Blackwell Science, LtdOxford, UKCEIClinical and Experimental Immunology0009-9104Blackwell Publishing Ltd, 2002 130 Original Article
Clin Exp Immunol 2002; 130:404–408
J. L. Bjersing et al. Ileal T cells and IgA B cells during vitamin A deficiency
Loss of ileal IgA+ plasma cells and of CD4+ lymphocytes in ileal Peyer’s patches of vitamin A deficient rats J. L. BJERSING*, E. TELEMO*, U. DAHLGREN* & L. Å. HANSON† *Department of Rheumatology and Inflammation Research, and †Department of Clinical Immunology, Göteborg University, Gothenburg, Sweden
(Accepted for publication 5 September 2002)
SUMMARY Child mortality in diarrhoeal disease is increased significantly by vitamin A deficiency in poor countries. The pathological mechanisms are not known in detail. However, in this paper we report that vitamin Adeficient Wistar rats had much reduced IgA + plasma cells in the ileal lamina propria (eightfold reduction from 470 cells/mm2, P = 0·009), as well as a prominent reduction of CD4 + cells in the parafollicular regions of ileal Peyer’s patches (reduction from 7200 to 105 cells/mm 2, P = 0·009). IL-2Ralpha-chain (CD25) positive lymphocytes in the ileal Peyer’s patches were also reduced significantly in vitamin A deficiency (from 1400 to 300 cells/mm2, P = 0·009). The density of CD8 cells tended to be increased relative to the control animals (from 5100 to 6000 cells/mm 2, not statistically significant). In conclusion, the marked decrease of lamina propria IgA + plasma cells may be one cause of the high diarrhoeal mortality in vitamin A deficiency. This, in turn, appears to be related to reduced numbers of activated or regulatory CD4+ T cells in Peyer’s patches. Keywords immunohistochemistry vitamin A deficiency
mucosal immunology
INTRODUCTION Vitamin A deficiency (VAD) is one of the main nutritional deficiencies in low-income countries and is associated with increased incidence of infectious diseases [1]. A major cause of increased child death due to VAD is increased mortality in diarrhoeal disease, as shown in several community studies [2–5]. Thus, supplementation with vitamin A has been shown to decrease child mortality rates strongly, with the most clear-cut effects on mortality in diarrhoea. The degree and extent of bacterial colonization of the small intestine in rats has been shown to be increased by VAD. Normal animals were colonized only in the ileum, while VAD rats were colonized in all parts of the small intestine. Penetration of gut bacteria to lymph nodes and blood as well as mortality in septicaemia was increased by VAD [6]. Intestinal IgA can neutralize bacteria, toxins and viruses in the gut lumen and may also act intracellularly [7]. VAD reduced the levels of serum and biliary IgA [8]. Mucosal IgA producing B cells responding to T celldependent antigens, such as bacterial exotoxins, are activated by T helper cells in the Peyer’s patches, go through an IgA switch, differentiation and clonal expansion. Class switching and Correspondence: J. L. Bjersing, Guldhedsgatan 10 A, 413 46 Göteborg, Sweden. E-mail:
[email protected]
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Peyer’s patches
small intestine
maturation into IgA producing plasma cells has been suggested recently to occur locally in the gut [9]. However, the B cells usually will migrate out from the Peyer’s patches via lymphatic vessels to the mesenteric lymph nodes, mature to plasma cells and finally home back to the intestinal lamina propria via the blood [10]. However, it is not clear by what mechanism VAD can affect intestinal IgA levels and contribute to an increased susceptibility to intestinal infections. Therefore, we wanted to investigate the effects of VAD on IgA+ plasma cells in the ileum of rats. Furthermore, we asked if changes of IgA+ cells and biliary IgA by VAD was caused by alterations in the T cell population of ileal Peyer’s patches. We have thus compared the ileal immunohistochemistry of vitamin A replete rats with rats on a VAD diet.
MATERIALS AND METHODS Animals Weanling male Wistar Furth rats (B&K Universal, Stockholm, Sweden) were fed a vitamin A-deficient or vitamin A-replete diet (Analycen, Lidköping, Sweden). The vitamin A-deficient pellets consisted of 24% casein de-vitaminized by heating at 105 ∞C for 5 days, 61% corn starch, 4% cellulose flour, a 1% mixture of all necessary vitamins except vitamin A, a 4% salt mixture and 6% peanut oil. The vitamin A-replete diet was identical except for the presence of 17 IU/g of vitamin A. All animals appeared to be healthy before sacrifice. © 2002 Blackwell Publishing Ltd
Ileal T cells and IgA B cells during vitamin A deficiency After sacrifice, 5 mm-long sections of ileum containing Peyer’s patches were embedded in OCT-medium (Sakura Finetechnical Co., Tokyo, Japan) and frozen in isopentane which had been precooled in liquid nitrogen. The tissue blocks were stored at - 70∞C prior to cutting into six mm-thick sections. The experimental work was approved by the local ethics committee for animal research.
Immunohistochemistry Sections were cut and then fixed in cold 50% acetone for 30 s in cold 100% acetone for 5 min and air-dried for at least 10 min. Depletion of endogenous peroxidase activity was performed with a PBS solution containing glucose, glucose oxidase and sodium azide. The sections were incubated overnight at 4 ∞C in a humid chamber, with mouse monoclonal antibodies directed against rat CD4 (W3/25) diluted 1/50, rat IL-2R-alfa chain - also known as CD25 (OX39 1/20 and Serotec MCA494G antibodies diluted 1/ 100 were used together) - or rat CD8 (Ox8, diluted 1/50), rat IgA (biotinylated, MCA191B, diluted 1/5000); and as negative control, a mouse anti-TNP antibody (03001D 1/50; Pharmingen, Stockholm, Sweden). All primary antibodies were diluted in PBSTween + 2% BSA. After washing the sections 3 ¥ 5 min in PBS, they were incubated with biotinylated horse antimouse IgG (BA-2001; Vector Laboratories, Burlingame, CA, USA ), for 1 h at room temperature. After three washings the sections were incubated for 30 min with streptavidin linked to horseradish peroxidase (streptABComplex/HRP, Dako, Glostrup, Denmark). For the colour reaction, they were washed again and incubated for 6–7 min in a filtered solution containing 0·18 mg/ml amino ethyl carbazole + 10% DMSO + 0·02 M sodium acetate at pH 5·5. All sections were counterstained with Mayer’s haematoxylin and mounted in Gurr Aquamount (BDH). After blinding of samples, the cells were counted using a computer supported image analysis system, Leica Q500MC (Leica, Cambridge, UK) All results were confirmed by a separate observer. Cells stained for IgA, CD4, CD25 or CD8 were recorded as cells/mm2 tissue. The epithelial areas of lamina propria were excluded when counting immune cells. Areas
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marked for counting were about 0·02 mm2 each and the distribution of cells in the lamina propria and between T cell areas was fairly homogeneous throughout each section.
Statistics Statistical significance was determined using a two-tailed Mann– Whitney U-test.
RESULTS In vitamin A-deficient rats, the reductions of IgA+ plasma cells in the ileal lamina propria were dramatic and statistically significant (P = 0·009; Figs 1 and 2). The median number of IgA+ plasma cells were reduced almost eightfold, from ca. 470 to 60 IgA+ cells/mm2. Neither vitamin A-deficient or vitamin A-replete animals showed any signs of infiltrating inflammatory cells into the gut mucosa (data not shown). To determine if the effects of vitamin A deficiency also acted on the regulation of intestinal plasma cells, T helper cells in the ileal Peyer’s patches were also studied. Indeed, the median number of CD4+ cells in the T cell areas (parafollicular regions) were reduced seventy-fold, from ca. 7200 to 105 cells per mm2 (Figs 3a,4). The CD4+ cells that were present in VAD rat ileum usually stained more weakly than in replete rats. As shown in Fig. 3b, IL-2 receptor alpha chain (CD25) positive lymphocytes in the T cell areas of ileal Peyer’s patches were also significantly
1200 P = 0·009
lgA+ cells/mm2
1000 800 600 400 200 0 VAD
NORM
Fig. 1. Number of IgA+ plasma cells per mm2 in ileal lamina propria. Each open ring represents one vitamin A-deficient rat (VAD); well-nourished control rats (NORM) are represented by filled squares. Statistical significance was determined by the Mann–Whitney U-test, P-values are indicated within the figure. Horizontal lines represent the median of each group.
Fig. 2. IgA+ plasma cells in ileal sections from a vitamin A-deficient rat (top) and a vitamin A-replete rat (bottom).
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reduced by VAD (from 1400 to 300 cells per mm 2, P = 0·009). Relative to all parafollicular cells (mainly T cells), CD25 cells decreased from 9·3% to 1·6%, P = 0·009 (Fig. 3c). The median size of the parafollicular regions tended to be reduced in VAD rats (not shown). In contrast, CD8+ in the Peyer’s patches tended to increase (from 5100 to 6000 per mm2, P = 0·12) in vitamin A deficiency (Fig. 3d). However, the size of parafollicular areas per section tended to decrease (41% reduction, P = 0·14). Therefore, total numbers of CD8 cells per section tended to be lower in VAD, while the CD8 : CD4 ratio and the density of CD8 cells tended to increase in VAD rats. In the ileal lamina propria as in the Peyer’s patches, CD4+ cells were significantly reduced in VAD (from a median of 1390 to 355 cells per mm2, P < 0·01). However, with regard to CD25+ cells (the median was reduced from 740 to 415 cells per mm 2, P = 0·12) or CD8 (from 900 cells to 960 per mm 2, not significant), there were no clear effects of VAD in the ileal lamina propria.
The literature describing the effect of VAD on the mucosal immune system is limited. However, it has been shown previously that antigen-specific and total IgA of serum and bile was much reduced in vitamin A-deprived rats [8]. According to another report, VAD led instead to increased biliary IgA as well as increased number of CD8+ cells while the number of IgA+ plasma cells in the jejunal lamina propria was reduced. The increase in biliary IgA in that study may have been caused by a higher total bacterial load during VAD, as the rats were colonized with ampicillin-resistant Escherichia coli and the natural gut flora was suppressed with antibiotics [6]. In IgA-deficient humans immune exclusion in the gut is clearly suboptimal, with increased influx of antigens. This has been found despite abundant intestinal IgM production, providing compensatory secretory IgM [11]. While the total number of bacteria in the small intestine as a whole was higher in VAD rats than in normal rats, it was not changed in the ileum [6]. Events in the ileum may therefore reflect more of the direct effects of VAD on the mucosal immune system and be less affected than in the upper small intestine by changes in bacterial load. In the present study, as in previous studies, VAD rats still appeared healthy because they were kept in a pathogen-free environment. However, when VAD rats are exposed to experimental infection they clearly have increased susceptibility to disease [6]. T cell activation is followed by early cell surface expression of the IL-2 receptor (CD25) and its interaction with IL-2 is a critical event in proliferation, differentiation and function of T cells [12]. In primates, 15% of lamina propria lymphocytes are CD25+ compared to
