-2851$/ 2) 9H W H U L Q D U \  6FLHQFH J. Vet. Sci. (2004), / 5 (3), 221–226 Mucosal mast cell-derived chondroitin sulphate levels in and worm expulsion from FcR γ -knockout mice following oral challenge with Strongyloides venezuelensis Denis Nnabuike Onah 1, *, Yukifumi Nawa 2 1 Department of Veterinary Parasitology & Entomology, University of Nigeria, Nsukka, Nigeria 2 Department of Parasitology, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan Mucosal mast cell-derived chondroitin sulphates (sulphated proteoglycans) were assayed in gut washings and homogenate of FcR γ -knockout (KO) and wild-type (WT) C57BL/6 mice challenged with Strongyloides venezuelensis in order to assess their possible role in secondary immunity against enteric nematodes. Groups of immune KO and WT mice were challenged by oral gavage with 300 infective larvae (L 3 ). Establishment of infection was assessed by daily faecal analysis to determine the number of eggs per gram of faeces (EPG) and by adult worm recovery on days 5 and 13 post challenge. Mucosal mast cell (MMC) counts were done on days 5 and 13 post challenge while MMC-derived chondroitin sulphates in gut washings (days 1 and 5) and homogenate (day 8) were assayed by high performance liquid chromatography (HPLC). Results showed that patent infection occurred in challenged KO but not WT mice despite significantly higher mastocytosis in jejunal sections of KO than WT mice ( p <0.001). Similarly but against prediction, significantly higher concentration of MMC-derived chondroitin sulphates was observed in gut homogenate of KO than WT mice ( p < 0.05). In contrast, significantly higher concentration of chondroitin sulphates was observed in gut washings of WT than KO mice ( p < 0.05). These results suggest that MMC in KO mice failed to release sufficient amount of sulphated proteoglycans into the gut lumen as did the WT mice, which may have been part of the hostile environment that prevented the establishment in and eventual expulsion of adult S. venezuelensis from the gut of WT mice following challenge. Key words: Strongyloides venezuelensis , mast cells, mucosal immunity, chondroitin sulphates, gut washings, mice Introduction The immune expulsion of adult Strongyloides species from the small intestine of mice and rats is associated with intraepithelial mast cell hyperplasia [1,2,7,14]. Mast cells are thought to mediate this expulsion by creating an environment hostile to the establishment and survival of gastrointestinal nematodes through inflammation-associated changes and release of inflammatory mediators [19]. The release of these mediators is induced by mast cell degranulation, which in turn is induced by mast cell activation triggered by cross-linking of the high affinity immunoglobulin Fc receptor (FcR) for IgE (Fc ε RI) with an antigen-IgE immune complex [3,8,11,13]. This type of acute inflammation, also known as type I hypersensitivity is therefore thought to depend on mast cells, its Fc ε RI and on IgE. Perturbation in any one of these three components should result in the loss of type I hypersensitivity responses [18]. This, in fact, is the case with Fc ε RI. To be expressed on the surface of cells and for signal transduction into the interior of the cell, Fc ε RI requires the homodimeric γ subunit of FcRs (FcR γ ) [5,10,17]. Ablation of the γ subunit by targeted gene deletion results in the loss of Fc γ RI expression on mast cells coupled with loss of mast cell functions including degranulation and mediator release in the mutant mouse [23]. Using these mice we showed that while the wild type (WT) counterparts were able to expel a primary Strongyloides venezuelensis infection, FcR γ - knockout (KO) mice failed to do so [16]. However, the confounding aspect of our study was that both intestinal mastocytosis and serum mouse mast cell protease-I (mMCP-I), the levels of which have negative correlation with nematode burden in sheep [22] were similar. Since FcR γ -KO mice fail to assemble Fc γ RI on their mast cells and therefore are unable to express mast cell degranulation and mediator release [23], we concluded that mMCP-1 may be released spontaneously without requiring the mast cell Fc γ RI cross-linking with the immune complex formed by the parasite antigen/IgE and that mMCP-1 is not involved in *Corresponding author Tel: +234-42-770106 E-mail: denisonah2003@yahoo.co.uk 222 Denis Nnabuike Onah, Yukifumi Nawa adult S. venezuelensis expulsion [16]. However, because mastocytosis has a strong association and often corresponds with the time of worm expulsion [14], we stated that mast cells must be involved in the process in a manner yet to be elucidated. We then speculated that failure of FcR γ -KO mice to expel S. venezuelensis might be related to inability of their