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Morphine Inhibits Mucosal Antibody Responses and TGF- mRNA in Gut-Associated Lymphoid Tissue Following Oral Cholera Toxin in Mice¹

Xiaohui Peng^*, John J. Cebra, Martin W. Adler^,, Joseph J. Meissler, Jr.^*, Alan Cowan^,, Pu Feng^* and Toby K. Eisenstein^2,*,

Departments of ^* Microbiology and Immunology and Pharmacology, and Center for Substance Abuse Research, Temple University School of Medicine, and Department of Biology, University of Pennsylvania, Philadelphia, PA 19104

	Abstract

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In this study, we investigated the effect of morphine on the mucosal immune system using fragment cultures of ileal segments, Peyer’s patches (PPs), and mesenteric lymph nodes. Mice were implanted s.c. with a morphine slow release pellet. Control groups received a naltrexone slow release pellet, a placebo pellet, or both a morphine and a naltrexone pellet. After 48 h, mice were orally immunized with cholera toxin (CT) and were boosted orally 1 wk later. Animals were sacrificed 1 wk after the booster immunization, and PPs, mesenteric lymph nodes, and ileal segments were cultured in 24-well plates for 12 days. Morphine resulted in a highly significant inhibition of CT-specific IgA and IgG production in fragment culture supernatants of all three tissues compared with placebo. Naltrexone blocked the reduction in Ab levels induced by morphine, indicating that the effect is opioid receptor mediated. Morphine did not significantly alter total IgA levels in any of the tissue culture supernatants. Morphine also inhibited CT-specific IgA and IgG levels in serum. By flow cytometry, morphine did not alter the lymphoid cell composition in PPs compared with placebo. The effect of morphine on TGF-, IL-5, and IL-6 mRNA expression in PPs and ileal segments was determined following oral immunization with CT. Morphine significantly decreased TGF- mRNA compared with that in the placebo group, and naltrexone blocked this effect. These results indicate that morphine inhibits Ag-specific IgA responses in gut-associated lymphoid tissue at least partially through the inhibition of TGF-, a putative IgA switch factor, in the gastrointestinal tract.

	Introduction

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Morphine has been shown to suppress NK cell activity (1, 2, 3), phagocytosis of Candida albicans by macrophages (4, 5), T cell proliferative responses to mitogens (6, 7, 8), and Ab production both in vivo and in vitro (9, 10, 11, 12). Morphine has also been shown to potentiate infections in mice, such as Salmonella (13) and toxoplasmosis (14), and to increase the replication of HIV in human cells infected in vitro (15). Many of the Ags encountered by the normal immune system gain access to the body via a mucosal surface. Protection of the mucosal surfaces is mediated in large part by local production of secretory IgA Abs (16). However, there are only two studies reported in the literature examining the modulatory effect of morphine on mucosal immune responses in vivo (17, 18). Dinari et al. (18) reported that morphine injection inhibited IgA responses to cholera toxin (CT)³ in gastrointestinal lavage fluids of mice. We have extended the observations of Dinari and shown that morphine administrated by slow release pellet in vivo inhibits subsequent CT-specific IgA responses by Peyer’s patches (PPs), mesenteric lymph nodes (MLNs), and ileal segments (ISs) placed ex vivo in organ culture. A potential mechanism for morphine-mediated inhibition of IgA responses was shown to be inhibition of TGF- mRNA.

	Materials and Methods

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Mice

Female, specific pathogen-free, C3HeB/FeJ, 6-wk-old mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and mouse chow and water were provided ad libitum. All mice were acclimatized for a minimum of 1 wk before experimentation.

