key: cord-0008444-zuw4xo9j
authors: McCabe, Michael J.; Lawrence, David A.
title: Lead, a major environmental pollutant, is immunomodulatory by its differential effects on CD4(+) T cell subsets
date: 2004-09-27
journal: Toxicol Appl Pharmacol
DOI: 10.1016/0041-008x(91)90129-3
sha: 4951f58031165962c732faf12e4ca14ffcf2848f
doc_id: 8444
cord_uid: zuw4xo9j

Studies were undertaken to address the necessity of B-T cell contact for the enhancement of B cell differentiation caused by the heavy metal lead (Pb). Membrane segregated cultures were used so that the influences of direct B-T cell contact and T cell factors on B cell differentiation could be independently evaluated. B-T cell contact was not absolutely required for Pb's enhancement of B cell maturation to antibody forming cells (AFCs); however, enhancement of the AFC response by Pb was optimal when B-T cell interactions were allowed. These results were corroborated by use of anti-L3T4 (mouse CD4) to block CD4(+) T cell-B cell interaction. Blockade of B-T cell contact with anti-L3T4 did not inhibit the enhancement of the AFC response by Pb. Additional experimentation showed that Pb enhanced the AFC response and Ig production in the presence of antigen-specific T cell help, suggesting that Pb enhances B cell differentiation by augmenting cognate help rather than by inducing a response to Pb-altered-self. In studies employing antigen-specific T cell clones, Pb was found to differentially modulate antigen presentation to T(H1) versus T(H2) T cell clones, in that T(H1) activation was inhibited and T(H2) activation was enhanced by Pb.

Genetic predisposition a n d e n v i r o n m e n t a l factors influence the induction of a u t o i m m u n e diseases (Schwartz a n d Rose, 1986) . Although clearly involved, the influence of environm e n t a l factors on a u t o i m m u n i t y is not u nderstood. Agents, including pathogens (Watanabe et al., 1983) , drugs ( H a h n et al., 1972), a n d chemicals (Pelletier et al., 1986) H e a v y metals, such as lead (Pb), i n d u c e pathophysiologic changes that affect m a n y organ systems (Goyer, 1986) i n c l u d i n g the i m m u n e system (Lawrence, 1985) . Pb exposure can cause h y p o i m m u n i t y leading to di-1 Present address: Karolinska Institutet, Department of Toxicology, Box 60400, S-104 01, Stockholm, Sweden. 13 m i n i s h e d host resistance to pathogens (Lawrence, 1981a) . In contrast, Pb also can augm e n t certain i m m u n e responses, perhaps c o n t r i b u t i n g to h y p e r i m m u n i t y against selfconstituents. Except for a few reports correlating o c c u p a t i o n a l Pb exposure with k i d n e y disease via an i m m u n e m e c h a n i s m ( W e d e e n et al., 1979 ; G a r c i a et al., 1980) , the possibility that Pb induces a u t o i m m u n i t y has n o t b e e n considered despite its i m m u n o m o d ulatory activities. Pb e n h a n c e s B cell differe n t i a t i o n in vitro a n d in vivo to T-cell-depend e n t antigens such as sheep erythrocytes (SRBC) (Koller et al., 1976; Lawrence, 1981a Lawrence, ,b,c, 1983 ; W a r n e r a n d Lawrence, 1986a) as well as to the T -c e l l -i n d e p e n d e n t polyclonal B cell activator, lipopolysaccharide (McCabe a n d Lawrence, 1990) , by an a m o u n t c o m p a r a b l e to the T-cell-derived cytokine addition, Pb simulates the activity of the T cell cytokine, interleukin-4, by increasing B cell expression of major histocompatibility complex class-II (Ia) molecules (McCabe and Lawrence, 1990) .

