Introduction

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Lead (Pb) is a ubiquitous environmental contaminant and belongs to the group of most toxic heavy elements in the atmosphere. In many countries, Pb poisoning continues to be a common occupational disease affecting several organ systems (Feldman and White, 1992; Gennart et al., 1992; Xuezhi et al., 1992). In the last 25 years, there has been an increased concern about the accumulation of lead in the environment. Many studies have demonstrated that Pb affects the function of a variety of cell types, including those of the nervous system (Cohen and Coryslechta, 1994; Struzynska and Rafalowska, 1994), the microvascular endothelium (Bressler et al., 1994), the kidney (Fowler et al., 1994), and the immune system (Luster et al., 1978; Lawrence, 1981, 1985; Fischbein et al., 1993; Cohen et al., 1994: McCabe, 1994). In vivo studies have shown that Pb is an immunotoxicant depressing humoral immunity (Koller and Kovacic, 1974; Luster et al., 1978), increasing host susceptibility to bacterial (Hemphil et al., 1971; Lawrence, 1981), and viral infections (Gainer, 1974).

The manner in which Pb affects the immune cells is not well understood. Although Pb treatment in vivo may result in immunosuppression, Pb has been observed to enhance lymphocyte proliferation in vitro (Shenker et al., 1977; Gaworski and Sharma, 1978; Lawrence, 1981; Warner and Lawrence, 1986; McCabe and Lawrence, 1990). This apparent discrepancy between the in vitro and in vivo findings may be due to complex interactions between tissues that form the in vivo targets of Pb, the ability of Pb to accumulate in various tissues, and/or the doses of Pb used in various studies. Moreover, immunosuppression and autoimmune-like conditions coexist in several diseases (Rosen, 1987). In this communication, we demonstrate that, depending on the concentration of Pb, in vitro proliferation of splenic lymphocytes is either stimulated or inhibited in the presence of Pb.

	Materials and Methods

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Animals. Pathogen-free male LEW (Lewis) and F344 (Fischer 344) rats were purchased from Harlan Sprague-Dawley Farms (Indianapolis, IN), housed in class-100 air quality rooms, and routinely monitored for common rat infections. Food (Lab Blox, Teklad, Madison, WI) and water were provided ad libitum, and animals 8 to 12 weeks of age were used in these studies.

Reagents. Phycoerythrin (PE)-conjugated monoclonal rat-specific antibodies to T cells (W3/13) and B cells (anti-IgM) were purchased from Serotec (Indianapolis, IN). PE or fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (mAbs) to CD4⁺ T cells (W3/25), CD8⁺ T cells (OX-8), and appropriate antibody isotype controls were purchased from PharMingen (San Diego, CA). DNase-free RNase, propidium iodide, and lead acetate trihydrate were obtained from Sigma (St. Louis, MO). Mouse antiphosphotyrosine mAb was obtained from Upstate Biotechnology (Lake Placid, NY).

Pb Treatment. We examined the effects of 0.1 to 1000 ppm (0.26 µM to 2.6 mM) Pb on lymphocyte responses. For some experiments, the maximally stimulating dose of Pb (50 ppm  131 µM) was used in order to observe effects that might otherwise have been missed.

Tissue and Cell Preparations. Rats were sacrificed by CO₂ inhalation and spleen (SP) cell suspensions were prepared as previously described (Razani-Boroujerdi et al., 1994). Briefly, spleens were pressed through stainless steel mesh and red blood cells were lysed by treatment with NH₄Cl solution. T and B cells were purified as described previously (Sopori et al., 1984). Briefly, SP cells (1 × 10⁸ cells) in 25 ml of complete tissue culture medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 10 µg/ml gentamicin, minimal essential medium nonessential amino acids, and polyvitamines) were depleted of macrophages by incubating the cells on a 100- × 15-mm glass Petri dish at 37°C for 1 h. T cells were purified by the passage of macrophage-depleted SP cells over a nylon-wool column (nylon-wool nonadherent cells). Enriched B cells were obtained from the macrophage-depleted population by a negative selection in which T cells were removed by panning on dishes coated with W3/13 mAb (Sopori et al., 1985). The purity of T and B cell preparations was about 90% by fluorescence-activated cell sorter analysis with an EPICS C (Coulter Electronics, Hialeah, FL) flow cytometer.

