INTRODUCTION

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THE SPONTANEOUSLY HYPERTENSIVE RAT (SHR) has been extensively used to study essential hypertension. By 12 wk of age, these rats exhibit elevated blood pressure and associated pathological changes in the vasculature (5, 11, 20, 26). The development of essential hypertension in the SHR has been attributed to a number of etiologies (20, 26), including one of immune origin (5, 11). In this regard, the SHR exhibited a number of immune deficiencies characterized by the production of autoantibodies cytotoxic for thymocytes (38), impaired T cell-dependent B cell responses (30, 39, 40), delayed allograft rejection of skin (40), and decreased mitogen-induced lymphoproliferative responses (29, 30, 40). Interestingly, these immune anomalies were reversible by implantation of thymic tissues from normotensive rats into the SHR, suggesting that these T cell dysfunctions accounted for the immune deficiencies in SHR (1, 24). Thymic engraftment also resulted in correction of hypertension, particularly in young SHR. Based on these observations, subsequent studies have attempted to determine whether the SHR immune system contributed to the development of hypertension and whether novel immunologically based therapeutics could be developed to correct hypertension in SHR (1, 24, 27).

To explore the relationship between hypertension and concomitant immune dysfunction in SHR, T cell responses to the T cell mitogen concanavalin A (Con A) were studied (29, 30, 39, 40). The SHR with established hypertension exhibited impaired T cell responses to mitogen; however, in young or in prehypertensive SHR, normal T cell proliferative responses were seen (30). No aberrant CD4-to-CD8 T cell ratios could be established in the SHR compared with a related normotensive Wistar-Kyoto rat (WKYR) (30). This association of concurrent development of elevated blood pressure and loss of lymphoid proliferative responses was in part attributed to the presence of NO production by macrophages (29). Macrophage depletion resulted in restoration of T cell responses to Con A (30), and the addition of the NO synthase inhibitor N^G-monomethyl-L-arginine (NGMA) produced similar results (29). Collectively, these observations suggested that T and B cell dysfunctions that developed as the SHR matured, i.e., during the developmental phase of hypertension, were secondary to the effects of other cell types and/or their soluble products.

Cytokines secreted by CD4⁺ T helper (Th) cells play a key role in the induction and the development of immune responses. In this regard, two distinct subsets of CD4⁺ Th cells have been identified based on the pattern of cytokines secreted and are referred as Th1 and Th2 type (21, 28). Th1-type cells secrete interferon- (IFN-), interleukin-2 (IL-2), and TNF- and control cell-mediated immune responses. In contrast, Th2 type cells secrete IL-4, IL-5, IL-6, IL-10, and IL-13 and regulate antibody production or humoral immunity. Induction of one particular pathway by CD4⁺ T cells is dictated by mode of immunization or infection (28), i.e., intracellular pathogens stimulate Th1 cells or cell-mediated immune responses, whereas extracellular pathogens stimulate Th2 cell responses. Furthermore, adjuvants can preferentially promote Th1- or Th2-type responses.

Studies have demonstrated the potency of cholera toxin (CT) as an adjuvant for both parenterally (45, 46) and mucosally administered antigens (14, 15, 45-47). As a result, elevated serum immunoglobulin (Ig) G and mucosal IgA antigen-specific antibodies are elicited to CT and coadministered antigens (7, 10, 14, 15, 45, 47). Marked elevations in serum IgG1 and transient IgE (15, 35) antibody responses were obtained in immunized mice, suggestive of a Th2 cell-dependent response as a result of the use of CT as adjuvant. This selective induction of a Th2 cell response was evident in a number of studies in which CT was shown to preferentially elicit Th2-type cytokines (15, 45-47). In fact, because of the route of delivery, i.e., oral delivery of CT, both Peyer's patch and splenic CD4⁺ Th cell responses showed elevations in numbers of antigen-specific IL-4- and IL-5-producing Th2 cells. After parenteral administration of CT, antigen-specific Th2 cells were induced as well as Th1 cells, evident by the induction of antigen-specific IFN--producing cells (45, 46). Regardless of the route of administration, CT has a predilection for the development of Th2-type responses. Based on this CT/Th2 cell paradigm, we hypothesized that if CT induces Th2 cell responses in rats, one may correct the SHR T cell dysfunction and permit T cell-dependent immune responses to a defined vaccine antigen, e.g., diphtheria toxoid (DT).

	MATERIALS AND METHODS

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Rat strains. Male SHR, WKYR, and Brown Norway rats (BNR) were obtained from Taconic Farms (Germantown, NY) at 10-12 wk of age and were maintained on Purina rat chow containing 1% NaCl (basal chow) and water ad libitum.

Lymphoid cell isolation. Spleens were excised aseptically and pressed through wire sieves to generate single cell suspensions. To obtain the mononuclear cell fraction, washed cell suspensions were applied to lymphocyte-rat density gradients (Accurate Chemical, Buffalo, NY) and centrifuged for 30 min at room temperature (29, 30). These splenic mononuclear cells (SMC) were removed from the interface and washed in complete media consisting of RPMI 1640 (Whittaker Bio-Products, Walkersville, MD) and 10% fetal calf serum (FCS; Hyclone, Logan, UT) plus supplements (GIBCO, Grand Island, NY) containing 100 U/ml penicillin, 100 µg/ml streptomycin, nonessential amino acids (0.1 mM), L-glutamine (0.2 mM), sodium pyruvate (0.1 mM), and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; 10 mM). Media, FCS, and supplements contained <0.025 ng/ml endotoxin.