For most antigens, B cells require T cell help for differentiation into antibody forming cells (AFCs). Helper (CD4 ÷) T cells act by two distinct mechanisms (Abbas, 1988) : cellto-cell interactions mediated by Ia restricted B-T cell collaboration and factor-mediated help fulfilled by T-cell-derived cytokines including interleukin-2, -4, -5, -6, and interferon-3, (IFN-7). Furthermore, factor-mediated T cell help has been categorized by the profile of cytokines present in supernatants of various CD4 ÷ T cell clones (Mosmann et aL, 1986) . Type 1 (Trfl) clones produce IL-2 and IFN-3,; whereas, THe clones produce IL-4, IL-5, and IL-6. THI and TH2-1ike cells, termed inflammatory and helper T cells, respectively, may exist in vivo (Bottomly et al., 1989) . Like their in vitro counterparts they differentially regulate immune responses through effector functions attributable to the cytokines that they secrete. Preferential perturbation of either subset may be protective or immunopathologic (Scott et al., 1989; Finkelman et al., 1986) .

This study was undertaken to determine which mode of T cell help is required for Pb enhancement of B cell differentiation and whether Pb can enhance B cell differentiation when antigen-specific T cell clones are the sole source of help. These studies suggest that Pb augments factor-mediated T cell help by enhancing factor production by T cells as well as B cell responsiveness to these factors. Furthermore, the results suggest that Pb enhances B cell differentiation by promoting cognate B-T cell interactions via heightened B cell surface Ia density rather than a T cell response to Pbaltered-self. Interestingly, a dichotomy between the influence of Pb on THt vs TH2 activities has been observed that may explain the inverse effects of Pb on host resistance and the posited influence on autoimmunity (Lawrence, 1985) .

Mice. Female BCF~ and BALB/c mice were obtained from Taconic Farms (Germantown, NY). C3H/HeJ, DBA/2J, C57BL/6 and CBA/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME) whereas, CBA/ N mice were obtained from the National Cancer Institute (Frederick, MD). Mice were housed in the AMC animal facility under SPF conditions and maintained on mouse chow and acidified water ad libitum until they were euthanized at 6-10 weeks of age.

Medium and reagents. M199 with Hank's BSS (M.A. Bioproducts, Walkersville, MD) supplemented with 5% FBS (Hyclone Laboratories, Logan, UT), 1 mM L-glutamine, 1 mM Na-pyruvate, 0.1 mM nonessential amino acids, 25 ug/ml gentamycin, 50 #M 2-mercaptoethanol, and NaHCO3 was used in all cultures.

A stock solution of 10 mM PbC12 (Fisher Scientific, Rochester, NY) was prepared in physiological saline and sterile filtered prior to dilution and addition to culture. Sheep erythrocytes, purchased from the Colorado Serum Co. (Boulder, CO) were stored in Alsever's preservative and washed 3× in BSS prior to use. Stock solutions of conalbumin (CA, Sigma Chemical Co., St. Louis, MO), keyhole limpet hemocyanin (KLH; Calbiochem, La Jolla, CA) and rabbit 3' globulin (RGG; Sigma) were prepared in physiological saline and sterile filtered prior to addition to culture. Anti-lg (Rabbit F(ab')2 anti-mouse F(ab')2) was purchased from Jackson ImmunoResearch (West Grove, PA).

Monoclonal antibodies. The following hybridoma cell lines were obtained from the American Type Culture Collection (Rockville, MD): HO-13.4 (anti-Thyl.2, a mouse IgM to all T cells); GK1.5 (anti-L3T4a, a rat IgG anti-CD4) and 53-6.72 (anti-Lyt2, a rat IgG anti-CD8). Cells were cultured as recommended by ATCC, supernatants were harvested, precipitated with (NH4)zSO4, dialyzed extensively into PBS pH 7.4, and sterile filtered. Each preparation was characterized by either titering for optimal staining as assessed by flow cytometry or cytotoxicity prior to use.