Assay for Mitogenesis. Proliferative responses were performed as previously described (Sopori et al., 1990). Briefly, in a final volume of 0.2 ml of complete medium, 2 × 10⁵ cells (SP, T, B, or T+B) were cultured in triplicates in flat-bottomed 96-well microtiter plates (Corning, NY) in the presence and absence of concanavalin A (Con A)/lipopolysaccharide (LPS) and indicated concentrations of Pb. Plates were incubated at 37°C in a 5% CO₂ atmosphere. After 48 h, cultures were labeled with 0.5 µCi of [³H]thymidine per well (New England Nuclear, Waltham, MA) and, after 24 h, cells were harvested using a Skatron cell harvester (Skatron, Sterling, VA). Samples were counted in a liquid scintillation counter. Proliferation results are presented as the mean cpm ± S.D. of triplicate cultures.

Allogeneic and Syngeneic Mixed Lymphocyte Reactions. The allogeneic mixed lymphocyte reaction (MLR) and syngeneic mixed lymphocyte reaction (SMLR) were determined essentially as described previously (Sopori et al., 1984). Briefly, for MLR, 2 × 10⁵ SP cells from LEW (RT1^l) were stimulated with 2 × 10⁵ of -irradiated (2000 rad) ACI (RT1^a) SP cells in a final volume of 0.2 ml in microtiter wells as described under the assay for mitogenesis. On day 4, cells were labeled with [³H]thymidine and harvested 16 h later. SMLR was carried out as the MLR cultures were except that LEW SP cells were stimulated with -irradiated (2000 rad) syngeneic SP cells.

Determination of Percentages of B Cells, T Cells, and T Cell Subsets by Flow Cytometry. To determine the percentage of lymphocyte populations, cells were stained with PE or FITC-labeled antibodies specific for a given lymphocyte subpopulation and then analyzed by flow cytometry as described previously (Razani et al., 1994). Briefly, in a V-bottom 96-well microtiter plate, 3 × 10⁵ cells were added to each well and washed twice with wash medium [phosphate-buffered saline (PBS) containing 5% fetal calf serum and 0.01% sodium azide]. Cells were pelleted and incubated on ice for 1 h with 5 µl of predetermined optimal concentration of PE or FITC-labeled mAb specific for various lymphocyte subpopulations. After washing, cells were resuspended in 200 µl of wash medium and analyzed by flow cytometry. At least 10,000 cells were scored for each analysis. Percentages of positive cells were calculated by subtracting the nonspecific (isotype controls) from the specific antibody fluorescent profiles.

Cell Cycle Studies. DNA content and cell volumes were determined by flow cytometry according to the protocol described by Kusewitt et al. (1992). Briefly, 2 × 10⁶/ml SP cells, T cells, or B cells were cultured at 37°C in the presence of indicated concentrations of Pb for various periods. Cells were pelleted and resuspended in 0.5 ml of cold PBS and permeabilized by adding, in a dropwise fashion, 4 ml of prechilled (20°C) 95% ethanol. Cells were stored at 20°C until further processing. To prepare cells for flow cytometric analysis, cells were washed with 4 ml of PBS at room temperature, resuspended in 0.5 ml of PBS, and treated with 10 µl of RNase (50 mg/ml; Sigma) and 20 µl of propidium iodide (1 mg/ml; Sigma). Cells were incubated in the dark for 30 min at 37°C and analyzed by a flow cytometer with excitation from an argon ion laser at 488 nm and detection at 610 nm. To eliminate doublets, cells were gated on peak red fluorescence. At least 10,000 cells were analyzed for each sample.

