Short communication
Acute effect of ethanol on IgA immunoreactive cells in the intestine-associated immune system
Mirela Budeè, Vera Todoroviæ, Neda Drndareviæ
Institute for Medical Research, Dr Subotiæa 4, PO B0X 102, 11129 Belgrade, Serbia &Montenegro Correspondence:Mirela Budeè, e-mail: mirelab@imi.bg.ac.yu
Abstract:
The purpose of this study was to investigate the acute effect of ethanol on mucosa-associated lymphoid tissue at the level of Peyer’s patches and the intestinal lamina propria in female rats and to determine whether this action of ethanol is modulated during the estrous cycle. Adult female rats showing proestrus or diestrus day 1 were treated intraperitoneally (ip) with ethanol (4 g/kg).
Untreated and saline-injected rats were used as controls. The animals were sacrificed by decapitation 0.5 h after ethanol administration. Immunoglobulin A (IgA) immunoreactive cells were analyzed by indirect immunohistochemistry using mouse anti-rat IgA and a Dako LSAB+ kit. The number of IgA-immunoreactive cells in Peyer’s patches was unaltered by ethanol treatment at both phases of the estrous cycle. However, stereological analysis revealed a significant increase in the number of IgA-immunoreactive cells (p < 0.01) in the intestinal lamina propria following acute ethanol administration at proestrus and on diestrus day 1. The results indicate that the intestinal lamina propria, the effector site of the mucosal immune system, can be affected by a single dose of ethanol at both phases of the estrous cycle.
Key words:
ethanol, IgA, Peyer’s patches, intestinal lamina propria, estrous cycle
Introduction
Clinical observations in humans and experimental studies in animal models have shown that alcohol, the most commonly abused substance, modulates both in- nate and acquired immunity [17, 21, 32]. Ethanol- induced changes include loss of lymphoid cells from the thymus, spleen, peripheral blood [16], lymph nodes [28], inhibition of accessory cell function of monocytes and myeloid dendritic cells [33] and of T-cell proliferation [6], suppression of primary antibody response [15] and splenic natural killer activity [36], as well as interference with cytokine networks [7].
It has also been reported that ethanol consumption can affect gut-associated lymphoid tissue (GALT) in
mice [18, 19, 29]. Whereas effects of chronic ethanol consumption have been studied by many investiga- tors, less is known about the acute immunomodula- tory effects of ethanol.
Previously, we have demonstrated that a single dose of ethanol affects the rat thymus [3, 4], spleen [5] and granulocytopoiesis [31]. To the best of our knowledge there are no earlier reports on the effect of acute ethanol treatment on the mucosal immune sys- tem. Therefore, we considered that it would be inter- esting to determine whether a single dose of ethanol could alter the mucosa-associated lymphoid tissue at the level of Peyer’s patches and the intestinal lamina propria in female rats. Bearing in mind gender differ- ences in the pharmacokinetics of ethanol in humans
Pharmacological Reports 2005, 57, 385`389 ISSN 1734-1140
Copyright © 2005 by Institute of Pharmacology Polish Academy of Sciences
fects of ethanol in rats [23, 31], we tested females at two phases of the estrous cycle, proestrus and diestrus day 1.
Materials and Methods
Experimental procedure
The experiments were conducted on female Wistar rats (obtained from the Breeding Colony of the Medi- cal Military Academy, Belgrade) weighing 220–300 g.
They were maintained in an environment of constant temperature, humidity and light (from 6 a.m. to 6 p.m.).
Chow and water were freely available. The estrous cycle was monitored every day by vaginal smear ex- amination. Rats showing proestrus or diestrus day 1 were treated ip with ethanol at 4 g/kg between 9.00 and 9.30 a.m. Untreated and saline-injected rats were used as control groups. The groups consisted of four to six rats. The animals were sacrificed by decapita- tion 0.5 h after the treatment. This protocol was ap- proved by our Animal Use Committee.
Blood ethanol determination
Ethanol concentrations were determined using the Sigma diagnostic procedure (No. 322-UV, Sigma Chemical Co., St. Louis, Mo) 0.5 h after administra- tion.