B cell preparation. B cells were enriched by treating splenocyte suspensions with two cycles anti-Thy 1.2 (HO-13.4, 1:10) plus rabbit complement to remove T cells. B cells obtained in this manner were routinely 90-95% slg + and < 1% Ly-1 +, and they did not respond to C0ncanavalin A (Con A) indicating that T cell contamination was functionally insignificant. Resting B cells were obtained by centrifugation of the B cell preparations into a discontinuous PercoU gradient with Percotl (Sigma) concentrations of 50, 60, 70, and 75%. The fraction of cells that layered at the 70% Percoll interface (sp gray 1.087 g/cm 3) was verified as resting by size (forward angle light scatter, FALS) and cell cycle analysis (acridine orange staining) by flow cytometry~ "Unseparated B cells" are defined as those B cells that were not further fractionated by the PercolI gradient separation, whereas "activated B cells" are defined as those that layered above 60% Percoll, indicating blastlike status. The AFC responses per 106 unseparated B cells and resting B cells were <50 and 0, respectively. Primary in vitro AFC assay. Spleens were aseptically removed and a single cell suspension was made by pressing the spleens between the frosted ends of two sterile microscope slides. Clumps were allowed to settle and the cell suspension was washed 1 × in BSS. Each spleen equivalent was resuspended in 5 ml ofPBS + 10% FBS + 0.1% NAN3, layered onto a 4-ml Ficoll-metrizoate density gradient, and centrifuged at 3000 rpm for 15 min at 22°C to remove erythrocytes, granulocytes, and dead cells. The lymphocyte-rich interface was collected, washed 3× in BSS and cultured as described (Mishell, 1967) . Briefly, 5 X 10 6 spleen cells/0.5 ml/well, + PbC12 + 25 #1 of 1% SRBC were cultured in 24-well cluster plates for 5 days in a special gas composed of 10% CO2, 7% 02, and 83% N2. On Day 5, cells of triplicate cultures were harvested and pooled, and the number of AFCs were enumerated by a slide modification of the Jerne plaque assay (Jerne and Nordin, 1963) . Only direct plaques were enumerated.

Membrane segregated cultures were designed as described (Eastman and Lawrence, 1984) . A 1.0-#m Nucleopore membrane was used to separate B cells at a density of 2.5 × 10 6 cells/upper chamber from splenocytes (approximately 50% B cells) at a density of 5 × 10 6 cells/ lower chamber. Both chambers contained 1% SRBC + 100 #M PbCl 2 or Pb-pretreated B cells (Pb-B cells; 100 #M PbC12, 45 min, 37°C, washed 3× prior to culture).

CBA/J mice were immunized intravenously with 5 × 106 SRBC. After 7 days, the spleens from these immunized mice were removed and T cells were obtained by nylon wool nonadherence (Julius et al., 1973) . T cells (10 7) were cultured with 10 7 irradiated (3300 rads) splenocyte feeders + 0.5% SRBC and 50 units/ml human IL-2 (Calbiochem) in 2 ml of medium in a six-well plate. The SRBC'T cells were maintained in culture under these conditions with weekly passage for 3 months before termination. SRBC'T proliferated (as measured by [3H]thymidine incorporation) in the presence of feeder cells and SRBC independent of the addition of exogenous IL-2. Furthermore, SRBC'T were specific for the eliciting antigen, SRBC, since they supported B cell differentiation into SRBC-specific AFCs but did not support AFC generation of B cells cultured with horse RBC or chicken RBC.

T cell clones. The T cell clones D10.G4.1 (Kaye et al.. 1983) , M264-37 (Mosmann et al.. 1986) , and HDK-1 (Cherwinski et al., 1987) were obtained from Dr. T. Mossman, DNAX Research Institute (Palo Alto, CA). D1.6 (Kurt-Jones et al., 1987) and CDC 25 (Tony et aL, 1985) were obtained from Dr. R. Noelle, Dartmouth Medical School (Hanover, NH). T cell clones were maintained and passaged as recommended by the suppliers and each clone was routinely assayed for mycoplasma contamination by the use of the Mycotect Test Kit, GIBCO (Grand Island, NY). Clones were maintained in medium plus 50 units/ ml human IL-2 without antigen or feeders for 7 to 14 days prior to use.

Measurement of lg in culture supernatants. Immulon 1, 96-well, flat-bottomed, ELISA plates (Dynatech Laboratories, Alexandria, VA) were used as the solid phase for the ELISAs. For the conalbumin-specific ELISA, 30 #g/0.1 ml/well conalbumin in 0.1 M HCO3 buffer, pH 8.6, was used as capture reagent. Supernatants were diluted 1: 4 in binding buffer (PBS + 0.1 mM EDTA, 0.25% BSA, 0.05% Tween 20, pH 7.0-7.4). Biotinylated goat antimouse IgM (Zymed Laboratories, San Francisco, CA) followed by alkaline phosphatase-conjugated streptavidin (Tago Immunologicals, Burlingame, CA) was used according to the suppliers specifications as developing reagents. Paranitrophenyl phosphate (Sigma) was used as substrate. The methodology for the polyclonal total IgM ELISA was essentially the same as the conalbumin-speciflc ELISA except that goat anti-mouse IgM (Zymed) was used as capture reagent and absorbance units were converted into IgM concentration by extrapolation from standard curves using mouse reference serum (ICN lmmuno-Biologicals, Lisle, IL) of known lgM concentration.