Inositol 1,4,5-Trisphosphate (IP₃) Assay. IP₃ was measured using [³H]inositol 1,4,5-trisphosphate radioreceptor assay kit (DuPont, Wilmington, DE), as described previously (Geng et al., 1996). Briefly, SP cells (1 × 10⁷/ml) were cultured in the presence of 50 ppm Pb or 5 µg/ml anti-cluster of differentiation 3 (CD3) for 0 to 10 min. The reaction was stopped by adding 0.2 volumes of ice-cold 100% trichloroacetic acid solution. Samples were incubated on ice for 15 min and centrifuged in cold for 1 min in a microfuge at 1000g. The supernatant was treated with a solution of 1,1,2-trichloro-1,2,2-trifluoroethane/trioctylamine (3:1) to remove trichloroacetic acid from the extracts. IP₃ was measured in the aqueous (top) layer by radioreceptor assay as described in instructions for the kit.

Tyrosine Phosphorylation Assays. Protein tyrosine phosphorylation was determined as described previously (Geng et al., 1996). Briefly, SP cells were incubated with different concentrations of Pb (0.1-100 ppm) at varying times (30 s to 48 h). The reaction was stopped with an excess of cold PBS and the cells were quickly pelleted, washed and lysed by a lysis buffer containing Tris HCl (10 mM, pH, 7.0), NaCl (50 mM), sodium orthovanadate (10 mM), tetrasodium pyrophosphate (50 mM), NaF (50 mM), iodoacetamide (2.3 mM), ZnCl₂ (5 µM), Nonidet P-40 (1%), phenlymethylsulfonyl fluoride (0.5 mM), and a concentration (5 µg/ml) of the following protease inhibitors: leupeptin, antipain, aprotinin, and pepstatin. Samples were stored at 70°C until the assay. Samples were thawed and boiled in a sample buffer containing 125 mM Tris (pH 6.8), 0.1% SDS, 25 mM dithiothreitol, and 0.01% bromphenol blue for 3 min. Samples were loaded on a 10% SDS-polyacrylamide gel electrophoresis along with Kaleidoscope Prestained Standards (Bio-Rad, Hercules, CA) and electrophoresed on a minigel system (Bio-Rad) at 150 V for 45 min. The gel was blotted onto a nitrocellulose paper, and the blot stained with Ponceu S (Sigma) to confirm the transfer of proteins from the gel to the paper. The blots were treated with 3% bovine serum albumin in Tris-buffered saline (pH 7.2) for 30 min to block nonspecific binding. Blots were incubated with mouse antiphosphotyrosine mAb overnight and washed and incubated with goat anti-mouse horseradish peroxidase-conjugated antibody for 2 h. Blots were washed and developed for horseradish peroxidase detection by either the chromogenic detection system (Renaissance 4CN plus; DuPont) or chemiluminescence (enhanced chemiluminescence Western blotting solutions; Amersham, Uppsala, Sweden).

Statistical Analysis. Statistical comparisons between different treatments were performed using a one-way analysis of variance. A Scheffe post hoc test was used to determine the significance among groups. These statistical procedures were performed using ABSTAT (Anderson-Bell Corp., Parker, CO).

	Results

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Pb Alters SP Cell Proliferation. Addition of Pb at concentrations from 0.1 to 200 ppm (0.1-200 µg/ml) to LEW SP cells significantly increased the incorporation of [³H]thymidine at doses above 0.5 ppm (Fig. 1). Maximal stimulation was observed with 25 to 50 ppm of Pb (Fig. 1A). However, the proliferative response dropped significantly below the background levels at Pb concentrations of >500 ppm, reaching >90% inhibition of the background response at 1000 ppm (Fig 1B). Similar results were obtained in F344 rats (data not shown). Thus, while the lower concentrations of Pb are immunostimulatory, higher concentrations inhibit SP cell proliferation.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Pb induces proliferation of unfractionated SP cells and cultures containing both T and B cells, but not isolated T cells and B cells. Cells were cultured in triplicate wells, under the conditions described in Materials and Methods, in the presence or absence of indicated Pb concentrations. Each data point in the figure represents the mean ± S.D. of six independent experiments. For each experiment, cells were pooled from two animals. To compare the results from different experiments, the background proliferative response (i.e., response in the absence of Pb) of each animal was assigned a value of 100%. The data have been graphed in two ways. A, expanded graph to visualize the stimulatory effects of low Pb concentrations. B, extended dose range to show the inhibitory effects of high Pb concentrations.

