining tissue nd bone chips. All cell wshes were done with RPMI 164 (GIBCO Lbortories) supplemented with 1 mm N-2-hydroxyethylpiperzine-N'- 2-ethnesulfonic cid (HEPES) nd 5% fetl clf serum. The percentges of lymphocytes nd other cell types were determined in cell suspensions from infected nimls. Cell smers were stined with Diff-Quick (Americn Scientific Products, McGw Prk, Ill.) nd exmined under light microscopy. There were no differences in the percentges of lymphocytes in ny of the cell suspensions from infected nd uninfected rts. The percentges of lymphocytes in infected or uninfected LEW rts were 87, 72, 88, 82, nd 86% in URN, NP, spleen, lung, nd LRN cells, respectively. In F344 rts, there were 88, 62, 86, 83, nd 87% lymphocytes in URN, NP, spleen, lung, nd LRN cells, respectively. The remining cells were mostly mcrophges, lthough 1 to 2% neutrophils were found in NP cell suspensions. Determintion of AFC. The ELISPOT ssy for M. pulmonis-specific AFC ws performed s previously described (33). Note tht the presence of neutrophils or pssive dsorption of ntibody does not ffect the results obtined with this ssy. Briefly, M. pulmonis cell lyste ntigen (14) ws incubted (5,ug of protein per well) overnight t 4 C in Millititer HH 96-well filtrtion pltes (ctlog no. STHAO961; Millipore Corp.). To minimize nonspecific binding, 1,ul of cell culture medium (RPMI 164, 1 mm HEPES, 1% L-glutmine,.2% NHCO3, 8,ug of gentmicin per ml, nd 5% fetl clf serum) ws dded to ech well, nd pltes were incubted t 37 C for t lest 1 h. Seril dilutions of cells were dded in triplicte to the pltes (1,ul per well) nd incubted for 4 h t 37 C in 5% CO2. The pltes were wshed with PBS nd then PBS-.5% Tween 2. To remove endogenous peroxidse ctivity, the pltes were incubted for 1 min with.5% H22 in PBS nd then wshed with PBS-.5% Tween 2. Mouse monoclonl nti-rt IgA (MARA-2; BSI/Serotec), nti-rt IgM (MARM-4; BSI/Serotec), horserdish peroxidse (HRP)-lbeled rt nti-mouse IgG, F(b)2 frgments (Jckson Immunoreserch Lbortories, Westgrove, P.), nd HRP-lbeled got nti-rt IgG ntibody (Kirkegrd & Perry Lbortories, Inc., Githersburg, Md.) were used to revel the ntibody rections. HRP-conjugted ntibody or monoclonl ntibodies specific for rt immunoglobulin clsses were plced into the pproprite wells t the mximum ntibody dilutions previously shown to hve miniml bckground in the ELISPOT ssy. After overnight incubtion t 4 C, the wells tht contined the monoclonl ntibodies were rected with HRP-conjugted rt nti-mouse ntibody for 5 h t room temperture. The pltes were then wshed with PBS, nd HRP substrte ws dded to ech well. The HRP substrte ws 25 mg of 3-mino-9-ethyl crbzole (Sigm Chemicl Co.) in 97 ml of.5 M sodium cette buffer (ph 5.) plus.4% H22; 3-mino-9-ethyl crbzole ws dissolved in 2 ml of N,N-dimethyl formmide. After the substrte rection, the pltes were thoroughly wshed with tpwter. The spots representing AFC were counted by using dissecting microscope illuminted indirectly with highintensity lmp. The cell concentrtions yielding bout 2 to 5 spots per well were counted nd verged. The AFC were expressed s the bsolute number of AFC in ech tissue or

3 VOL. 59, 1991 ANTIBODY RESPONSES TO M. PULMONIS INFECTION URN c3 1 I l- U. IL C, Dys Post-Infection Dys Post-Infection FIG. 1. Development of nti-m. pulmonis ntibody responses in LEW nd F344 rts. IgG AFC responses in LEW () nd F344 () rts re shown. At the 3-dy time point, no or very few AFC were detected in ny tissue. Similr results were obtined for the other ntibody subclsses. The dt re expressed s the mens + stndrd devitions of IgG-producing AFC/16 cells within the indicted tissues. AFC/16 cells; the numbers of AFC were corrected for the percentge of lymphocytes in the initil cell popultion. Sttistics. All experiments were repeted t lest twice. Sttisticl nlysis ws performed by using the SYSTAT version 5. progrm (Systt, Inc., Evnston, Ill.) on Mcintosh SE computer. The dt were nlyzed by nlysis of vrince nd Person correltion fter logrithmic trnsformtion of the dt (1), followed by post hoc tests for multigroup comprisons when pproprite. A probbility (P) of less thn.5 ws ccepted s significnt. RESULTS Antibody responses in LEW nd F344 rts. In previous studies, LEW rts developed higher-thn-expected ntibody responses, reltive to those of F344 rts, to M. pulmonis fter infection (31). Subsequently, the URN were described s mjor site of ntibody production in infected F344 rts (33). To further exmine the development of ntibody responses in rts with MRM, the numbers of AFC in lymphoid tissues were determined in LEW nd F344 rts t 3, 7, 14, 21, nd 28 