Antigen presentation assays. Triplicate cultures containing the T cell clones (104/well) and appropriate antigen and antigen presenting cells (irradiated B cells or splenocytes, 5 × 10a/well) were set up in 96-well round bottom plates _+ PbCI2. Culture wells, pulsed with 1 #Ci [3H]thymidine for the final 24 hr of culture, were harvested (Skatron, Lierbyen, Norway) 72 hr after initiation, and radioactivity was counted by liquid scintillation spectroscopy.

Statistical analysis. Differences between various treatment and control groups were evaluated for by Student's t test.

Pb enhances both factor-mediated and cognate T cell help for AFC generation. The independent influences of B-T cell contact and soluble T cell factors on B cell differentiation were evaluated using membrane segregated cultures. As shown in Table 1 , PbCI2 significantly increased the number of SRBC-specific AFCs from both the upper and lower chambers of membrane segregated cultures. Optimal enhancement (sixfold) of the AFC response of upper chamber B cells was obtained when PbC12 was added to both chambers (Table 1, Row 1); however, the addition of Pbpretreated B cells to the upper (Row 2) or lower (Row 3) chambers yielded two-and threefold increases respectively in the AFC response of b After 5 days in culture, cells from both chambers were harvested and AFCs were enumerated. Values represent the ratio of the number of AFC detected in the indicated chambers compared to the number of AFC detected in identical control cultures without any Pb treatment. AFC/106 recovered cells for the controls were: Row 1-3, splenocytes plus B cells in the bottom (low) chamber (760 + 151, n = 4) plus B cells in the upper (top) chamber (1094 ---107, n = 4); Row 4, splenocytes plus resting B cells in the low chamber (336 + 50, n = 3) with resting B cells in the top chamber (98 ___ 15, n = 3); Row 5, splenocytes plus activated B cells in the low chamber (777 + 116, n = 3) with activated B cells in the top chamber (2553 + 383, n = 3). Student's t test was employed to examine statistically significant differences between the Pb treatments and the controls. * Indicates p < 0.001. ** Indicates p < 0.01. upper chamber B cells. Pb-pretreated B cells placed in the upper chambers did not significantly modify the AFC response of splenocytes in the lower chambers suggesting that the Pb did not dissociate from the upper chamber B cells and modulate the lower chamber response. Overall, the data suggest that Pb augments the AFC response by enhancing the production of putative helper T-cell-derived factors from lower chamber cells as well as the responsiveness of B cells to these factors. In addition, it is likely that Pb enhances the production of T-cell-derived factors by potentiating B-T cell interaction (i.e., cognate help). The magnitude of the enhancement in the upper chambers correlated with the activation stage of the responding B cells; that is, Pb increased the AFC response of resting B cells (Row 4) to a greater extent than unseparated B cells (Row 1) or activated B cells (Row 5).

To substantiate that Pb enhanced B cell differentiation by cognate T cell help, splenocytes obtained from CBA/N mice which require B-T interaction for B cell differentiation (Sher, 1982) were studied. As with splenocytes from "normal" mouse strains, including CBA/J, DBA/2J, BALB/c, BCF1, and C3H/HeJ, Pb increased the AFC response of CBA/N splenocytes (Table 2) .