Pb Increases Proliferation of Both T and B Cells. To determine whether Pb preferentially stimulated a subpopulation of lymphocytes, the percentages of B cells, T cells, CD4⁺ T cells, and CD8⁺ T cells were determined in SP cells cultured with 0.1 to 50 ppm Pb for 3 days. Data presented in Table 1 (for a dosage of Pb with highest mitogenic effect) show that, in spite of significant increases in proliferation (see Fig. 1), percentages of these subpopulations were not significantly altered after Pb exposure, indicating that the Pb treatment increased the proliferation of these lymphocyte subsets to the same extent. Similar results were obtained in cell cultures exposed to lower Pb treatments (not shown).

Pb-Induced Proliferation Requires Participation of Both T and B Cells. Unlike unfractionated SP cells, proliferation of enriched T and B cell fractions was not significantly stimulated by Pb (Fig. 1, A and B). Nevertheless, in the absence of Pb, the T cell fraction did respond to Con A (not shown), indicating that the lack of response to Pb is not due to limiting numbers of accessory cells in the T cell fraction. However, the proliferative response to Pb was restored after the T and B cell fractions were combined (Fig. 1). Thus, the Pb-induced proliferative response of lymphocytes may require the presence of both B and T cells. Moreover, in the presence of -irradiated SP cells, both T cells (Fig. 2A) and B cells (Fig. 2B), exhibited a significant proliferative response to 50 ppm Pb. Similar but less pronounced proliferation was obtained with lower Pb levels (0.5-5 ppm, not shown). Furthermore, while purified -irradiated B and T cells could replace the SP cells in stimulating the proliferation of purified T and B cells, respectively, addition of -irradiated macrophages did not restore this response (Fig. 2). These results suggest that both T and B cells are essential for Pb-induced proliferation and that Pb may cause an increased interaction between T and B cells. Since stimulation of T cells with syngeneic B cells represents the SMLR response (Savage et al., 1993), these results suggest that Pb enhances SMLR.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Pb stimulates the SMLR response. T cells (A) and B cells (B) from the LEW rat spleen were cultured in the presence of -irradiated syngeneic cells: spleen (SPX), macrophages (MX), B cells (BX), or T cells (TX). Where indicated, Pb was added to a final concentration of 50 ppm. Cells were harvested on day 4 after pulsing with [3H]thymidine (see Materials and Methods). Data bars, mean ± S.E.M. from six separate experiments as described in Fig. 1.

Pb Stimulates the MLR. Figure 3 shows that 50 ppm Pb dramatically increases the MLR response of LEW (RT1^l) SP cells to -irradiated (2000 rad) SP cells from a major histocompatibility complex-disparate strain ACI (RT1^a) (ACIX). In conjunction with the results shown in Fig. 2, these results suggest that Pb stimulates both the MLR and SMLR responses.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Pb enhances the allogenic MLR response. LEW SP cells were stimulated with -irradiated (2000 rad) ACI spleen cells (ACIX) in the presence or absence of 50 ppm Pb. Experimental conditions are as described in Materials and Methods. Data bars, mean ± S.E.M. from six different experiments (one or two rats/experiment).