Drug treatment, oral immunization, and fragment cultures

Mice were anesthetized with isofluorane and implanted s.c. with a 75-mg morphine slow release pellet. Control groups received a 30-mg naltrexone pellet, a placebo pellet, or both a morphine and a naltrexone pellet. Naltrexone is a morphine antagonist. (This method of continuous administration of morphine to prevent episodes of withdrawal is common (6, 19).) All pellets were obtained from the National Institute on Drug Abuse (Rockville, MD). After 48 h, mice were deprived of food for 2 h and then given 0.25 ml of a solution containing eight parts HBSS and two parts 7.5% sodium bicarbonate by gastric intubation to neutralize stomach acidity. After 30 min, mice were orally immunized by intubation with CT (10 µg in 0.25 ml; List Biological Laboratories, Campbell, CA) in PBS. The mice, under anesthesia, were boosted orally 1 wk later. Mice were sacrificed 1 wk after the booster immunization, and PPs, MLNs, and ISs were harvested, washed, and cultured in 24-well plates in 1.0 ml of Kennett’s complete medium for 12 days in an atmosphere of 90% O₂ and 10% CO₂ at 37°C (20). Tissues from three or four mice per treatment were used in separate cultures in each experiment. Individual culture supernatants were harvested and frozen at -70°C before assay for Ab production by ELISA.

ELISA for CT-specific and total Ab

Each well of 96-well ELISA plates was coated overnight at 4°C with CT (1 µg/ml). Plates were washed three times with PBS supplemented with 0.5% Tween 20 and blocked with 3% BSA in PBS for 1 h at room temperature. Following three washes with PBS/Tween 20, serial 2-fold dilutions of culture supernatants (100 µl/well) were added to sample wells and incubated for 4 h at room temperature. Plates were washed three times with PBS/Tween 20 and were incubated overnight at 4°C with 100 µl of a 1/500 dilution of alkaline phosphatase-labeled goat Ab specific for mouse IgA, IgG, or IgMµ (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Following a thorough washing, p-nitrophenyl phosphate was added, and the plates were incubated at room temperature for 60 min. OD at 405 nm was determined using an automated microplate reader. For total Ab, plates was coated overnight at 4°C with goat anti-mouse IgA (Southern Biotechnology Associates, Birmingham, AL). Fifty microliters of undiluted culture supernatants was added to the sample wells, and the amount of total IgA was assayed using the developing anti-mouse IgA Ab described above. Total IgA concentrations were calculated from a standard curve made by using different concentrations of purified mouse IgA (Southern Biotechnology Associates).

Flow cytometry

Mice were implanted s.c. with a morphine pellet, a naltrexone pellet, a placebo pellet, or both a morphine pellet and a naltrexone pellet. Forty-eight hours later, mice were given CT (10 µg/ml) orally, then a booster 7 days later. Seven days after the booster, mice were sacrificed, and PPs were harvested. Cells from PPs (1 x 10⁶/ml) were stained with either PE-labeled Abs specific for mouse CD3 and CD11b or FITC-labeled Ab specific for mouse surface Ig (BD PharMingen, San Diego, CA). Cells were incubated with Abs for 30 min at 4°C in the dark. Following two washes with PBS, cells were fixed with 1% paraformaldehyde and analyzed with a FACScan. Two cohorts in each group were analyzed.

RT-PCR

Mice were implanted with a morphine pellet, a naltrexone pellet, a placebo pellet, or both a morphine and a naltrexone pellet. Forty-eight hours later, mice were given CT (10 µg/ml) orally, then a booster 7 days later. Seven days after the booster, mice were sacrificed, and PPs and ISs were harvested. Total RNA was extracted using RNAzol B according to the manufacturer’s instructions (Tel-Test, Friendswood, TX). RNA (1–3 µg) was reverse transcribed using Superscript II RT (Life Technologies, Gaithersburg, MD) and random hexamer primers (Promega, Madison, WI). The cDNA samples were then subjected to PCR analysis. Primers for hypoxanthine phosphoribosyl transferase, TGF-, IL-5, and IL-6 were purchased from Stratagene (La Jolla, CA). cDNA was amplified for 35 cycles (94°C for 40 s, 60°C for 20 s, 72°C for 40 s, and a final extension at 72°C for 10 min) using Taq polymerase (Roche, Indianapolis, IN). PCR products were analyzed by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. PCR blots were densitometrically scanned and quantified using NIH Image software.

Statistics

Statistical analysis was conducted using ANOVA following a rank transformation due to nonnormality, followed by Dunnett’s test vs control. A p < 0.05 was considered to be significant.