To further elucidate whether B-T cell contact was required for Pb's enhancement of B cell differentiation into AFCs, the ability of Pb to enhance the AFC response under conditions where B-T cell contact was prevented by the use of monoclonal antibodies with specificity for T cell surface molecules that are involved in B-T cell interaction was examined. As demonstrated in Fig. 1 , anti-L3T4 reduced the control AFC response by 75% but insignificantly lowered the AFC response in the presence of Pb. In contrast, anti-Lyt2, which blocks suppressor cell activity, enhanced the AFC response in both the presence and absence of Pb. By reducing B cell-CD4 ÷ T cell interactions, anti-L3T4 reduces the activation of helper T cells; however, in the presence of Pb, Note. Splenocytes from the indicated mouse strains were cultured at 5 × 106 cells/well plus SRBC in the presence or absence of 100/.tM PbC12. After 5 days, the cells were harvested and AFCs were enumerated. The values are the mean of triplicate wells (SD < 15%) and are representative of four separate experiments.

despite the reduced cognate interaction, factormediated help remained enhanced. Hence, the effect of diminished cognate help by the anti-L3T4 is negated. Furthermore, the efficiency of the anti-L3T4 blockade could be dimin-ished since Pb increases the density of the CD4 ligand (i.e., Ia) on B cells (McCabe and Lawrence, 1990 ). Enhancement of the AFC response by Pb in the presence ofanti-Lyt2 supports the view that Pb immunopotentiation is due to the enhancement of helper T cell activities or direct effects on B cells and not due to the inhibition of suppressor T cell functions.

The effect of Pb on Ig production in the presence of antigen specific T cell help. The nature of the effect of Pb on B-T cell interaction was unclear. Conceivably, Pb could alter the B cell surface (i.e., create a neoantigen) so that T cells bearing the appropriate clonotypic T cell antigen-specific receptors could then recognize Pb-altered-self antigens and respond accordingly. Considering this possibility, the influence of Pb on B cell differentiation in cultures containing B cells, antigen, and antigen-specific T cell clones as the sole source of T cell help was tested. In contrast to the heterogeneous polyclonal repertoire of T cell receptor specificities of splenic T cells (potentially including those with specificity for a possible Pbaltered-self antigen), T cell clones having unique antigen specificities were used to cir- Splenocytes were cultured at 5 × 106 cells/well plus SRBC _+ PbC12. Monoclonal anti-L3T4 and anti-Lyt2 at 2.5 ~tg/ml or 5 #g/ml of nonspecific Rat IgG were added to individual wells at culture initiation. After 5 days, the cells were harvested and AFCs were enumerated. The results represent the mean _+ SEM from three experiments (actual AFC/106 cells for the saline Rat IgG control was 108 _+ 24). The values above the bars represent p values (t tests) for comparisons of AFC responses in the presence of blocking antibodies (anti-L3T4 or anti-Lyt2) _+ Pb versus the AFC responses in the presence of Rat IgG + Pb. cumvent the complication of Pb potentially activating putative Pb-altered-self reactive T cells.

Initially, a SRBC primed T cell line was tested in cocultures containing B cells and SRBC +_ 100 #M PbC12. Pb significantly enhanced (twofold) the AFC response in these cultures (Fig. 2) arguing against a Pb-alteredself phenomenon. To further substantiate this claim, other antigen-specific T cell clones were utilized. The THE clone, D10.G4.1, which is specific for either CA/H-2 k or an allo-reactive epitope of H-2 b, was cultured _+ 10 or 100 #M PbC12 with either C3H/HeJ (H-2 k) B cells plus CA or C57BL/6 (H-2 b) B cells plus anti-Ig. In the CA/H-2 k B cell system, optimal help for CA-specific IgM production was obtained with 10 4 D10.G4.1 cells per culture. At this cell density, l0/~M Pb significantly increased CAspecific IgM production (Fig. 3a) . This result has been corroborated using an additional TH2 cell clone, CDC 25, which has specificity for RGG in association with H-2 d (data not shown). As with the CA/H-2k-specific stimulation of the D10.G4.1 cells, Pb enhanced polyclonal IgM production in the allo-stimulated system (Fig. 3b) . In contrast to this influence on allo-stimulated polyclonal TH2 activity, Pb inhibited the polyclonal IgM response of BALB/c (H-2 d) B cells cultured with anti-Ig and the H-2 d allo-specific THI clone M264-37 (Fig. 3c) .