CD4⁺ T cells were purified by negative selection using a Cellect Rat CD4 T cell kit (Biotex Laboratories, Edmonton, AB) by a procedure suggested by the manufacturer. This procedure yielded >93% CD3⁺, CD4⁺, and CD8 T cells. To obtain feeder cells, 1 × 10⁷ SMC/ml in RPMI 1640 with 0.025 M HEPES and 3% BSA (tissue culture grade; Sigma Chemical, St. Louis, MO) were incubated with 10 µg/ml OX-34 (anti-CD2) antibody (PharMingen, San Diego, CA) for 1 h on ice. After a washing step, cells were resuspended in 1:10 dilution of low-Tox-M baby rabbit complement (Accurate) for 1 h at 37°C. Cells were subsequently washed and layered onto lympholyte-rat density gradient to obtain the mononuclear cell fraction. Feeder cells isolated by this method routinely were <5% CD3⁺.

IFN- and IL-4 enzyme-linked immunosorbent assay. For the IFN- enzyme-linked immunosorbent assay (ELISA), a cross-reactive hamster monoclonal anti-mouse IFN- antibody (Genzyme, Boston, MA) at 2.0 µg/ml was used to coat Maxisorp Immunoplate II microtiter wells (Nunc, Roskilde, Denmark) overnight at room temperature. This antibody has been previously shown to inhibit 100% of rat IFN- activity (29). After a blocking step, samples and varying dilutions of recombinant rat IFN- (GIBCO) were incubated overnight at 4°C. After complete washing, a 1:200 dilution of biotinylated polyclonal rabbit anti-rat IFN- antibody (Bio-Source International, Camarillo, CA) was added and incubated overnight at 4°C. Wells were again washed, and a 1:800 dilution of a horseradish peroxidase-conjugated goat anti-biotin antibody (Vector Laboratories, Burlingame, CA) was incubated for 90 min at 37°C. After extensive washing, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium (ABTS) substrate (Moss, Pasadena, CA) was added to determine sample concentrations derived from a standard curve using recombinant rat IFN-.

For the IL-4 ELISA, a goat anti-mouse IL-4 antibody (R&D Systems, Minneapolis, MN) at 20 µg/ml was used to coat Nunc Maxisorp microtiter wells overnight at room temperature. After a blocking step, samples and varying dilutions of recombinant rat IL-4 (Genzyme) were incubated overnight at 4°C. Subsequent to washing, a 1:200 dilution of a mouse monoclonal anti-rat IL-4 antibody (Harlan Bioproducts for Science, Indianapolis, IN) was incubated for 2 h at 37°C, and a 1:1,000 dilution of a horseradish peroxidase-conjugated goat anti-mouse IgG1 antibody (Southern Biotechnology Associates, Birmingham, AL) was incubated for 90 min at 37°C. After extensive washing, wells were incubated with ABTS substrate (Moss) until color development. Optimal densities were measured using a Bio-Tek Instruments plate reader (Winooski, VT) at 415 nm.

Reverse transcription-polymerase chain reaction for detection of rat IFN- mRNA. To demonstrate the induction of IFN- responses by CD4⁺ T cells on stimulation with Con A, reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect IFN--specific mRNA levels. PCR primers for rat IFN- and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, were purchased (Clontech Laboratories, Palo Alto, CA). SHR and WKYR SMC (5 × 10⁶/ml) were cultured for 2 days either in media alone, with 5.0 µg/ml Con A (Sigma), or with 5.0 µg/ml Con A plus 100 µM NGMA (Calbiochem, La Jolla, CA). After culture, CD4⁺ T cells were isolated, and cells were subsequently suspended in 2 ml Tri-Reagent (Molecular Research Center, Cincinnati, OH) to isolate total RNA. RNA isolation and RT-PCR assay were similar to that previously described (42). Total RNA was quantified, and the mRNA was reverse transcribed into cDNA by incubation at 42°C for 45 min, using random hexamers and RT (Perkin Elmer Cetus, Norwalk, CT). The cDNA was amplified by PCR in a thermal cycler (Perkin Elmer) for 35 cycles. For amplification of cytokine cDNA, each cycle consisted of a 45-s, 95°C melting step, a 2-min 65°C hybridization step, and a 3-min 72°C elongation step. RT-PCR was used to analyze the relative abundance of specific cytokine mRNA, and dilution analysis was performed to ensure that mRNA used for amplification was at a concentration in the linear range for PCR product formation, and not near or at saturation. PCR with the primer sets utilized in this study did not generate any PCR products in the absence of the RT step, indicating that these preparations lacked DNA contaminants.