	Results

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Morphine inhibits IgA and IgG responses to CT in gut-associated lymphoid tissue (GALT)

The effect of morphine on mucosal Ab production using fragment cultures of ISs, PPs, or MLNs was investigated. Mice were anesthetized and implanted s.c. with a 75-mg morphine slow release pellet. Control groups received a 30-mg naltrexone pellet, a placebo pellet, or both a morphine and a naltrexone pellet. After 48 h, mice were orally immunized with CT (10 µg/mouse), then were boosted 1 wk later. Mice were sacrificed 1 wk after the booster immunization, and PPs, MLNs, and ISs were harvested and cultured in 24-well plates for 12 days. Culture supernatants were harvested and assayed for Ag-specific Ab production by ELISA. As shown in Fig. 1, IgA (Fig. 1A) and IgG (Fig. 1B) Abs specific for CT were detected in PP, MLN, and IS fragment culture supernatants taken from placebo- and naltrexone-pelleted mice. However, morphine dramatically inhibited CT-specific IgA and IgG production in PPs, MLNs, and ISs compared with placebo. Naltrexone, the morphine antagonist, blocked the reduction in Ab levels induced by morphine, indicating that the effect is opioid receptor mediated. CT-specific IgM was not detected in any of the tissue culture supernatants.

View larger version (49K): [in this window] [in a new window]   FIGURE 1. The effect of morphine on CT-specific IgA and CT-specific IgG production in ISs, MLNs, and PPs. Mice were implanted s.c. with morphine, naltrexone, placebo, or morphine plus naltrexone pellets and were orally immunized and boosted 1 wk apart with CT (10 µg/mouse). Supernatants of organ cultures of ISs, MLNs, and PPs were assayed for Ab production 12 days later by ELISA. A, IgA-specific Ab responses to CT; B, IgG-specific Ab responses to CT. Results are expressed as the mean ± SD. Tissues from three mice given morphine or four mice in other groups were cultured individually. *, p > 0.05; **, p > 0.005. This experiment was conducted three additional times, and similar results were obtained.

View larger version (32K): [in this window] [in a new window]   FIGURE 2. The effect of morphine on total IgA responses. Culture supernatants obtained as described in Fig. 1. were assayed for total IgA production by ELISA. Results are expressed as the mean ± SD. Tissues from three mice given morphine or four mice in other groups were cultured individually. This experiment was conducted three additional times with similar results.

The capacity of morphine to affect systemic immune responses after oral CT immunization was investigated by measuring CT-specific Ab isotypes in serum. Sera of individual mice were collected 7 days after the booster immunization and assayed for Ag-specific Ab production by ELISA. IgA, IgM, and IgG specific for CT were detected in placebo- and naltrexone-treated groups (Fig. 3). Morphine significantly decreased CT-specific IgA and IgG levels in serum, and naltrexone blocked the suppression. Morphine did not significantly alter CT-specific IgM levels.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. The effect of morphine on CT-specific Ab production in serum. Groups of animals were treated as described in Fig. 1. Serum was collected 1 wk after the booster immunization and was frozen at -70°C before assay for CT-specific Ab production by ELISA. Individual sera were titrated, with three mice in the morphine group and four mice in each of the other groups. Results are expressed as the mean ± SD. **, p > 0.005.

Flow cytometry was used to determine whether morphine inhibits CT-specific Ab production by reducing B cell numbers in PPs. Cells were taken from the PPs of mice treated with morphine and immunized with CT as described in the experiments in Figs. 1–3. As shown in Table I, morphine did not significantly alter the percentage of B cells, T cells, or macrophages in PPs compared with those in placebo-treated animals or other control groups.