Pb Stimulates the Entry of Lymphocytes into the Cell Cycle. We examined the effects of various Pb concentrations on the kinetics of entry of splenocytes into the cell cycle. Treatment of SP cells with 50 ppm Pb significantly increased the entry of these cells into the S (area 2) and G₂-M (area 1) phases of the cell cycle within less than 6 h of Pb exposure (Fig. 4). This unusually fast cell-cycle response suggests that Pb treatment may trigger the progression of preexisting G₁ cells into the cell cycle. Thus, Pb may stimulate the transition of lymphocytes from the G₁ to the S phase of the cell cycle. The magnitude of the effect obtained with 0.5 to 12 ppm Pb treatment was smaller (not shown). However, at Pb concentrations of 500 and 1000 ppm, there was a significant increase in the cell population with less than 1× DNA content (Fig. 4, area 4), suggesting increased death of cells at these Pb concentrations. These results further support the inference that, although lower Pb concentrations (<200 ppm) are mitogenic, higher concentrations may be lymphotoxic.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Pb treatment stimulates the progression of G1 cells into the cell cycle. LEW SP cells were cultured in medium alone (BK), 0.5 µg/ml Con A, and 50 and 1000 ppm Pb. Histograms represent the distribution of cells in the various phases of the cell cycle after 6 h of incubation. Areas 1, 2, and 3 correspond to the cell cycle phases of G2-M, S, and G0-G1, respectively. Area 4 represents cells with less than 1× DNA content (apoptotic). Con A- and low Pb- (50 ppm) treated cells have significantly (p < .05) higher numbers of cells in the G2-M and S phases of the cell cycle relative to BK. High Pb (1000 ppm) treated cells show a significant (p < .001) increase in the apoptotic fraction (area 4) compared with BK.

Pb Augments the Response of Spleen Cells to T and B Cell Mitogens. In the presence of 50 ppm Pb, the proliferative response of SP cells to the T cell mitogen, anti-CD3 antibody, and the B cell mitogen, LPS, is significantly increased (Fig. 5). Smaller increases in anti-CD3 and LPS-induced proliferation were observed with lower Pb levels (0.5-5 ppm, not shown). In the presence of 50 ppm Pb, Con A typically induced greater spleen cell proliferation than in the absence of Pb. However, this effect was not statistically significant (not shown). Thus, Pb may enhance the response of lymphocytes to relatively weak mitogens.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Pb (50 ppm) increases proliferation of LEW SP cells in response to anti-CD3 antibody (A) and LPS (B). Solid line (control) and dotted line (with Pb). Each data point represents the mean ± S. D. of four to five experiments (n = 8).

Pb Increases IP₃ Levels in Splenocytes without Stimulating Protein Tyrosine Kinase (PTK) Activity. One of the earliest events in the antigen-induced activation of lymphocytes is an increase in the PTK activity, which in turn activates phospholipase C (PLC) catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) to IP₃ and diacylglycerol (Cambier et al., 1994; Chan et al., 1994). To determine whether Pb stimulated lymphocyte proliferation through a similar pathway, we determined the concentration of IP₃ in SP cells following exposure to 50 ppm Pb or anti-CD3. Results presented in Fig. 6 indicate that, within 5 to 7 min, Pb significantly increased IP₃ levels in these cells and the magnitude of response was similar to that obtained with anti-CD3. However, as seen by Western blot analysis of tyrosine phosphorylation (Fig. 7), unlike treatment with anti-CD3 or anti-IgM, there is no significant increase in the PTK activity in Pb-treated SP cells over the 0-time controls. These results suggest that Pb may stimulate the PLC activity through a mechanism that is independent of PTK activation and is, therefore, different from the antigen receptor-mediated lymphocyte activation.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Pb increases IP3 production by SP cells. LEW SP cells were incubated with 50 ppm Pb or anti-CD3 antibody (5 µg/ml) for 0 to 10 min at 37°C. Lysates were assayed for IP3 concentrations. Values represent an average of five experiments. IP3 levels were significantly (p < .05) elevated by both treatments.

View larger version (104K): [in this window] [in a new window]   Fig. 7.   Pb does not stimulate the activity of PTKs in SP cells. SP cells were incubated with anti-CD3 (5 µg/ml), anti-IgM (5 µg/ml), and indicated Pb concentrations for 5 min. The lysates were examined for PTK activity by Western blot analysis. Similar results were obtained with longer Pb treatments (10 min to 24 h). BK, response in the absence of Pb; mw, molecular weight of standards.