dys fter intrnsl infection with M. pulmonis. The experiment ws done t lest twice until dt from totl of six nimls per rt strin were obtined for ech time point, except for four nimls t 21 dys postinfection. Consistent with the results from previous studies (8, 1), gross lung lesions were observed in bout hlf of the LEW rts t 21 nd 28 dys fter infection, wheres no gross lung lesions were found in F344 rts. At lest one uninfected niml per rt strin ws used s control t ech time point; no nti-m. pulmonis responses were seen in these nimls. In both LEW nd F344 nimls, the URN ppered to be the initil site of ntibody production fter infection (Fig. 1). Few or no AFC were found in ny tissue 3 dys fter infection with M. pulmonis. At 7 dys postinfection, there ws lrge increse in the number of AFC/16 cells in URN, wheres the other tissues hd only minor levels (P <.5). There ws no significnt difference due to ntibody isotype in ny of the tissues t 7 dys postinfection. The level of AFC ctivtion continued to increse in URN but not s drmticlly s in the first 7 dys. In ll other tissues, there were lrge increses in AFC ctivtion by 14 dys fter infection, with less rpid increses in AFC ctivtion t lter times. As shown in Fig. 2, the upper respirtory nodes were mjor site of totl ntibody production in both LEW nd F344 rts (P <.5). There ws no sttisticl difference in the totl mount of ny ntibody isotype produced in the URN of LEW nd F344 rts. However, LEW rts hd drmticlly higher numbers (greter thn 6 times) of totl AFC in the LRN thn did F344 rts (P <.5). This ws true for ll clsses of ntibody. There ws lso higher number of totl AFC in the URN of LEW rts (P <.5), but it ws less thn double tht found in F344 rts. There ws lso slightly higher number of totl IgG-, IgA-, nd IgE-producing AFC in LEW lungs thn in F344 lungs (P <.5), nd the totl number of IgM-producing AFC in lungs ws close to being significntly different (P =.52). Thus, the reltive levels of the ntibody responses in the LRN were the mjor difference in the distribution of ntibody responses in infected LEW nd F344 rts. However, other tissues, especilly the URN, of LEW rts lso hd higher numbers of totl AFC.

4 3718 SIMECKA ET AL. INFECT. IMMUN. b d is H4 5 '-4 d * IgE * IgG * Igl l] IgG C u '-i 4J E co d zuuuuv I I I 15C 1n_ 5- n IF v 14 C 4 * Igl IgG d ci '-4 E 8F o Lung NP * IgE O IgG FIG. 2. Contribution of different tissues to totl nti-m. pulmonis ntibody response. The totl number of AFC in tissues from LEW (, b) nd F344 (c, d) rts t 14 dys postinfection. The distribution of ntibody responses ws similr t lter time points. The stcked br chrts indicte the totl number of AFC in ech tissue, including the brekdown of the isotypes. Note the different scles used when showing ll tissues (, c) nd only lungs nd nsl pssges (b, d). Nonspecific ctivtion of B cells in infected rts. Previous studies (1, 18, 19, 37) suggested tht nonspecific ctivtion nd/or recruitment of B cells my occur in rts infected with M. pulmonis. To exmine this possibility, LEW nd F344 rts were immunized intrperitonelly with 125,ug of KLH nd rested for 2 to 3 weeks, llowing the ntibody response to KLH to subside (dt not shown). Hlf of these rts then were intrnslly infected with M. pulmonis, nd the remining control rts remined uninfected to mesure the residul nti-klh response. Two weeks fter infection, the numbers of IgG-producing cells specific for KLH or M. pulmonis were determined in ll lymphoid tissues. Significntly higher KLH responses were found in both infected LEW nd F344 rts s compred with those in the control nimls (Fig. 3). This ws true whether the dt were expressed s totl AFC (s shown) or AFC/16 cells. The only KLH-specific AFC in the uninfected (control) nimls were found in the LRN. No significnt difference ws found between the responses in LEW nd F344 rt strins due to the extremely lrge vrition in these experiments. There ws no significnt difference in the numbers of KLH-specific AFC in the URN, LRN, nd NP in infected nimls, wheres the lung contined smller numbers of AFC. Spleens hd lower, but not sttisticlly significnt (P =.53), numbers of AFC. The distribution of M. pulmonisspecific AFC ws similr to those described bove. However, the distribution of AFC specific for M. pulmonis did U not correlte well with the tissue distribution of KLHspecific AFC. Thus, nonspecific ctivtion nd/or recruitment of B cells occurs in both LEW nd F344 rts fter infection with M. pulmonis. DISCUSSION Most studies on respirtory immunity nd disese hve exmined only the presence of ntibody in nsl or trcheobronchil secretions or the distribution