Immunization protocol. Rats were given subcutaneous injections of a mixture of DT (200 µg/rat) kindly provided by Lederle-Praxis Biologicals. (Rochester, NY) and CT (2.0 µg/rat; Sigma) in phosphate-buffered saline (PBS). This antigen mixture was given three times at 1-wk intervals, following a similar regimen used for mice (10). Rats were killed by CO₂ asphyxiation 3-7 days after the last immunization.

Serum DT- and CT-B-specific antibody production by ELISA. Antibody responses were monitored by ELISA using a previously adapted method for detecting murine anti-tetanus toxoid and anti-CT-B antibody responses (10, 47). Briefly, DT (0.75 µg/well) in PBS was added to wells of Maxisorp Immunoplate II microtiter plates (Nunc), and CT-B (0.5 µg/well; Sigma) in PBS was coated onto Falcon (Microtest III) 96-well assay plates (Becton Dickinson, Oxnard, CA) overnight at 4°C. Serum samples were serially diluted in ELISA wash buffer (PBS + 0.5% BSA + 0.5% Tween-20), added to wells and incubated overnight at 4°C. Subsequently, wells were rinsed with wash buffer, and detection antibodies were added for 90 min at 37°C. For the detection of antigen-specific antibodies, dilutions of 1:1,000 were used for alkaline phosphatase-conjugated goat anti-rat IgG-specific antibody (Southern Biotechnology Associates) and alkaline phosphatase-conjugated rabbit anti-rat IgM antibody (Zymed Laboratories, South San Francisco, CA). Specific antibody reactivity was determined by the development of a color reaction on addition of nitrophenyl phosphate (Sigma) substrate in 0.1 M carbonate buffer, pH 9.5. Optical densities were measured using a Bio-Tek Instruments plate reader at 405 nm. IgG subclass reactivities to DT or to CT-B were evaluated by using horseradish peroxidase-conjugated monoclonal antibodies to rat IgG1, IgG2a, IgG2b, and IgG2c (Zymed). Specific reactivities were determined by the addition of 100 µl/well of 0.1 mg/ml of ABTS (Sigma) in 0.1 M citrate buffer, pH 4.5, and 0.01% H₂O₂, and absorbance was read at 415 nm. End-point titers were expressed as the reciprocal dilution of the last sample dilution giving an absorbance 0.1 OD unit above the OD₄₀₅ and OD₄₁₅ of negative controls after a 15-min incubation.

T cell cultures. To assess the proliferative responses by SHR, WKYR, and BNR cells to DT and CT, DT and CT-B were used to coat latex microspheres using a modified procedure of a previously described method (44, 47). Briefly, 1 ml (in 0.5-ml aliquots) of Polybead-hydroxlylate latex microspheres (1.0 µm; Polysciences, Warrington, PA) was washed twice with sterile 0.1 M sodium bicarbonate buffer, pH 8.8, at 10,000 g for 12 min at 4°C. The washed microspheres were resuspended in 0.8 ml of the same buffer, and 0.4 ml of 100 µg of DT or CT-B in sterile 0.5 M tris(hydroxymethyl)aminomethane · HCl, pH 8.0, were added slowly to microspheres with continuous mixing. Antigen was allowed to adhere to the microspheres by incubating for 24 h at room temperature by continuous rocking. Subsequently, microspheres were washed twice with sterile PBS, and beads were resuspended in RPMI 1640 with 0.025 M HEPES and 500 µg/ml gentamycin.

SMC or CD4⁺ T cells plus irradiated feeder cells were suspended in complete media and plated in 96-well microtiter dishes (Costar, Cambridge, MA) at 2 × 10⁵ cells/well in a final volume of 0.1 ml containing varying DT- or CT-B-coated microspheres-to-cell ratios (0.25:1 to 50:1) for proliferation assays. For cytokine production, CD4⁺ T cells and irradiated feeder cells were cultured with 20:1 and 2:1 DT or CT-B beads. For the anti-cytokine antibody-treated cultures, a cross-reactive hamster monoclonal anti-mouse IFN- antibody (Genzyme) previously shown to inhibit 100% of rat IFN- activity (29) was added. A mouse monoclonal anti-rat IL-4 antibody (Harlan Bioproducts for Science) was also used in this study. For the cytokine-treated cultures, 100 U/ml of recombinant rat IL-4 (Genzyme) or 100 U/ml of recombinant rat IFN- (GIBCO) was added to the cultures. Cells were cultured for 3-5 days at 37°C and 5% CO₂ in air. For the proliferation assays, cells were pulsed with 0.5 µCi/well of [³H]thymidine (New England Nuclear, Wilmington, DE) during the last 16 h of culture. Subsequently, cells were harvested onto glass fiber filter disks to detect incorporated radioactivity and counted using a Beckman LS6000IC scintillation counter (Fullerton, CA). The data are presented as the mean value obtained from quadruplicate wells in counts/min (±SD).