Morphine inhibits TGF- mRNA expression in GALT

Cytokines such as TGF-, IL-5, and IL-6 are important in IgA production (21, 22, 23, 24, 25). To address the mechanisms of IgA inhibition by morphine, we investigated whether morphine inhibits cytokine responses in GALT. Experiments were conducted to determine the effect of morphine on TGF-, IL-5, and IL-6 mRNA expression in PPs and ISs following oral immunization with CT. Total RNA was extracted from PPs, ISs, and MLNs, and RT-PCR was conducted using specific primers for TGF-, IL-5, and IL-6. As shown in Fig. 4A, TGF- mRNA was detected in placebo and naltrexone groups in the PPs and MLNs. Interestingly, morphine dramatically decreased TGF- mRNA compared with that in the placebo group. The specificity of the morphine effect was tested using the opioid receptor antagonist naltrexone. Naltrexone blocked the inhibition of TGF- mRNA by morphine, indicating that these effects are mediated by classical opioid receptors. Fig. 4B shows the pooled densitometry data for TGF- in three experiments. Neither IL-5 nor IL-6 mRNA was detected in any of the tissues examined, nor was IL-4, IL-10, or IFN- mRNA detected. In the three experiments, only faint TGF- mRNA bands were detected in any of the groups in the MLNs (data not shown).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. The effect of morphine on TGF- mRNA expression in ISs and PPs. Groups of animals were treated as described in Fig. 1. ISs and PPs were harvested 1 wk after booster immunization. Total RNA was isolated and reverse transcribed to cDNA. A, cDNA was used as a template for PCR using primers specific for TGF-, IL-5, and IL-6. The PCR products were analyzed on 2% agarose gels and visualized by ethidium bromide staining. Results of a representative experiment are shown. M, morphine; M+N, morphine plus naltrexone; N, naltrexone; P, placebo; HPRT, hypoxanthine phosphoribosyl transferase. B, PCR blots for TGF- were densitometrically scanned and quantified using NIH Image software. Results are expressed as the mean ± SD of blots from three separate experiments. **, p > 0.005.

	Discussion

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Opioids have profound antidiarrheal and constipating actions. Opioid receptors present in the circular muscle and submucosal plexus of the intestine are classically thought to mediate the inhibition of intestinal motility (26, 27, 28). The discovery of opiate binding sites in the mucosa has added another dimension to our understanding of how opioids influence gut stasis. Opiate binding sites in the mucosa have been localized to the basal portion of villous and crypt cells of the intestinal epithelium (29). Three different opioid receptors, µ, , and , have been cloned (30, 31, 32, 33, 34). Both the µ opioid receptor (MOR) and the opioid receptor subtypes have been shown to mediate opioid-induced decreases in gastrointestinal transit time in mice (35, 36). The role of MOR in gastrointestinal transit was extensively investigated using MOR knockout mice (37) and by administration of specific MOR Abs or antisense oligodeoxynucleotides to MOR (38). Both approaches showed that MORs are major mediators of intestinal motility. A agonist has also been shown to delay gastrointestinal transit time in the guinea pig (39).

Despite the knowledge that opioids profoundly affect gut physiology, there is very limited literature on the effect of opioids on the secretory immune system in the intestinal tract. GALT comprises the PPs, MLNs, and large numbers of lymphoid cells scattered throughout the lamina propria and epithelium of the intestine. Secretory IgA is thought to play a major role in protection against pathogens in the mucosa. The lamina propria and the epithelial layer of the small intestine have been studied as major sites where IgA is produced and transported (40). Our data show that large amounts of IgA are produced in ISs in culture from the placebo group, which was orally inoculated with CT. However, morphine dramatically suppressed CT-specific IgA levels, and naltrexone blocked the inhibition. Moreover, morphine apparently suppressed Ag-specific Ab production by inhibiting B cell function, not by reducing B cell number, at least in PPs. These results are consonant with those of Carr et al. (17) and Dinari et al. (18). Both groups found that opioids inhibit IgA responses in the gastrointestinal tract. Carr et al. (17) reported that -endorphin suppressed Con A-stimulated IgA, IgG, and IgM production by PPs. We have extended the work of Dinari et al. (18) and shown that Ag-specific IgA secretion is diminished in three major types of tissues associated with GALT: the PPs, MLNs, and isolated ISs containing lamina propria. Our use of organ culture definitely establishes that the IgA measured originates in the GALT. Decreased IgA responses were evident even though no morphine was added to the medium used for the organ cultures, indicating that the inhibiting effect of the drug possibly prevents generation of Ag-specific IgA-secreting cells, rather than blocks the capacity of plasma cells to secrete IgA. This hypothesis is supported by our finding that morphine decreases the expression of TGF- mRNA in PPs. The observation that the anti-cholera IgA responses can be reduced without affecting the total amount of IgA probably reflects the fact that the anti-cholera Abs represent only a small fraction of total IgA.