mast cells to degranulate and release inflammatory mediators other than mMCP-1 [16]. Sulphated goblet cell mucins [7] and experimental introduction of glycosaminoglycans of the type produced by mucosal mast cells [12] have been shown to prevent the establishment of S. venezuelensis infection and mediate its expulsion from infected mice. Chondroitin sulphates are the major proteoglycans contained by mucosal mast cells in mouse [25]. In this study therefore, we assayed the amounts of chondroitin sulphates in gut homogenate and washings of FcR γ -KO mice and their WT counterparts following oral challenge with S. venezuelensis . This was in order to ascertain (i) if differences exit in their concentrations in the gut of KO and WT mice and (ii) whether differences in worm establishment and expulsion following challenge might be related to any differences in their concentrations within the gut niche occupied by the parasite in the two mouse types. Materials and Methods Animals FcR γ -knockout C57BL/6 male mice of 8 weeks old were generous gifts from Professor Toshiyuki Takai and Dr. Masao Ono (Tohoku University, Sendai, Japan). Age- matched specific pathogen free wild type C57BL/6 mice of the same sex were purchased from Japan SLC (Shizuoka, Japan). All animals were kept in our laboratory animal unit for 2 weeks to acclimatize before use at 10 weeks old. Before and during the period of the experiment they were supplied with feed and water ad libitum. Parasite The strain of S. venezuelensis originally isolated from a brown rat in Okinawa Prefecture, Japan [6] and established as a laboratory strain [20] was used in this study. Infective third stage larvae (L 3 ) used for infecting experimental animals orally (oral gavage) were obtained from the lungs of mice given primary infection and sacrificed on day 3 post infection. Briefly, 10 C57BL/6 male mice were each infected with 5000 L 3 of S. venezuelensis obtained by the filter-paper faecal culture method and sacrificed on day 3 post-infection by anaesthetic overdose using diethyl ether. The lungs were removed by dissection, shredded with fine- tipped thumb forceps in fine-meshed coffee strainers placed in a beaker of warm PBS and incubated at 37 o C for 3 h. Emerged lung L 3 were recovered by centrifugation, re- suspended and washed three time in fresh warm (37 o C) PBS, counted and adjusted to 1500 lung L 3 /ml. Infection of experimental animals All animal groups for challenge were primed with 2000 L 3 subcutaneously 30 days before challenge. For uniform treatment, every mouse in both the challenge and challenge control (primary infection) groups was treated orally with 20-mg/kg mebendazole (Sigma, St. Louis, MO) on day 26 post priming to get rid of any residual adult worms from the priming dose. On day 30 post priming, each animal in the challenge and control groups was infected with 300 lung- recovered L 3 contained in 200 µ l PBS and introduced directly into the stomach using a stomach needle with blunt oval tip. Experimental protocol Thirty knockout (KO) and thirty wild-type (WT) mice were used for the experiment. Twenty each of the KO and WT mice were primed as stated above. For challenge, primed KO and WT mice with 10 each of their naïve controls were infected as described above. The challenge groups were sacrificed 5 mice each on day 1, 5, 8 and 13 post challenge for sample collection while 5 each of their controls were sacrificed on day 5 and 13 post infection. Daily faecal egg counts expressed as eggs per gram of faeces (EPG) and adult worm recovery to establish whether patent infection occurred were carried out until day 13, and on day 5 and 13 post infection respectively in both challenge and control animals. In the challenge animals only, histology for MMC numbers was carried out on day 5 and 13, while various chondroitin sulphates and mouse mast cell protease- 1 (mMCP-1) in gut washings were assayed on day 1 and 5 post challenge. Chondroitin sulphates in gut homogenate were assayed on day 8 post challenge. EPG and adult worm recovery As stated above, faecal samples for daily EPG was collected only from individual animal of the groups sacrificed on day 13 post challenge. To ascertain worm establishment, adult worms were recovered from sacrificed challenge and control animals on day 5 and 13 post infection. The entire small intestine of each sacrificed animal was isolated and processed for adult worm recovery. The methods used for EPG and adult worm recovery, were as previously described [9,20]. Histology and serology Jejunal pieces were taken and histological sections prepared, stained and mucosal mast cells were enumerated as previously described [16] on day 5 and 13 post challenge. Also samples of gut washings as described below were collected on days 