Immunohistochemistry
After sacrifice, five successive Peyer’s patches of the small intestine were excised beginning from the ce- cum, fixed in Bouin’s solution and embedded in par- affin. Immunohistochemistry was performed on 6mm thick paraffin sections. After deparaffinization and dehydration, the endogenous peroxidase was inacti- vated by incubation with 0.3% hydrogen peroxide in methanol. Following further dehydration and washes in phosphate-buffered saline (PBS), the slides were incubated for 10 min with 10% normal goat serum in a humid chamber at room temperature. Monoclonal mouse anti-rat IgA antibody (ICN Pharmaceuticals)
a biotinylated secondary antibody reacts with several peroxidase-conjugated streptavidin molecules was employed for amplification using a DAKO LSAB+/HRP kit. Diaminobenzidine tetrahydrochloride (DAB) was used for the visualization of immunoreactive cells. Fi- nally, the nuclei were counterstained with Mayer’s he- matoxylin.
Stereological analysis
Surface area (At) of the intestinal lamina propria and Peyer’s patches was estimated at an objective magni- fication of × 25 under a Galen III (Cambridge Instru- ments) microscope using the multipurpose test system M 168 [34] and calculated according to the equation:
At= Pt31/2/2 d2
where Pt= number of points falling on the tissue com- partment and d = length between two test points. The immunoreactive cells were counted at an objective magnification of 40× and the number of IgA+ cells was expressed per area unit of Peyer’s patches and the intestinal lamina propria.
Statistical analysis
Data are expressed as means ± standard error of the mean. Student’s t-test was used for comparison of the mean values between two groups.
Results
The blood ethanol levels were similar at proestrus (34 ± 0.71 mmol/l) and diestrus day 1 (31.11 ± 2.54 mmol/l) 0.5 h after the administration fulfilling the criterion for intoxication (> 21.7 mmol/l).
Histological examination revealed that acute etha- nol treatment produced marked local mucosal hypere- mia and edema in the rat small intestine. Analysis of the tissue specimens prepared from animals at proes- trus displayed the same histological pattern as the ani- mals at diestrus day 1.
To test the effect of acute ethanol treatment on mucosa-associated lymphoid tissue, the number of
IgA-immunoreactive cells was determined in Peyer’s patches and the intestinal lamina propria at both phases of the estrous cycle. In the control untreated rats, the distribution pattern of IgA-immunoreactive cells showed that they were numerous in the follicles
of Peyer’s patches. Those cells exhibited surface im- munoreactivity. In addition, IgA positive cells were also observed in the subepithelial area as well as in the mantle-zone and interfollicular area. In those ar- eas, IgA positive cells expressed cytoplasmatic immu- noreactivity. In the alcohol-treated rats, the staining pattern of Peyer’s patches for IgA positive cells was similar to that in control rats.
Stereological analysis revealed no significant dif- ference in the number of B cells in Peyer’s patches that expressed IgA between control and ethanol- treated rats at proestrus and diestrus day 1 (Fig. 1).
Unlike Peyer’s patches, a significant increase in the number of IgA-immunoreactive cells (p < 0.01) was found in the intestinal lamina propria following acute ethanol treatment compared with the control groups (Fig. 2). This effect of ethanol was similar at both phases of the estrous cycle. To determine the effect of the intraperitoneal injection, rats treated with saline in the same volume as for ethanol, were also used as controls, but the numbers of IgA-immunoreactive cells were similar to those observed in untreated rats at proestrus and day 1 of diestrus.
Discussion
In the present study, the effect of a single dose of etha- nol on the rat GALT was evaluated. Intraperitoneal administration of alcohol was chosen on the basis of our previous work [3, 4, 5, 31]. At autopsy, we exam- ined Peyer’s patches at different time points: 0.5, 3, 6 and 20 h after ethanol administration. As the changes in Peyer’s patches were most prominent 0.5 h following ethanol treatment, it seemed reasonable to use this time point for the morphometric study. Female rats were used because they are more susceptible to im- munomodulatory effects of ethanol than males [13].
We have shown previously that estrous cycle can modulate the actions of ethanol on a granulocytic cell line [31] and adrenal cortex [23].
Bearing in mind these results, rats showing proes- trus or diestrus day 1, i.e. phases of the estrous cycle characterized by different levels of sex hormones, were treated with ethanol. Furthermore, this experi- mental approach was also initiated because of the well-known interactions of gonadal hormones with the immune system [12, 25].
Ethanol and IgA
Mirela Budeè et al.