Statistical analysis. Data were analyzed using Student's t-test and one-way analysis of variance Tukey test. Values were considered significantly different if P was <0.05.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

SHR show a propensity for IFN- production, which in turn stimulates elevated levels of NO. Previous studies (29, 30, 40) have established that the SHR showed diminished lymphoproliferative responses upon mitogen stimulation, and this diminished capacity was attributed to macrophage-generated NO (29). The addition of the NO synthase inhibitor NGMA resulted in the restoration of mitogen-induced lymphoproliferative responses and reduced NO production (29). To demonstrate that the mitogen-induced NO production was attributed to increased IFN- production, SHR and WKYR SMC cultures were stimulated with Con A. As shown in Fig. 1A, increased IFN- production was evident compared with unstimulated cultures derived from SHR and WKYR SMC. On stimulation with Con A, SMC from SHR generated 3.3-fold greater IFN- production than similarly stimulated WKYR SMC. The addition of NGMA to the SHR cultures resulted in a 100% increase in IFN- production versus Con A-stimulated cultures. No significant change in IFN- levels was observed on NGMA addition to WKYR cultures. This evidence suggests that the SHR exhibits a propensity for IFN- production. This was further substantiated at the mRNA level by RT-PCR, and it was shown that the changes in IFN- secretion were due to alteration in CD4⁺ T cell IFN- mRNA (Fig. 1B). On the basis of these observations, we queried whether CT, a known Th2-IL-4 inducer, would reverse the observed immune suppression in the SHR in an antigen-specific fashion.

View larger version (17K): [in this window] [in a new window]   View larger version (48K): [in this window] [in a new window]   Fig. 1.   Concanavalin A (Con A)-stimulated splenic mononuclear cells (SMC) from unimmunized spontaneously hypertensive rats (SHR) showed enhanced interferon (IFN)- production compared with similarly treated Wistar-Kyoto rat (WKYR) SMC cultures. SHR and WKYR SMC were cultured alone or with Con A (5.0 µg/ml) in the absence or presence of 100 µM nitric oxide synthase inhibitor NG-monomethyl-L-arginine (NGMA) for 2 days. Culture supernatants (A) were assessed for IFN- production by a rat IFN--specific enzyme-linked immunosorbent assay (ELISA). Addition of NGMA to SHR cultures enhanced IFN- production while having minimal effect on WKYR cells. Data are depicted as average of 3 experiments ± SE and were subjected to 1-way analysis of variance followed by a Tukey test. * P < 0.002 compared with unstimulated SHR cultures; ** P < 0.003 compared with unstimulated WKYR cultures;  P < 0.05 compared with Con A-treated SHR cultures; P <0.004 compared with Con A plus NGMA-treated SHR cultures. CD4+ T cells were purified and assessed by reverse transcription-polymerase chain reaction (RT-PCR) for the production of IFN- mRNA (B). Augmentations observed in IFN- production were also reflected at the mRNA level by SHR (S) and WKYR (W) CD4+ T cells. Total RNA was isolated and subjected to RT-PCR to analyze for the production of mRNA for IFN-. PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining. Depicted are PCR products for IFN- [288 base pairs (bp)] following culture in media (M) alone, with Con A (CA), or with Con A and NGMA (CA + N). Depicted also are PCR products for the housekeeping gene G3DPH (983 base pairs) to monitor loading of sample RNA and PCR reactions.

CT reverses DT-specific antibody unresponsiveness in SHR. To test the hypothesis that the potent adjuvant CT could reverse the immune suppression seen in adult SHR, groups of SHR and WKYR were immunized with DT alone or in combination with CT, and antibody responses to DT were determined. The SHR and age-matched WKYR received three subcutaneous doses of 200 µg DT/dose at 1-wk intervals, and serum from each rat was individually collected 3 wk later. The maximum serum IgG anti-DT titer obtained in SHR was ~1:2,000 and was fivefold less than similarly dosed, age-matched WKYR, which produced immune sera IgG titers of 1:10,000 (Fig. 2). Serum IgM antibody responses by SHR were also depressed, as evidenced by a 16-fold difference in IgM anti-DT titers when compared with WKYR (Fig. 2). However, coadministration of 2.0 µg CT with DT resulted in a pronounced rise in serum IgG titers of ~1:40,000, which represented a 20-fold elevation in antigen-specific serum IgG responses compared with SHR receiving only DT (Fig. 2). This IgG anti-DT titer induced by CT coimmunization was similar in magnitude to that obtained with DT- and CT-immunized WKYR. Likewise, a rise in SHR IgM anti-DT antibody responses was also evident (Fig. 2). Both the WKYR and SHR also produced elevated IgG anti-CT-B antibody titers (1:1 × 10⁵). Thus these results suggest that the SHR was capable of responding to both DT and CT-B antigens, and the depressed IgG and IgM anti-DT antibody titers obtained in DT-immunized SHR was the result of an active inhibitory mechanism.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Coimmunization with cholera toxin (CT) of SHR results in the reversal of the depressed immunoglobulin (Ig) M and IgG antibody responses to diphtheria toxoid (DT). Beginning at 12 wk of age, SHR and WKYR (3/group) received a 200-µg dose of DT on days 0, 7, and 14 by the subcutaneous route. The antibody titer was averaged for each group and is expressed as mean ± SD. Compared with WKYR, SHR showed weak IgG and IgM anti-DT antibody responses at day 21 that were approximately 1 log less than WKYR antibody responses when analyzed by a DT-specific ELISA. After coimmunization with 2.0 µg of CT/rat in age-matched controls, CT boosted both SHR IgG and IgM anti-DT antibody responses with similar magnitude as with the DT- and CT-immunized WKYR. Likewise, similar anti-CT-B antibody titers were obtained in both the SHR and WKYR when analyzed by anti-CT-B ELISA. This evidence shows that CT coimmunization can restore normal antibody responses in SHR. A: anti-DT. B: anti-CT-B.