of AFC within the lower respirtory trct (5, 12, 15, 21, 23, 28, 35, 36); few studies hve considered ntibody responses in both the upper nd lower respirtory trcts (4, 27). However, the elucidtion of the immune mechnisms present in ll tissues of the respirtory trct is of gret importnce for the development of optiml vccintion regimens s well s for understnding the mechnisms of pthogenesis of vrious infectious nd llergic diseses. The results from our lbortory suggest tht the upper respirtory trct plys mjor role in the development of ntibody responses in some respirtory infections. In n erlier study (33), we demonstrted tht the upper respirtory trct is the mjor site of ntibody production in F344 rts 28 dys fter infection with M. pulmonis. In the present study, we exmined the production of ntibody responses in LEW nd F344 rts fter intrnsl infection with M. pulmonis. In both LEW nd F344 rts, the URN were the initil

5 VOL. 59, 1991 ANTIBODY RESPONSES TO M. PULMONIS INFECTION 3719 * Infected 7- Control 6- < 5- - LEW rts b r T 3-1- Spleen URN LRNJ N'P Lung LRN NP Lung Tissue Tissue FIG. 3. Nonspecific ctivtion of B cells in M. pulmonis-infected nimls. LEW nd F344 rts were primed with KLH nd rested before infection with M. pulmonis. There ws significntly higher number of KLH-specific IgG AFC in LEW () nd F344 (b) rts infected with the orgnism s compred with those in control nimls, which were not infected. The dt re expressed s the mens ± stndrd devitions. site of ntibody production fter infection. By 14 dys fter infection, AFC were found in other tissues. As in our previous study (33), the URN remined mjor site of AFC through the 28-dy period in both LEW nd F344 rts. These results suggest tht URN my be significnt source of primed or ctivted B cells, which migrte to other stimulted (infected) tissues. Previous studies from our lbortory hve compred serum ntibody responses in LEW nd F344 rts fter immuniztion with M. pulmonis ntigen (29) nd intrnsl infection with the orgnism (31). After immuniztion with nonreplicting orgnisms, F344 rts produce higher levels of serum IgG, prticulrly the IgG2b subclss, thn do LEW rts (29). In contrst, infected LEW rts develop serum ntibody responses, including IgG2b responses, equl to or greter thn those of F344 rts (31). However, the subclss distribution of the ntibody response is consistent with the immuniztion studies. IgG2b contributed the most to the totl IgG in infected F344 rts, wheres IgGl nd IgG2 were the mjor components of the LEW IgG response. From these results, we suggested tht the totl IgG response to M. pulmonis in infected LEW rts is much higher thn expected becuse of infection without ffecting the IgG subclss distribution. In the present study, there ws mjor difference between LEW nd F344 rts in the number of M. pulmonis-specific B cells in the drining lymph nodes of the lungs (LRN). LRN from infected LEW rts contined lrger proportion of M. pulmonis-specific B cells (AFC/16 cells) thn did LRN from F344 rts. This difference ws mplified by difference in the totl number of cells recovered from LRN fter infection, resulting in much higher number of AFC in LEW rts compred with tht in F344 rts. There ws lso lrger ntibody response in the lungs nd URN of LEW rts. The ntibody response within the lungs ws minor compred with tht of the LRN. Thus, the results from the present study re consistent with the higher-thn-expected serum ntibody responses in infected LEW described in the previous studies (31) nd suggest tht the ntibody production in the LRN nd URN contributes significntly to the differences between LEW nd F344 responses. Although erlier in vivo nd in vitro studies suggest tht polyclonl ctivtion of B cells cn occur s result of M. pulmonis infection (18, 19, 37), the present study is the first to demonstrte tht polyclonl ctivtion of B cells does ctully occur in rts with MRM. More KLH-specific IgG AFC were found in nimls immunized with KLH nd infected 2 weeks lter with M. pulmonis thn were found in similrly immunized, uninfected nimls. This ws true for both LEW nd F344 rts. Unfortuntely, we cnnot directly compre the levels of polyclonl ctivtion of B cells in these two strins becuse the numbers of primed B cells probbly differ. F344 rts produce higher levels of serum IgG to KLH thn do LEW rts fter immuniztion (29), supporting the ide tht there ws probbly difference in the number of KLH-primed B cells before infection with M. pulmonis. Nonspecific recruitment of AFC to lymphoid tissues during MRM my lso occur. Bice et l. (2) demonstrted tht B cells re recruited to the lung without regrd to ntigen