Pb differentially affects antigen presentation to TIC1 and Tn2 T cell clones. In a previous study, Pb (noncytotoxic doses) has been shown to inhibit antigen presentation (as measured by IL-2 production) to the ovalbumin-reactive T cell hybridoma, DO11.10 (Smith and Lawrence, 1988 ). The DO11.10 resembles a TH1like cell and similarly to the Tm clones (Fig.  3c) its activity was suppressed by Pb. In contrast to the inhibition of THI activation, our present results show that Pb enhances TH2 cell activity and the accompanying B cell responses (Figs. 3a and 3b) . To further substantiate these selective and diverse effects of Pb on helper T cell activities, the effects of Pb on several Tnl and TH2 clones were tested in antigen presen-

4000. tation assays. Pb inhibited antigen presentation by B cells to the KLH/H-2d-restricted Tm clone HDK-I (Fig. 4a) . The opposite result was obtained with D10.G4.1 (TH2 clone), in that Pb enhanced its antigen-specific (CA/H-2 k) response (Fig. 4b) . Using additional THI and TH2 clones, D1.6 and CDC25, respectively, which are both specific for RGG associated with H-2 d, Pb inhibited the THI proliferative response induced by RGG presented by splenocytes (Fig. 5a) and B cells (Fig. 5b) , but Pb enhanced the Try2 proliferative response (Fig. 5c ).

The present study was undertaken to determine the mechanisms by which the heavy metal Pb enhances T-cell-dependent B cell differentiation. Although Pb is known to enhance the in vitro production of antibody (Lawrence, 1981a,b; Warner and Lawrence, 1986a) and directly activate B cells (McCabe and Lawrence, 1990) , the influence of Pb on B-T cell interactions has not been clearly delineated. It was discovered ( Table 1) direct B-T cell contact was not permitted, although optimal enhancement of the AFC response by Pb occurred when B-T cell contact was permitted. Hence, it appears that Pb enhances both cognate helper T cell activity as well as factor-mediated T cell help. Furthermore, with regard to the effects of Pb on factormediated T cell help, it appears that Pb influences the production of factors by T cells, which likely is related to the effect of Pb on cognate interactions and subsequent T cell activation. Presently, the identities of the T cell factors, which may be any of the well-characterized interleukins or combinations thereof, are not known. It has been previously reported that Pb can enhance in vitro generation of lymphokdnes by T cells (Warner and Law-rence, 1988) . Pb also appears to enhance B cell responsiveness to the T cell helper factors, in that the upper chamber Pb-pretreated B cell response (in the absence of cognate T cell help) was enhanced. This suggests that Pb may influence B cell expression of surface receptors for particular interleukins. At present, this is a matter of conjecture since we have not measured Pb effects on interleukin receptor density on B cells. It is conceivable that Pb could modulate the surface density of interleukin receptors on B cells since Pb has been shown to modulate the cell surface density of other important molecules on B cells such as Ia, CD23, and sIgD (McCabe and Lawrence, 1990) . The ability of Pb to increase B cell surface Ia density is consistent with the interpre- tation that Pb promotes cognate T cell help, in that Ia molecules mediate B-T cell interactions (Katz et al., 1975) and increased Ia expression has been indicated to enhance the immune response (Matis et al., 1983) . Experiments to determine whether factor-receptor systems are influenced by Pb should be undertaken to shed more light on how Pb enhances the responsiveness of the B cell component of the immune system. The present understanding of how B cell differentiation is controlled by endogenous factors and normal cellular interactions, let alone how environmental factors such as Pb may influence these processes, is far from clear. However, it has become clear that after antigen-specific stimulation of the appropriate B cell and T cell clones, the ensuing immune response leading to antibody production is controlled by an intricate network of nonspecific factors and cellular interactions. Our data suggest that environmental factors such as Pb can influence the immune response by modulating these nonspecific regulatory components rather than creating neoantigens that may be recognized in a specific way by the appropriate lymphocytes. This influence by environmental factors on the regulation of the immune response perhaps is more difficult to envision than a direct modification of "self," but it is likely to be of paramount importance with respect to the influence of toxicants on autoimmune disease and host resistance to pathogens and neoplasia. Furthermore, such modulations may be more ubiquitous, in that they may result from modification of similar biochemical processes in different organ systems.