1 and 5 post challenge and analysed for mMCP-1 concentration by ELISA as described [16]. Proteoglycan assay in gut washings and homogenate The entire small intestine isolated from each of the Chondroitin sulphates and worm expulsion in FcR γ -knockout mice 223 sacrificed animal on the days specified above was washed out twice with a total volume of 10 ml sterile distilled water. Each sample was then centrifuged at 350 g for 10 min to remove debris. The supernatant was frozen until used. For gut homogenate, approximately 15 cm of the upper small intestine was cut off, washed out as above and minced using a homogeniser (Polytron homogeniser, Kinematika AG, Littau, Switzerland). The minced samples were suspended in 5 ml sterile PBS, centrifuged as above and the supernatant was frozen until used. For the assay of the various chondroitin sulphates, each sample was removed from the freezer, thawed under room temperature and was then processed and analysed by high-performance liquid chromatography according to the methods of Yoshida et al. [24] and Shinmei et al. [21]. Statistical analysis Data were compiled and subjected to descriptive statistics while differences between group-means were obtained by the Students t -test using Microsoft Excel Statistical Toolpack. Differences at p = 0.05 were considered significant. Results Eggs per gram of faeces (EPG) The mean daily EPG of animals sacrificed on day 13 post challenge are presented in Fig. 1. All control WT and KO animals as well as all challenged KO mice persistently shed eggs in their faeces until the day of sacrifice. In contrast, there were no eggs in the faeces of all challenged WT mice. One significant observation however, is that the oral route of infection with lung-recovered L 3 does not seem to be an efficient means of establishing patent infection judging from the level of EPG of the animals (less than 1000 at peak EPG) when compared with the EPG of mice infected by the subcutaneous route in which peak EPG usually runs in tens of thousands [15,16]. Adult worm recovery Consistent with the EPG result, control WT and KO as well as challenged KO mice developed persistent patent S. venezuelensis infection. The mean number of adult worms recovered from animals sacrificed on days 5 and 13 are shown in Fig. 2. As expected, no adult worms were recovered from any of the challenged WT mice on both days, which agreed with the zero EPG recorded for this group. Again, the very few number of adult worms recovered from these mice indicate that oral implantation of third stage larvae is not very efficient in establishing patent infection. Intestinal mast cell numbers and serum mMCP-1 concentration Mast cells were enumerated in jejunal sections prepared on days 5 and 13 post oral challenge. Results (Table 1a) show that significantly higher numbers of mast cells were counted in KO than WT mice on day 5 ( p < 0.01) and day 13 ( p < 0.001). On the other hand, there was no significant difference ( p > 0.05) in the amount of mMCP-1 concentration in gut washings of challenged KO and WT mice on days 1 and 5 post challenge (Table 1b). Sulphated proteoglycan concentration in gut washings and homogenate Results of the assay for various sulphated proteoglycans (Chondroitin sulphates A, C, D, E, and total chondroitin sulphate) in gut washings of challenged KO and WT mice sacrificed on days 1 and 5 are presented in Figs. 3a and 3b respectively. The results show that significantly higher concentrations of the chondroitin sulphates occurred in the gut washings of WT than KO mice on these days ( p < 0.05). In contrast, significantly higher concentrations of these Table 1. (a) Mucosal mast cell number/10 villus crypt unit (Mean ± SD) in FcR γ -KO and WT mice challenged orally with 300 lung- recovered S. venezuelensis L 3 Day PC Animal MMC/10VCU ± SD p Value 5 KO 320 ± 51 WT 156 ± 22 < 0.01 13 KO 456 ± 91 WT 057 ± 10 < 0.001 (b) Mean ± SD of mMCP-1 level (ng/ml) in gut washings of FcR γ -KO and WT mice challenged orally with 300 L 3 of S. venezuelensis mMCP-1 Concentration (ng/ml) Day 1 Day 5 KO 91 ± 36 205 ± 82 WT 85 ± 28 191 ± 56 p Value > 0.05 > 0.05 224 Denis Nnabuike Onah, Yukifumi Nawa sulphated sugars were obtained in gut homogenate preparations of KO than WT mice on day 8 post-challenge ( p < 0.05, Fig. 3c). Discussion Primary infection of mice with S. ratti and S. venezuelensis results in the development of a strong immunity, which completely aborts a patent infection following secondary challenge of the animals with the parasites [4,20]. This strong immunity depends on CD4 + T cells which make Th 2 cytokines and induce mast cell- dependent gut inflammatory responses and changes in gut physiology, all of which act in concert to create an environment hostile to worm establishment