Immunization of rats with DT and CT elicits IgG2a and IgG1 antibody responses to DT and CT-B. Additional groups of SHR and WKYR were immunized with DT and CT to assess IgG subclass responses induced as a consequence of this immunization regimen. Included in this analysis was the BNR to ensure that the observed differences in antibody responses of rats given the combined DT and CT vaccine was not due to inherent differences in major histocompatibility complex class II peptide recognition. One week after the last immunization, the various groups were analyzed for serum anti-DT and anti-CT-B antibody responses. Each rat strain responded with a similar magnitude of IgM and IgG antibody titers to both DT and CT-B (Fig. 3). There was no species-specific segregation of IgG subclass responses to either DT or CT-B in SHR, WKYR, or BNR (Fig. 3). Each strain showed elevated IgG2a and an order of magnitude lower IgG1 antibody titer to both DT and CT-B (Fig. 3). Furthermore, there was no variation in the magnitude of the IgG2a or IgG1 antibody titers among the three species. No detectable levels of IgG2b or IgG2c anti-DT or anti-CT-B antibodies were obtained in any of these three rat strains tested. Based on these IgG subclass responses, induction of rat IgG2a and IgG1 antibodies have been previously shown to be indicative of Th2 cell involvement because these IgG antibodies were generated subsequent to Th2 cell-dependent nematode infections (17, 41).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Coimmunization with CT resulted in elevated IgG2a and IgG1 antibody responses to both DT and CT-B in SHR, WKYR, and Brown Norway rats (BNR). Each rat group received three immunizations at 7-day intervals with DT and CT (3 rats/group) as described in Fig. 2 legend. DT- and CT-B-specific serum antibody titers at day 21 were averaged for each species and are shown ±SD. Immune IgG2a antibody titers were the predominant IgG subclass induced to both DT and CT-B. This immunization scheme was equally effective for each rat strain tested because each species responded to the same degree with regard to IgG antibody subclass response, and this evidence suggests that SHR can respond to DT when appropriately immunized. A: IgG. B: IgM. C: IgG2a. D: IgG1.

CT reverses immune suppression of DT-specific T cell proliferative responses to DT in SHR. To determine the type of CD4⁺ Th cell response elicited by CT coadministration, optimal in vitro T cell proliferative responses were examined. SHR and WKYR received three immunizations, either with DT alone or in combination with CT. Total SMC were isolated and stimulated in vitro, with varying ratios of DT or CT-B absorbed to latex beads (referred to as DT beads and CT-B beads, respectively). SHR immunized with DT only harbored T cells that failed to proliferate to DT beads in vitro. In contrast, the SMC from WKYR subjected to a similar immunization regimen responded to the DT beads in a dose-dependent fashion (Fig. 4). The SMC from both rat strains failed to proliferate to CT-B beads as expected because they were not immunized with CT. The coadministration of CT during immunization greatly augmented the ability of the SHR to respond to DT. Immune SMC from DT- and CT-immunized SHR proliferated in a dose-dependent fashion to levels similar to those obtained by DT- and CT-immunized WKYR (Fig. 4). In fact, the coadministration of CT reversed unresponsiveness of SHR and augmented responses to both DT and CT-B. Although the SMC from DT- and CT-immunized SHR were able to proliferate in response to the CT-B beads, the magnitude of induced proliferation was not as great as that obtained with SMC from similarly immunized WKYR (Fig. 4).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Coimmunization with CT resulted in the reversal of the depressed SHR T cell proliferative response to DT. SHR and WKYR were immunized as described in Fig. 2. Rats were killed 4 days after their last immunization, and SMC were isolated. SMC were stimulated for 5 days with varying ratios of cells to antigen-absorbed latex beads. During the last 16 h of culture, cells were pulsed with 0.5 µCi of [3H]thymidine to monitor its incorporation [expressed as counts/min (cpm) ± SD]. SHR immunized with DT only showed no proliferative responses to DT or CT-B beads, whereas WKYR immunized with DT only showed a dose-dependent proliferative response to DT, but not to CT-B. After coimmunization with CT, the SHR showed a dose-dependent proliferative response to DT of equal magnitude as identically treated WKYR. DT- and CT-coimmunized WKYR showed greater CT-B proliferative responses than similarly treated SHR. The results presented are representative of 3 separate experiments and show that CT coimmunization reverses the T cell dysfunction in SHR. A: DT. B: CT-B.