It seems odd that a highly toxic metal enhances humoral immunity. Nonetheless, enhancement of inappropriate immune responses certainly are not beneficial since they may progress to autoimmune disease. Furthermore, enhancement of particular responses may result in dysregulation of the appropriate responses or may prevent them from occurring altogether. This is especially interesting in the cases of immune responses dominated by one or the other helper T cell subset (Tin and THe cells). Modulation of Tin vs TH2 activity in vivo has been implicated in various immunopathological states, most strikingly with protozoal and helminth infections (Scott et al., 1989) or the nematode Nippostrongylus brasiliensis (Finkelman et al., 1986) . In ad- dition to the categorization of TH1 and TH2 cells based on their intefleukin activities (Mosmann et al., 1986) , these CD4 + T cell subsets differ in several other important aspects. Recent information indicates that the biochemistry of T HI cell activation differs from that of Tin cells (Gajewski et al., 1990; Munoz et al., 1990; Betz and Fox, 1991) which may be mechanistically related to the differential effects found with Pb. The differential effects of Pb on the CD4 + T cell subsets may involve cAMP since these subsets have been reported to have differential sensitivities to cAMP (Munoz et al., 1990) , and Pb and cAMP have been reported to synergistically modulate lymphocyte activity (Lawrence, 198 lc) . Additional studies are needed to elucidate the biochemical mechanism(s) whereby Pb affects lymphocytes and TH1 and TH2 cells in particular. The observed differential effects of Pb on the Tm and TH2 clones may provide some clues about the interleukin activities that Pb influences in regard to the enhancement of antibody production. Since TH2 clones are considered to be the optimal helpers for B cell responses (Janeway et al., 1988) , it is not surprising that Pb enhances TH2 activity and B cell differentiation to AFC formation and Ig production. Tm clones and their associated cytokines can influence B cells, in that they can to a limited extent support polyclonal but not antigen-specific B cell differentiation. Pb did not impart on Tul clones the capacity to support antigen-specific B cell differentiation (data not shown). Furthermore, in contrast to its influence on allo-reactive polyclonal Tm activity, Pb inhibited the limited polyclonal B cell response supported by the Tm clone, M264-37, a result consistent with the fact that Tm cells, which aid only at a high cell density, are not efficient helpers of B cell differentiation. In light of the differential effects of Pb on the helper T cell subsets, it is interesting to note that Tm cells require a higher density of "processed antigen" to be presented for their activation (Janeway et aL, 1988) .

The differential influence of Pb on TH1 vs TH2 cells may explain, in part, the diverse in vivo effects of Pb, e.g., suppression of host resistance to Listeria monocytogenes (Lawrence, 1981 a) and other pathogens (Lawrence, 1985) yet the postulated ability of Pb to promote autoimmunity by its immunopotentiating effects. Listeria, an intracellular pathogen, is controlled mainly by Tm cells in vivo. If Pb decreases Tm activity in vivo less macrophage stimulatory factors (e.g., IFN-3,) would be produced. In addition to impairing macrophage activation, Pb has been shown to directly inhibit macrophage development (Kowolenko et al., 1989) . On the other hand, Pb can directly activate B cells (McCabe and Lawrence, 1990; Lawrence, 1981c) , and enhanced B cell: Tm cell interactions might lead to dysregulated B cell responsiveness and autoimmunity. Features of autoimmune disease induction include aberrant expression of Ia and cytokines as well as lymphoid infiltration of target tissues due to altered interactions between the lymphoid subsets (Schwartz and Rose, 1986) . Autoimmune disease incidence increases with aging. Interestingly, age-related changes in cytokine production by T helper cell subsets recently have been reported (Kubo and Cinader, 1990) . Pb involvement in immune-mediated nephritis has been suggested (Wedeen et al., 1979; Garcia et aL, 1980) ; however, data directly implicating Pb in autoimmune disease is not available. Autoimmune disease induced by mercury has been documented (Hirsch et al., 1982; Pelletier et al., 1985) . Our data suggest that Pb can promote immune dysfunctions characteristic of autoimmune disease induction. Inasmuch as little is known about the etiology of autoimmune diseases, a role for toxic agents such as Pb warrants consideration.

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This work was supported by NIH Grant ES03179.