Ig isotype switching by B cells is a biologically important feature of the humoral immune response. Cytokines such as TGF-, IL-5, and IL-6 play an important role in IgA responses. TGF- induces surface IgM⁺ B cells to switch to sIgA expression (21, 41, 42). IL-5 and IL-6 have been shown to be the cytokines that are most effective for induction of IgA synthesis (22, 23, 24, 25). To study the mechanisms by which morphine suppresses Ag-specific IgA responses, experiments were carried out to examine whether morphine influences cytokine responses in the gastrointestinal tract. Our data show that morphine significantly suppresses TGF- mRNA expression in PPs and ISs. In a preliminary experiment TGF- levels were reduced 42.5% in PP cells taken from morphine-treated mice and cultured in single-cell suspension compared with levels in cells from the three control groups, as determined by ELISA. A decrease in the availability of TGF- may account for the suppression of Ag-driven IgA by morphine.

Our findings were unexpected, in that opioids have been linked to increased production of TGF-. Thus, Murtaugh et al. (43) reported that antiserum to TGF- blocked suppression of the respiratory burst induced by methadone when it was added in vitro to porcine PBMCs, implying that TGF- release was responsible for the opioid-mediated down-regulation of macrophage function. Also, Chao et al. (44) showed that morphine potentiated TGF- release from human PBMC stimulated with LPS and IFN-. These observations fit with the general concept that TGF- is anti-inflammatory (45) and immunosuppressive. However, in the gastrointestinal tract TGF- has pleiotropic effects. It is produced by cells designated Th3, which are thought to mediate both the switch to IgA production as well as oral tolerance (46). At present we cannot reconcile our observations showing down-regulation of TGF- in the GALT following morphine treatment with the studies by Murtaugh (43) and Chao (44) showing that opioids increased TGF- production in PBMCs. There may be complex regulatory pathways engaged in our paradigm of in vivo morphine administration that will require additional experimental approaches to dissect. Our data do present an internally consistent picture, in that TGF- suppression correlates with suppression of induction of IgA Ab to a specific Ag (CT) presented via the oral route.

A large body of evidence shows that drug abusers have an increased incidence of infections (47, 48, 49). Opioids have been shown experimentally to sensitize to infectious agents that enter the body via the gastrointestinal tract, such as Salmonella (13, 50) and Shigella (51), and to induce sepsis (52). Although there is no direct evidence that opioids enhance HIV infection via the intestinal mucosa, it has been shown that opioids increase the replication of HIV and viral expression in human cells infected in vitro (15, 53). HIV is known to infect M cells (54) in addition to other cells in the human intestinal tract (55). The present studies raise the possibility that the immunosuppressive activity of opioids in the gastrointestinal tract may contribute to HIV pathogenesis via the intestinal route. A further implication of our data is that oral vaccines might be less effective in heroin addicts.

	Acknowledgments

 
We thank Dr. Han Qing Jiang for help with the technique of tissue fragment cultures and Dr. John P. Gaughan for statistical analysis.

	Footnotes

 
¹ This work was supported by National Institute of Drug Abuse Grants DA11134 and DA06650.

² Address correspondence and reprint requests to Dr. Toby K. Eisenstein, Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. E-mail address: tke{at}astro.ocis.temple.edu

³ Abbreviations used in this paper: CT, cholera toxin; IS, ileal segment; GALT, gut-associated lymphoid tissue; PP, Peyer’s patch; MLN, mesenteric lymph node; MOR, µ opioid receptor.

Received for publication November 14, 2000. Accepted for publication July 19, 2001.

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