0 20 40 60 80 100 120 140 160 180 200
diestrus day 1 NumberofIgA-ircells/mm2 ofPeyer'spatches
proestrus
ethanol saline controls
Fig. 1.Number of IgA-immunoreactive (ir) cells at proestrus and di- estrus day 1 in Peyers patches in ethanol-, saline-treated and control rats. Values are the mean ± SE
0 200 400 600 800 1000 1200 1400
1600 **
**
diestrus day 1 NumberofIgA-ircells/mm2 oflaminapropria
proestrus
ethanol saline controls
Fig. 2.Number of IgA-immunoreactive (ir) cells at proestrus and diestrus day 1 in intestinal lamina propria in ethanol-, saline-treated and control rats. Values are the mean ± SE. Significantly different from corresponding control rats, ** p < 0.01
the mucous membrane surface [2]. Thus, IgA- immunoreactive cells in Peyer’s patches and the intes- tinal lamina propria were analyzed following acute ethanol treatment. Cell-migration studies in rats con- firmed the importance of Peyer’s patches as sites of generation of antigen-specific IgA precursors which then migrate to the gut lamina propria [1, 10] where they undergo terminal differentiation into plasma cells. Although a majority of data still suggest that Peyer’s patches are the inductive sites of mucosal im- munity and lamina propria is the effector site, these functional distinctions are not absolute [20, 35] because class switching and differentiation to IgA-producing cells can occur in the gut lamina propria in mice [9].
Our results revealed that a single, hypnotic dose of ethanol (4 g/kg, ip) induced no significant changes in the number of B cells that express IgA in Peyer’s patches at both phases of the estrous cycle. However, the number of IgA-immunoreactive cells was signifi- cantly increased during ethanol intoxication at the level of the intestinal lamina propria, at the proestrus as well as on diestrus day 1. The levels of estradiol and progesterone following acute ethanol treatment at proestrus and diestrus day 1 were reported previously using the same experimental model [31]. No signifi- cant difference was found in the number of IgA- immunoreactive cells between proestrus and diestrus day 1 in our study. This confirms the results of McDermott et al. [22] who reported that the intestinal localization of the precursors of IgA plasma cells was unaltered during the estrous cycle. In addition, phases of the estrous cycle have a little or no influence on the mucosal and systemic specific antibody response in- duced after vaginal and intraperitoneal immunization with protoxin Cry1Ac from Bacillus thuringiensis in mice [24]. Our findings cannot be directly compared to others because there are no studies on the acute ef- fect of ethanol on the mucosal immune system.
Using flow cytometry, it has been shown that chronic ethanol consumption significantly decreased the number of CD8+ and IgA+ cells in the intestinal lamina propria in female mice [18]. Further studies demonstrated a significant decrease in the total number of cells and in the absolute number of T and B cells in Peyer’s patches after chronic ethanol treat- ment [19]. Morever, it has been reported that ethanol feeding resulted in profound loss of GALT lymphoid
that chronic ethanol ingestion reduced cellular and hu- moral components of the GALT in male rats, possibly by cell loss as a result of ethanol-induced apoptosis [30].
Excessive consumption of ethanol can interfere with the IgA system at various levels. High levels of serum secretory IgA were found in patients with se- vere alcoholic cirrhosis of the liver [26]. Elevated lev- els of secretory IgA in serum that were reversible after ethanol withdrawal were also demonstrated in pa- tients with non-cirrhotic alcoholic liver disease. Se- rum secretory IgA levels were as discriminative as gamma-glutamyl transferase activity in distinguishing between chronic alcoholic patients without cirrhosis and non-alcoholic subjects [27]. In addition, alcohol- induced liver disease is also associated with the depo- sition of IgA in the kidney glomeruli, superficial cap- illaries of the skin and along hepatic sinusoidal mar- gins [2]. In vitro exposure to either 1% or 5% ethanol led to impairment of IgA transcytosis [8].
Although the mechanisms underlying the increased number of IgA-immunoreactive cells in the intestinal lamina propria in this study are not known, these re- sults clearly demonstrate that a single dose of ethanol can modulate the effector site of the mucosa- associated immune system at proestrus and diestrus day 1. Further studies in this field are necessary to elucidate this effect of ethanol.
Acknowledgments:
This work was supported by a grant from the Ministry of Science, Development and Technology of Serbia. We are very grateful to Mrs. Leposava Jovanoviæ and Mrs. Sneana Markoviæ for their excellent technical assistance.
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Received:
December 3, 2004; in revised form: March 15, 2005.
Ethanol and IgA
Mirela Budeè et al.