CT augments CD4⁺ T cell responses in SHR, WKYR, and BNR. In addition to the SHR and WKYR, BNR were also assessed for their ability to respond to CT, especially because this rat strain has been identified to be particularly sensitive to Th2 cell-dependent responses (19), in contrast to the SHR, which is more prone to Th1 cell-dependent responses (29). Before determination of whether Th1 or Th2 cytokine responses were elicited as a result of CT coimmunization, studies were performed to assess whether CD4⁺ T cells were responsible for the antigen-specific T cell proliferative responses following in vitro stimulation with antigen. CD4⁺, CD8 T cells from DT- and CT-immunized SHR, WKYR, or BNR were cocultured with their respective T cell (OX-34)-depleted feeder cell population in the presence of varying doses of DT or CT-B beads. Our results revealed that the isolated immune CD4⁺, CD8 T cells were responsible for the proliferative responses because depletion of CD4⁺ T cells abrogated in vitro proliferative responses (data not shown). To directly demonstrate that the responding T cell population was indeed of a CD4⁺, CD8 phenotype, the CD4⁺ T cells derived from either the SHR, WKYR, or the BNR were cocultured with their respective feeder cells. Proliferative responses were induced in a dose-dependent fashion to both DT or CT-B beads (Figs. 5 and 6). Although the BNR SMC did proliferate with a higher overall stimulation index (SI), their corresponding cocultured CD4⁺ T cells responded with similar SI to DT beads as cocultured SHR or WKYR CD4⁺ T cells (Fig. 5). Likewise, similar proliferative responses to CT-B were obtained with cocultured CD4⁺ T cells from immune SHR, WKYR, or BNR. For optimal stimulation, a feeder cell population was required, as evidenced by the SI for each T cell population tested (Figs. 5 and 6). Taken together, these results show that the adjuvant, CT, enhanced CD4⁺ T cell immunity to both DT and CT-B in SHR.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   CD4+ T cells are the effector T cell population responsible for the proliferative responses to DT obtained from SHR (A), WKYR (B), and BNR (C) coimmunized with DT and CT. CD4+ T cells were isolated from immune rats by negative selection and were costimulated with irradiated CD2-depleted feeder cells. CD4+ T cells were cultured for 5 days alone or with 2:1 or with 20:1 DT beads to CD4+ T cells. The level of incorporated [3H]thymidine was expressed as a stimulation index: cpm of DT-stimulated cells/cpm of unstimulated cells. Minimal stimulation was obtained for CD4+ T cells in the absence of feeder cells or with feeder cells alone. This evidence shows that proliferative responses to DT were attributed to CD4+ T cells. Data presented are representative of 3 separate experiments. A: SHR. B: WKYR. C: BNR.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   CD4+ T cells are the effector T cell population responsible for the proliferative responses to CT-B obtained from SHR, WKYR, and BNR coimmunized with DT and CT. Cultures containing either SMC or CD4+ T cells were analyzed as described in Fig. 5 legend. Level of incorporated [3H]thymidine was expressed as the stimulation index (experimental/control). Minimal stimulation was obtained for CD4+ T cells in the absence of feeder cells or with feeder cells alone. This evidence clearly demonstrates that the proliferative responses to CT-B were attributed to CD4+ T cells. Data presented here are representative of 3 separate experiments. A: SHR. B: WKYR. C: BNR.

Induction of IL-4-producing CD4⁺ T cells by coadministered CT reverses the immune suppression in SHR. To test whether IL-4 or IFN- was induced upon antigen stimulation, CD4⁺ Th cell cultures derived from DT- and CT-immunized SHR and BNR were assessed for cytokine secretion by ELISA. Stimulation of SHR CD4⁺ T cells with DT or CT-B beads induced IL-4 with minimal IFN- production (Fig. 7). Treatment with an anti-IFN- antibody resulted in enhanced IL-4 production by both DT- and CT-B-stimulated CD4⁺ T cells. Anti-IL-4 antibody treatment had a marginal effect on IFN- production by DT- and CT-B-stimulated SHR cultures (Fig. 7B). In the case of BNR, both IL-4 and IFN- production were found after stimulation of antigen-specific CD4⁺ T cells (Fig. 7, C and D). The levels of IL-4 produced by the BNR were similar to those seen in the SHR. Treatment with an anti-IFN- antibody resulted in increased IL-4 production by DT- and CT-B-stimulated BNR CD4⁺ T cell cultures. In a similar fashion, increased IFN- production was obtained on anti-IL-4 antibody treatment in both DT- and CT-B-stimulated CD4⁺ T cell cultures. This evidence suggests that mixed Th1 and Th2 cell subsets were induced in the BNR to both DT and CT after our parenteral immunization regimen. When a similar immunization scheme was applied to the SHR, the elicited responses were clearly Th2 cell dominant.