specificity when n inflmmtory stimulus is present. In our study, significnt number of KLH-specific AFC were found in the URN fter infection. Antibody responses to KLH were never found during this study in URN fter primry intrperitonel immuniztion (dt not shown). In ddition, we found tht primed B cells develop in the URN only fter intrnsl immuniztion with n ntigen (32). Overll, these dt suggest tht nonspecific recruitment of B cells to URN, nd probbly other tissues, occurs during the pthogenesis of MRM. The mechnisms of nonspecific ctivtion or recruitment of B cells during MRM re presently unknown. One likely possibility is tht cytokines ssocited with specific immune nd/or inflmmtory responses ply role. B-cell chemotctic fctors re present in delyed-type hypersensitivity lesions (17) nd my be present in the lesions ssocited with MRM. In ddition, polyclonl ctivtion of B cells cn occur in vivo fter pssive trnsfer of ctivted Th2 helper T lymphocytes, which produce interleukin-4 (34). Most likely, other cytokines contribute to the polyclonl ctivtion nd nonspecific recruitment of B cells during disese. In ddition to cytokines, the direct interction of M. pulmonis nd B cells my contribute to the effects seen during MRM. M. pulmonis membrnes re mitogenic for B cells in vitro (2, 24). We hve lso found tht mjor cell surfce ntigen of M. pulmonis is chemotctic for nive B cells (25). Thus, cytokine nd/or M. pulmonis interctions with B cells most likely result in the ctivtion nd recruitment of these cells. In summry, ntibody responses first develop in the URN fter infection of rts with M. pulmonis. The URN remins mjor site of ntibody production throughout the course of

6 372 SIMECKA ET AL. disese. Since infected LEW rts hd equl or higher numbers of AFC compred with those in infected F344 rts, these results suggest tht the level of locl ntibody production lone, like tht of serum ntibody (31), is not responsible for the decresed susceptibility of F344 rts to MRM. The differences in totl ntibody responses pper to be due to the greter numbers of cells recovered from the tissues of infected LEW rts compred with those in F344 rts, suggesting tht LEW rts my hve greter production of chemotctic fctors. Our dt lso demonstrte tht nonspecific ctivtion nd recruitment of B cells occur during the development of MRM. Although further studies re needed, the mechnisms involved in the nonspecific effects on B cells probbly influence the distribution nd levels of specific ntibody responses nd thus my ctully contribute to the development of disese. ACKNOWLEDGMENTS We thnk Cecil Czerkinsky for his invluble id in the estblishment of the ELISPOT ssy in our lbortory. We lso thnk Ginger Gmbill, Dvid Ansrdi, nd Sin Shojee for their excellent technicl help during the course of these studies. This work ws supported by Public Helth Service grnt HL19741 to Gil H. Cssell from the Ntionl Hert, Lung, nd Blood Institute. Jerry W. Simeck ws Prker B. Frncis Fellow in Pulmonry Reserch. Suznne E. Ross ws supported by Public Helth Service trining grnt T32HL7553 to Gil H. Cssell from the Ntionl Hert, Lung, nd Blood Institute nd is currently Prker B. Frncis Fellow in Pulmonry Reserch. REFERENCES 1. Armtrdge, P Sttisticl methods in medicl reserch. Blckwell Scientific Publictions, Oxford, Englnd. 2. Bice, D. E., M. A. Degen, D. L. Hrris, nd B. A. Muggenburg Recruitment of ntibody-forming cells in the lung fter locl immuniztion is nonspecific. Am. Rev. Respir. Dis. 126: Brdford, M A rpid nd sensitive method for the quntitition of microgrm quntities of protein utilizing the principle of protein-dye binding. Anl. Biochem. 72: Brdley, P. A., F. J. Bourne, nd P. J. 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7 VOL. 59, 1991 ANTIBODY RESPONSES TO M. PULMONIS INFECTION 3721 Upper respirtory trct is the mjor site of ntibody production in mycoplsml induced disese. Reg. Immunol. 2: Spinell, S., G. Milon, nd M. Hontebeyrie Joskowicz A CD4+ TH2 cell line isolted from mice chroniclly infected with Trypnosom cruzi induces IgG2 polyclonl response in vivo. Eur. J. Immunol. 2: Tylor, G., nd C. J. Howrd Clss-specific ntibody responses to Mycoplsm pulmonis in ser nd lungs of infected nd vccinted mice. Infect. Immun. 29: Wldmn, R. H., C. S. Spencer, nd J. E. Johnson Respirtory nd systemic cellulr nd humorl immune responses to influenz virus vccine dministered prentlly or by nose drops. Cell. Immunol. 3: Willimson, J. S., J. K. Dvis, nd G. H. Cssell Polyclonl ctivtion of rt splenic lymphocytes fter in vivo dministrtion of Mycoplsm pulmonis nd its reltion to in vitro response. Infect. Immun. 52: