Abstract
The effects of cyclosporin A (CY) and cyclophosphamide (CPS) on Peyer’s patches (PP) were studied in Wistar rats, exposed in utero and neonatally or during adult age. In one study, pregnant dams received 5 or 15 mg/kg bw/day CY from gestation day 6 to day 21 of lactation. In two other studies, animals were exposed at young adult age: female rats received orally 5 or 20 mg/kg/day CY or 5 or 10 mg/kg bw CPS for 4 weeks; males received orally 5 mg/kg bw CPS for 4 weeks, or a single iv injection of 50 mg/kg bw CPS. Upon in utero and neonatal exposure, the numbers of grossly observed PP were increased in male pups from the high-dose CY dams at 70 days of age. Exposure to high-dose CY at adult age only tended to decrease the numbers of PP; germinal center development was reduced in the PP from the middle segment of the small intestines, as examined microscopically. Exposure to both doses CPS at adult age reduced the numbers of PP and reduced germinal centre development and the number of lymphocytes in all compartments of PP. It was concluded that the effects of CPS and CY could be established by counting the number of grossly visible PP and by microscopic observation of PP, provided that regional differences of PP were taken into account. Moreover, the type of effects of an immunotoxic agent may vary with age of exposure.
Introduction
The mucosae of the respiratory and gastrointestinal tract, and the skin are often the first sites of contact with antigens and noxious substances. The mucosa of the intestinal tract forms a weak mechanical barrier because of its absorptive function, unlike skin and the mucosae of the oral cavity, esophagus, and nonglandular stomach. However, it is provided with an elaborate specific defense system. Immune-competent cells play a crucial role in this host defense, and are organized in lymphoid structures such as Peyer’s patches (PP), cryptopatches, and follicles or are disseminated diffusely in the epithelium and lamina propria (Lefrancois and Puddington, 1995; Brandtzaeg and Pabst, 2004).
The examination of the effects of xenobiotics on immune-competent cells of the gastrointestinal tract has been focussed on PP. PP are the inductive sites of the immune responses against antigens from the gut and they recruit IgA-secreting plasma cells to be disseminated to the lamina propria (reviewed by Schmucker, 2002). A few compounds have been reported to alter PP morphology and function, as assessed mainly by the intestinal IgA response. The immunosuppressive drugs azathioprine, cyclosporin A (CY), and cyclophosphamide (CPS) decreased the cellularity and size of various compartments and inhibited germinal center development in PP of young rats, mice, or rabbits (Cozon et al., 1991; Cuff et al., 1998; ICICIS Working Group, 1998; Fujimura and Owen, 2002; Schulte et al., 2002;). CPS also decreased the intestinal IgA secretion (Cozon et al., 1991). The immune-potentiating fungicide hexachlorobenzene increased the presence of high-endothelial venules in the interfollicular compartment of PP in young rats (Schulte et al., 2002).
Reports are scarce of effects on the morphology of PP. This may partly depend on the selection of patches for microscopic examination. For example, azathioprine at 25 mg/kg bw by oral gavage for 28 days decreased the number of grossly visible PP in Wistar rats, but the patches selected at necropsy and examined microscopically were normal (Bruder et al., 1999). This observation led to the counting of grossly visible PP as a parameter to evaluate immunotoxic effects, and it raised the question whether regional differences exist in sensitivity of PP to immune-modulating agents. In mammals like pigs, horses, and humans, 2 types of PP, the jejunal and ileal PP, have been observed with different developmental, morphologic and functional characteristics (reviewed by Makala et al., 2002; Yasuda et al., 2004), and it is conceivable that the two types of PP would react differently to immunotoxicants. Different types of PP have not been recognized in rodents, although a single large patch in the neigbourhood of the ileocecal junction is most prominent and is reported to appear grossly in rats at about 12 days of age (Hummel, 1935). However, clusters of CD4+ lymphocytes were observed along the gut already at day 16–17 of gestational age in mice (Adachi et al., 1997). Therefore, PP may be especially sensitive to immune-modulating agents when exposure takes place prenatally and shortly after birth. In that period, regional differences in sensitivity of PP may be prominent, because of differences in developmental rate of PP. Moreover, immunotoxic effects induced during the perinatal period may be long-lasting whereas changes induced in adults are mostly reversible (Dietert et al., 2003; Hussain et al., 2005).
The effects of the immunosuppressive drugs CY and CPS on PP were studied in Wistar rats of 10–13 weeks of age, which were exposed during young adult age. In addition, the effects of CY on PP were studied in Wistar rats of 4 or 10 weeks of age, which were exposed in utero and via the milk.
Materials And Methods
Test Compounds
Cyclophosphamide (CPS), or endoxan, was purchased from Baxter BV, Utrecht, the Netherlands. Cyclosporin A (CY) was purchased from Sigma-Aldrich Chemie BV, Zwi-jndrecht, the Netherlands. CY and CPS were dissolved in respectively raffinated olive oil (Chempri, Raamdonksveer, the Netherlands) and demineralized water (oral gavage) or saline (iv injection).
Animals and Dosing
The animals used were Wistar outbred (Crl:(WI)WU BR) rats, obtained from colonies under SPF-conditions at Charles River Wiga GmbH, Sulzfeld, Germany. They were kept under controlled conditions and given water and food (Rat and Mouse No. 3 Breeding Diet, RM3; SDS Special Diets Services, Witham, England) ad libitum. The welfare of the animals was maintained in accordance with the general principles governing the use of animals in toxicity experiments of the European Communities (Directive 86/609/EEC) and Dutch legislation (The Experiments on Animals Act, 1997).
PP were counted in the small intestines of untreated Wistar rats. In addition, PP were counted in Wistar rats from 3 immunotoxicity studies with CY in olive oil, CPS in water or with 1 of the 2 vehicles (Table 1). In study I, 7-week-old female Wistar rats were treated daily by oral gavage with 5 or with 20 mg/kg bw CY or 5 or 10 mg/kg bw CPS or their respective vehicle controls, during 4 weeks. Seven-week-old males were treated with 5 mg/kg CPS. In study II, 12-week-old male Wistar rats were given a single iv injection with 50 mg/kg bw CPS, 1 week before necropsy. The 1-week interval between injection and necropsy was based on the time-effect relationship observed in the spleen upon a single injection with CPS (Kuper et al., 2000).
In study III, pregnant Wistar rats were treated by oral gavage with 5 or 15 mg/kg bw CY, or the vehicle olive oil, from day 6 of pregnancy up to day 21 of lactation. The doses were based on doses in a comparable immunodevelopmental study in which immune function changes were observed in the offspring (Hussain et al., 2005). The offspring were therefore exposed to CY during most part of the gestation period and during the lactation period, via the mothers. Thereafter, the offspring was kept untreated till the animals were 4 weeks (30 days) or 10 weeks (70 days) old. The times of necropsy were chosen on the basis of so-called immunodevelopmental landmarks (Dietert, 2005), which are currently explored for their use in guidelines addressing developmental toxicity (Ladics et al., 2005).
Experimental
At necropsy, PP were counted per entire small intestinal tract. In the untreated female rats and in the intravenous study (study II) the distribution of grossly visible PP along the small intestines was examined by dividing the intestines into segments of 20 cm each and numbering the segments from proximal to distal; the number of PP was counted in each segment.
PP in the untreated females were numbered from proximal to distal and sampled in rings of intestines, closely cropped around the patches, for microscopic examination. In immunotoxicity study I, two PP were sampled in closely cropped rings of intestine, namely one PP in the middle part of the intestines and the large patch within 10 cm distance from the cecum. In study II, so-called Swiss rolls (Moolenbeek and Ruitenberg, 1981) were prepared from each 20 cm intestinal segment of one animal per group.
This was done by dividing the segments with their PP into two pieces of 10 cm each, opening them longitudinally at the mesenteric side and rolling the parts around a stick with the mucosa outwards. The cropped rings of intestines and the Swiss rolls were fixed in neutral phosphate-buffered formalin and embedded in paraffin. Three 3–6 μm step-sections were taken from each intestinal ring, the first through the center of the patch and the other two at a distance of 40 μm each. A similar procedure was followed with the Swiss rolls, the first section taken throughout the center of the Swiss roll and the distance between the sections being 100 μm. The sections were stained with hematoxylin and eosin.
Statistics
Body and organ weights were analyzed by one-way analysis of covariance, followed by Dunnett’s multiple comparison tests or by Student’s t-test. The numbers of Peyer’s patches were analysed by a two-way ANOVA, followed by Bonferroni-corrected pairwise t-tests.
Results
Untreated Wistar Rats
At gross observation, PP appeared as small round to oval patches, slightly elevated above the surface of the intestines, at the anti-mesenteric side. Quite occasionally, PP were represented also as small indentations/pits (as confirmed by microscopy). The PP did not have a fixed localization but occurred quite haphazardly along the intestinal tract, except for a large patch observed at ca. 5–10 cm from the ileocecal junction. The number of PP was highest in the middle section of the small intestines, in segments II to IV (Table 2). The rats did not have PP within 2 cm from the ileocecal junction.
Microscopic examination of intestinal rings with PP demonstrated that the number and size of follicles/PP increased from anterior to posterior (Table 2). The number of follicles with germinal centers or secondary follicles (germinal center development) and their size increased also from anterior to posterior. Large interfollicular areas were found in some PP in the second half of the intestines.
Young Adult Wistar Rats: Effects of Orally Administered CY and CPS on PP
In the 28-day oral gavage study (study I), CY did not induce an effect on the relative thymus and spleen weights or on the number of grossly visible PP, although the number of PP tended to decline in the 20 mg/kg bw CY females (p < 0.9, Bonferroni-corrected pairwise t-test) (Table 3). Microscopic examination did not demonstrate an effect of low- or high-dose CY on the large PP close to the cecum (Table 4). PP sampled from the middle part of the intestines, at about 50 cm from the stomach (corresponding with segment III), exhibited a decreased number and size of germinal centres in the high-dose CY females (Figures 1A–1B) in most but not all animals.
CPS reduced the organ weights and the number of grossly visible PP significantly in males and females (Table 3). Gross examination at necropsy revealed not only less PP but also much smaller PP in the low- and high-dose CPS groups. In the 5 mg/kg bw CPS females, occasionally PP were seen as groups of small white dots, especially in the proximal part of the small intestines, or as vesicles, especially in the middle part. However, the one large PP in the distal part (described above for untreated rats) appeared unaffected in size in most animals.
In the 10 mg/kg bw CPS, PP were hardly seen at all, even the large patch in the distal part was considerably reduced in size. The diameter of the small intestines of these CPS animals was decreased markedly and the tissue itself was friable. Microscopic examination revealed a reduction in the number of small lymphocytes in the follicular and interfollicular compartments and a reduction in the size of the compartments; in a few animals of the 5 mg/kg bw CPS, the compartments of the patches close to the cecum appeared of comparable size as the controls but germinal centers were small, not well-developed (i.e., no distinct dark and light zones) or absent (Table 4). The patches in the middle part were affected most in both dose groups (Figures 1C–1F).
At the individual animal level, an association was observed between the highest thymus weight and the highest number of PP in the CY- and CPS-treated groups, indicating non-or low-responders, with the exception of the 10 mg/kg bw CPS females in which thymus weight and PP were markedly affected in all animals.
Young adult Wistar Rats: Effects of Intravenously Administered CPS on PP in Different Segments of the Small Intestines
A single iv injection with 50 mg/kg bw CPS significantly reduced the number of PP, as observed 1 week after the injection (Table 5). The size of the individual PP was also reduced; the PP in the distal part of the small intestines were seen as groups of discrete white dots, similar to those observed in the 5 mg/kg bw CPS given during 28 days by oral gavage (see previously). The reduction was most prominent in the more distal part of the small intestines.
Microscopic examination of the PP in Swiss rolls revealed a decrease in the number of small lymphocytes, especially in the follicular compartment and most PP were devoid of germinal centers. The Swiss rolls permitted examination of the cryptopatches/lymphocyte-filled villi, which were unaffected by CPS, both in number (30–35 per intestinal tract) and in histology.
Offspring/F1-Generation of Wistar Rats: Effects of CY on PP
Exposure to 15 mg/kg bw CY from day 6 of the gestation period up till weaning at day 21 did not induce statistically significant effects in the number of PP in the small intestines of the 30-day-old offspring (Table 6). In the 70-day-old offspring, the number of PP was increased in the 15 mg/kg males. The large patch in the proximal part was unaltered, the PP in the distal and middle section of the small intestines were slightly more prominent in the male offspring of mothers treated with 15 mg/kg bw CY.
Discussion
Peyer’s patches (PP) play an important role in the local immunity of the small intestines of mammals. Their number varies distinctly between species, from about 100–300 in man to 5–9 in rabbits (Makala et al., 2002). In the present study, 18 (11–25) PP were counted in 11 to 13-week-old untreated or vehicle-treated Wistar rats, which is comparable to numbers of PP in rats reported elsewhere (Hummel, 1935) and slightly more than observed in BN rats of the same age and housed under the same conditions (12 PP, 9–15; Kuper, unpublished data). The number of PP in rats was found to be quite stable with age from young adult onwards (Schmucker et al., 2002), neither affected by bacterial load as observed in several species nor by diet, in contrast to intestinal IgA that was significantly influenced (Ikeda et al., 2002; Makala et al., 2002). The absence of PP within 2 cm from the ileocecal junction in the untreated rats (Table 2) implies that they did not possess ileal PP, if the ileum in rats is considered to be only 1–2 cm long (according to the definition of the ileum as the part of the small intestines reached by the ileocecal ligament).
CPS given by gavage to young adult Wistar rats at doses of 5 and 10 mg/kg bw during 4 weeks or given by single intravenous injection of 50 mg/kg bw reduced the number and size of PP and reduced the number of lymphocytes in all compartments of PP. B-lymphocytes in PP may be particularly vulnerable to CPS (Cuff et al., 1998). CY given by gavage at doses of 5 and 20 mg/kg bw during 4 weeks to young adult rats induced considerably less effects in PP than CPS. Even at the high dose, CY only tended to decrease the number of PP and marginally reduced the development of germinal centers.
Comparable effects of CY on the B-lymphocyte compartment of PP have been described by others (reduction of B cells in mouse, Cozon et al., 1991; reduced follicle cellularity and disappearance of germinal center in rat, Haba et al., 1991; reduction of height of domes and height and width of follicles in rat, Fujimura and Owen, 2002). Effects of CY on the interfollicular (T cell-dependent) compartment, namely decreased cellularity, have been found as well (ICICIS, 1998; Schulte et al., 2002).
Whereas CY exposure in young adult rats tended to reduce the number of PP and reduced germinal center development in PP, the number of PP in rats exposed during gestation and lactation was unchanged in 4-week-old rats and increased in rats at 10 weeks of age. Such contrasting results in animals exposed after weaning versus animals exposed perinatally have been found also with other immune parameters. For example, CY was immunosuppressive when administered to rats after weaning but induced autoimmune-like phenomena when rats were exposed before weaning (Hussain et al., 2005). Differences in early and late effects have been observed also with dexamethason. Dexamethason administered during gestation reduced the IL-2 production in 5-week-old rats but stimulated the production in rats at 13-weeks of age (Dietert et al., 2003). This indicates that the developing mucosal immune system may react quite different to immunotoxic agents than the established mucosal system.
The increase in number of PP in 10-week-old rats, exposed to CY via the mother, occurred in the absence of effects on weight and histology of the thymus and spleen (Tegelenbosch-Schouten and Wolterbeek, 2005). This indicates that PP were sensitive to CY, and that counting them is a sensitive method for detecting immunotoxicity. It remains questionable if counting is more sensitive than microscopic examination of PP. The effects of cyclophosphamide (CPS) in the present study were prominent, both when determined by counting as well as by microscopic observation of PP. The high-dose CY on the other hand only tended to decrease the number of PP and the microscopically observed change, i.e., reduction in germinal center development, was marginal.
Germinal center development was reduced in the PP taken from the middle part of the intestines but not of the large PP close to the cecum. Moreover, the reduction could only be established by using step-sections because germinal centers are often absent or only small in PP in the proximal and middle region of the intestines (Table 2). Thus the sensitivity of microscopic examination depended on appropriate and standardized selection of PP, which is hampered by the haphazard occurrence of most PP in rat.
Based on counts in different segments of the intestinal tract (Table 4), the PP in the distal part of the small intestines appeared quite sensitive to CPS and CY, with the exception of the large patch close to the cecum. This is in accordance with the observation that the distal part of the small intestines in rodents was a major site of entrance of a variety of substances (penetration of Salmonella enteritidis, Carter and Collins, 1974; absorption of paraquat, Steffen and Konder, 1979; uptake of luminal oligopeptides, Haupt et al., 2002). Therefore, counting PP approximately within 40 cm from the ileocecal junction in combination with microscopic examination of a few PP may be a sensitive method to detect immunotoxicity to PP.
In summary, PP in young adult rats were affected negatively by CPS and only minimally by CY. In contrast, CY, when given in utero and via the milk, induced an increase in the number of PP when rats were 10 weeks of age. It was concluded that counting the number of grossly visible PP and microscopic observation of PP were of equal sensitivity, provided that regional differences of PP were taken into account. Moreover, the type of effect of an immunotoxic agent may vary with age of exposure.