View larger version (40K): [in this window] [in a new window]   Fig. 7.   T helper (Th) 2-type responses were induced after in vitro stimulation of immune CD4+ T cells from DT- and CT-immunized SHR (A and B) and BNR (C and D) with DT or CT-B resulted in the production of interleukin (IL)-4. CD4+ T cell cultures as described in Fig. 6 legend were stimulated with 10:1 DT or CT-B beads for 3 days in the presence or absence of a hamster monoclonal anti-mouse IFN- (-IFN-) antibody (10 µg/ml) or a mouse monoclonal anti-rat IL-4 (-IL-4) antibody (10 µg/ml), and supernatants were collected to assess IFN- and IL-4 production by cytokine-specific ELISA (U/ml of cytokine/1 × 106 CD4+ T cells). IL-4 (A) was induced by DT or CT-B stimulation of SHR CD4+ T cells with minimal production of IFN- (B), whereas both IL-4 (C) and IFN- (D) were stimulated by BNR CD4+ T cells. Increased IL-4 production was noted after treatment of antigen-stimulated SHR or BNR CD4+ T cell cultures with (A and C) anti-IFN- antibody, and no significant differences in IFN- production were noted after treatment of SHR cultures with anti-IL-4 antibody (B). In contrast, increased IFN- production was noted in BNR CD4+ T cell cultures after treatment with anti-IL-4 antibody. This evidence demonstrates that the reversal of the immune suppression in the SHR by CT coimmunization was mediated by the stimulation of Th2 cells producing IL-4. Data presented here are representative of 2 separate experiments. * P < 0.001.

To further substantiate the dominance of Th2 cell responses, the role of exogenous addition of IFN- or IL-4 to antigen-stimulated SHR and BNR CD4⁺ T cell cultures was assessed. The addition of recombinant rat IL-4 to either SHR or BNR cultures resulted in enhanced CD4⁺ T cell proliferation when stimulated either with DT or CT-B beads (Fig. 8). In contrast, the addition of recombinant rat IFN- resulted in the inhibition of CD4⁺ T cell proliferation during antigen stimulation. This provides further evidence that the CD4⁺ Th cell responses after CT coadministration were of a Th2 type. Although both Th1 and Th2 cell responses were noted to DT and CT-B in these rats, the addition of IFN- to cultures did not result in any enhancing effect on the CD4⁺ T cell proliferation, suggesting that the DT- and CT-B-specific Th1 cells participate to a lesser extent in the immune response, and this exogenous addition of IFN- to the antigen-driven cultures may have suppressed IL-4-producing CD4⁺ T cells.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Addition of recombinant rat IL-4 and not recombinant rat IFN- resulted in enhanced T cell proliferation by CD4+ T cells from DT- and CT-immunized SHR (A) and BNR (B) after in vitro DT or CT-B stimulation. SHR and BNR CD4+ T cell cultures were stimulated with 10:1 DT or CT-B beads as described in Figs. 5 and 6 and in the presence or absence of exogenous recombinant cytokine. Increased cell proliferation (stimulation index) by rat IL-4 (100 U/ml) or decreased cell proliferation by rat IFN- (100 U/ml) was significant (* P < 0.05 for DT-triggered cells; ** P < 0.05 for CT-B-triggered cells) when recombinant cytokines were added during antigen stimulation. Depicted data are representative of 3 separate experiments.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

It is now established that an active immunosuppressed state exists in the adult SHR (1, 24, 29, 30, 38-40), and in this study we have shown that the SHR responded poorly to a highly immunogenic vaccine protein, e.g., DT. The cytokine environment plays a crucial role in the development of immune responses, and Th1 type cells producing IFN- were shown to be involved in the immunosuppressed SHR (29). Thus DT and CT as adjuvant were coadministered to SHR to address whether a change in the type of Th cell environment could affect their hyporesponsiveness. The present study has shown that this immunosuppressed state can be reversed by the adjuvant effect of CT with the subsequent induction of Th2-type responses.

The precise mechanisms of the immune dysfunction in SHR strain have not been well defined, although this immune deficiency has been associated with hypertension. In support of this, young SHR (4 wk of age), which do not exhibit signs of hypertension, show normal B and T cell responses to mitogenic stimulation (30). The immune hyporesponsiveness of adult SHR is not likely due to an increase in T suppressor cells because SHR do not exhibit an imbalance in their CD4-to-CD8 T cell ratios (30). We have demonstrated that the suppressive activity in adult SHR was mediated by splenic macrophages because the depletion of this population or the addition of NO synthase inhibitors to SMC cultures reversed the unresponsiveness to the T cell mitogen Con A (29, 30). Of interest, the induction of NO production by macrophages was enhanced by IFN-, and we have shown that anti-IFN- antibodies inhibited this effect by 50% (29). Thus we hypothesized that the nominal immune hyporesponsiveness observed in adult SHR was likely to be a cytokine network biased toward Th1-type cells producing IFN-.

To date, defining the Th1/Th2 paradigm in the rat has been limited to specific pathological immune disorders for Th2 cell-dependent responses, e.g., helminth infections (17, 31, 41) or mercury chloride treatment (16, 32), and for Th1 cell-dependent responses, e.g., autoimmune disorders (33, 37, 43) and allograft rejection (2, 34). This was in large part due to the limitation of reagents specific for rat cytokines. However, such reagents have recently become available (36), and we can now ascertain the mechanisms involved in Th cell-mediated immunity following vaccine and adjuvant delivery. These studies will allow a more detailed inquiry to determine the immune dysfunction that occurs in the SHR and the corrective effects of the adjuvant CT.