	Discussion

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Results presented herein suggest that Pb is a weak mitogen for lymphocytes and stimulates the proliferation of both T and B cells. At higher concentrations (>200 ppm), however, Pb is immunosuppressive, causing inhibition of background proliferation. While the enhancement of lymphocyte proliferation has also been reported by others (Shenker et al., 1977; Gaworski and Sharma, 1978; Lawrence, 1981; Warner and Lawrence, 1986; McCabe and Lawrence, 1990), our results indicate that Pb has no significant effect on isolated populations of T and B cells. However, addition of -irradiated B cells to T cells or -irradiated T cells to B cells reconstituted the Pb-induced proliferative response of these cell cultures; addition of -irradiated macrophages was ineffective in restoring this response. We have previously demonstrated that the rat SMLR response is dependent on the interaction between T cells and B cells resulting in the proliferation and differentiation of both cell types (Sopori et al., 1990; Savage et al., 1993). Thus, Pb may facilitate T cell-B cell interaction accounting for the enhancement of the SMLR and MLR responses (i.e., responses where T cell-B cell interaction plays a pivot role in the response) (Singer and Hodes, 1983; Savage et al., 1993). The ability of Pb to enhance the SMLR response could potentially play a role in the increased frequency of some autoimmune diseases observed in Pb-exposed individuals (Wedeen et al., 1979).

To understand the mechanism through which Pb treatment increases lymphocyte proliferation, we investigated the effects of Pb on the progression of SP cells into the cell cycle. Culturing of SP cells with 50 ppm Pb caused a significant progression of cells into the S and G_2-M phases of the cell cycle within 6 h. This relatively fast Pb-induced entry of lymphocytes into the S phase suggests that Pb may stimulate the progression of the splenic lymphocyte fraction, which is in the G₁ phase at the time of cell isolation. This could also explain the manner through which Pb enhances proliferation of lymphocytes to relatively weak mitogens (i.e., anti-CD3, LPS); in the rat, LPS is a relatively poor B cell mitogen. The ability of heavy metals to induce cell cycle progression in murine splenocytes was also reported by others (Warner and Lawrence, 1986)

One of the earliest events in the antigen-mediated activation of lymphocytes is an increase in PTK activity (Cambier et al., 1994; Chan et al., 1994). An important consequence of this activation is tyrosine phosphorylation of PLC leading to hydrolysis of PIP₂ into diacylglycerol and IP₃ (Berridge, 1993; Robey and Allison, 1995). IP₃ causes an increase in the intracellular calcium level (Berridge, 1993), leading to a progression of G₀-G₁ cells into the S phase of the cell cycle (Crabtree and Clipstone, 1994). Because Pb enters into cells and binds to commonly used indicators for measuring ionized calcium, Pb interferes with Ca⁺⁺ detection by standard methods (Schanne et al., 1989). Therefore, instead of measuring Ca⁺⁺ we determined whether Pb treatment had any effect on PIP₂ metabolism by measuring the intracellular IP₃ levels. As shown in Fig. 6, within minutes of Pb treatment, lymphocytes had significantly higher levels of IP₃, suggesting that Pb stimulates PLC activity. An increase in IP₃ levels in Pb-treated rat astrocytes has been reported by Dave et al. (1993). However, unlike activation of lymphocytes through ligation of antigen receptors, the Pb-mediated increase in PLC activity in SP cells does not appear to depend on the activation of PTK activity. Therefore, it is unlikely that increased IP₃ levels in Pb-treated cells result from the activation of PLC. [Although the modulation of PTK by Pb⁺⁺ has not been reported, the inhibitory effects of Pb⁺⁺ on other protein kinase activities (e.g., protein kinase C) is well documented (Saijoh et al., 1988; Rajanna et al., 1995).] The other known pathway for IP₃ synthesis involves the G-protein-dependent activation of PLC (Gilman, 1987); at present, we have no direct evidence to suggest that Pb stimulates the G-protein-dependent signaling pathway in lymphocytes. Nonetheless, our results suggest that the lymphoproliferative effects of Pb may result from increased IP₃ synthesis which is independent of antigen receptor activation.