SHR exhibited impaired IgM and IgG antibody responses following parenteral immunization with the protein vaccine DT. This hyporesponsiveness was reflected by low CD4⁺ T cell proliferative responses to DT, which contrasted with responses of normotensive WKYR (from which SHR were derived), which exhibited normal antibody and DT-specific CD4⁺ T cell proliferative responses. Thus, in addition to the reported hyporesponsiveness to mitogenic stimulation (30), SHR exhibits diminished immune responses to highly immunogenic vaccines in vivo. Coimmunization of SHR with DT and CT as adjuvant reversed the hyporeactivity of SHR to DT and resulted in increased serum antibody titers, which reached levels identical to the anti-DT antibody titers measured in WKYR and BNR receiving the same combined vaccine. Furthermore, DT-specific proliferative responses by CD4⁺ T cells were also restored by coimmunization of SHR with DT and CT, suggesting that the restoration of immunity in SHR was mediated by T cells. Studies in mice have established that CT supports Th2-type responses with subsequent IgE antibodies and IgG1 subclass responses (10, 15, 35, 47). Evaluation of IgG subclass responses in SHR immunized with DT and CT revealed DT- and CT-B-specific antibody responses of IgG2a and IgG1, but an absence of antibodies of IgG2b or IgG2c subclasses. Past studies have suggested that the rat IgG2a and IgG1 responses are associated with Th2 cell-dependent responses analogous to murine IgG1 responses, on the basis of the elevation in antigen-specific IgG2a antibodies in parasite-infected rats (17, 31, 41). This is further supported by a marked nucleotide homology between mouse 1 and rat 2a and 1 genes (3), which again supports the notion that rat IgG2a and IgG1 antibodies are associated with Th2 cell-dependent, IL-4-supported antibody responses. However, definitive proof must await in vitro studies with the recombinant cytokines and B cell-induced switches to IgG subclasses.

To further substantiate the hypothesis that CT-induced Th2 cells can reverse the immune suppression in SHR, we found that IL-4 was induced after immunization with the combined DT and CT vaccines. In fact, IL-4 was present in culture supernatants of both SHR and BNR CD4⁺ T cells from immunized rats after in vitro restimulation with DT or CT-B. Furthermore, IL-4 levels in culture supernatants exceeded IFN- levels produced by both rat strains, supporting the notion that the adjuvant CT elicits Th2 cell-dependent responses (15, 45-47). The predominant Th2-type response induced by CT in SHR was also confirmed by the finding that minimal IFN- levels were present in culture supernatants and by the observation that treatment of CD4⁺ T cell cultures with anti-rat IL-4 antibody did not enhance IFN- production. Furthermore, as one study suggested, IFN- secretion may be in part regulated by IL-4 (16). Both anti-DT- and anti-CT-B-specific CD4⁺ T cells from BNR showed increases in IFN- and IL-4 secretion after treatment with anti-IFN- and anti-IL-4 antibodies, respectively. We observed that the addition of recombinant rat IL-4 augmented DT- and CT-B-specific CD4⁺ T cell proliferative responses in SHR and BNR. On the other hand, recombinant rat IFN- greatly suppressed these antigen-specific responses in both strains. These results suggest that a costimulation of both antigen-specific Th1 and Th2 cells occurs and are consistent with previous reports in which IL-4 stimulated Th2 cells and IFN- stimulated Th1 cells, whereas reciprocal treatment diminished T cell responses (8, 23, 36). Furthermore, the potential of IL-4 to enhance antigen-specific proliferation of CD4⁺ T cells provides additional support to the notion that CT preferentially induced Th2 cell subsets. Thus, as observed in murine studies (15, 45-47), SHR and normotensive rats coimmunized with CT preferentially develop Th2-type cells in response to DT and to CT-B. However, the development of Th2-type responses may be different in rats because, after our immunization scheme, we did not observe the transient serum IgE antibody responses reported in mice (15, 35).

In summary, coimmunization with the protein vaccine DT and the adjuvant CT resulted in reversal of immune suppression in the SHR and both restoration of antigen-specific CD4⁺ T cell proliferation and elevated antibody responses to DT. In terms of cytokine production, coimmunization with CT clearly promoted Th2-type cells producing IL-4. Studies are currently underway to determine the precise mechanisms involved in the antigen-specific immune dysfunction in the SHR in the absence of adjuvant promoting Th2-type responses.

Perspectives

From this study, it is clear that the SHR exhibited impaired immunity to the highly immunogenic antigen DT. To mount an immune response to DT, the addition of the adjuvant, CT, reversed the immune suppression to DT, and the SHR became responsive and mounted a CD4⁺ Th2 (IL-4)-dependent immune response. Thus the SHR has the capabilities to mount a normal immune response if appropriately stimulated and to reverse the SHR's tendency to elevated IFN- production, which in turn stimulates elevated levels of NO, which suppresses immunity. It is still unclear why the SHR has the propensity for generating elevated levels of IFN- when compared with the normotensive strains WKYR and BNR. What remains to be determined are the molecules (or mode of communication) exchanged between the vascular and immune systems to resolve the issue of whether the observed immune suppression obtained with the SHR is directly linked to its hypertension. Current studies are in progress evaluating what is the juncture between hypertension and immune suppression.