in and their eventual expulsion from the intestinal niche [15]. Our prediction was that an important part of this inflammatory response and change in gut physiology resulting in worm expulsion is the presence of sulphated proteoglycans released by degranulating mast cells. Therefore, if FcR γ -KO mice are unable to undergo mast cell degranulation and mediator release [23], the enabling hostile environment for the ablation of secondary infections will be lacking in primed KO animals. Consequently, if sulphated proteoglycans play a role in worm expulsion, then the introduction of migrating L 3 into a hostile intestinal environment in primed KO and WT mice would most likely result in a patent infection in the former but not in the latter. Results of this study largely support our predictions. First, significantly higher concentrations of chondroitin sulphates were present in gut washings of WT, in which the challenge infection was sterile than in KO, in which persistent patent infection was established. Secondly, there was no significant difference in the concentrations of mMCP-1 in gut washings taken from both mouse types irrespective of the fact that significantly higher numbers of intraepithelial mast cells were counted in KO than in WT animals. Taken together, it can be inferred that (a) mMCP-1 may be released by mast cells in a manner not dependent on degranulation i.e., not requiring the antigen-IgE-Fc ε R cross- linking and triggering, (b) mMCP-1 may not be important in the mucosal immune mechanisms resulting in the expulsion of adult S. venezuelensis from the gut and (c) mucosal mast cell-derived glycosaminoglycans (chondroitin sulphates) are apparently involved in and therefore play a role in the prevention of the establishment of adult S. venezuelensis in and their eventual immune expulsion from the gut. These agree with the suggestions of Onah et al. [16] regarding mMCP-1 and its possible role in worm expulsion as well as with their speculation that FcR γ -KO mice are probably unable to expel primary S. venezuelensis infection as a result of failure of their mast cells to degranulate and release mediators other than mMCP-1. In fact, that significantly higher concentrations of chondroitin sulphates were obtained from the homogenized gut tissue of KO (which contained more mast cells) than WT is an added support that the intraepithelial mast cells in KO contain them but are not releasing them in enough quantities into the gut lumen to effect worm expulsion. In addition, we have similar evidence that when immune KO and WT mice are challenged by subcutaneous introduction of 3000 S. venezuelensis L 3 , fewer larvae are recovered from the lungs of KO than in WT animals 3 days later yet patent infection F ig. 1. Daily numbers (mean ± SD) of eggs per gram of faec es ( EPG) in immune and control FcRγ-KO and WT mi ce c hallenged orally with 300 lung-recovered L 3 of S. venezuelens is a nd sacrificed on day 13 post challenge. F ig. 2. Number of adult worms (mean ± SD) recovered from t he s mall intestines of immune and control FcRγ-KO and WT mi ce c hallenged orally with 300 lung-recovered L 3 of S. venezuelens is a nd sacrificed on days 5 and 13 post challenge. Chondroitin sulphates and worm expulsion in FcR γ -knockout mice 225 occurs only in the latter animals. In this infection protocol serum mMCP-1 was also similar but sulphated proteoglycans were found to be significantly higher in the WT than KO animals. Moreover, our results in this study agree with and support those of Maruyama and his associates [12] who showed that glycosaminoglycans of the type produced by mucosal mast cells significantly inhibited the invasion and establishment of adult S. venezuelensis implanted into the duodenum of naïve mice. In conclusion, defective secondary immunity against S. venezuelensis in FcR γ -KO mice is associated with significant decrease in the amount of mast cell-derived chondroitin sulphates released into the gut lumen, suggesting that the sugars are intimately associated with worm expulsion. It is our opinion that extensive further studies into the role of sulphated sugars of the type produced by mucosal mast cells in parasitic gastrointestinal nematode expulsion is essential and worthy of support as they seem attractive candidates for anthelmintic drug investigation and development. Acknowledgments We thank Mrs. Eri Ono for excellent technical assistance F ig. 3. (A) Chondroitin sulphate (ChS) concentration ( µ g/ml) (mean ± SD) in gut washings of FcR γ -KO and WT mice challenged oral ly w ith 300 lung-recovered L 3 of S. venezuelensis and sacrificed on day 1 post challenge. ChS-A:E = Chondroitin sulphate A-E; U s- C h = Unsulphated chondroitin. (B) Chondroitin sulphate (ChS) concentration ( µ g/ml) (mean ± SD) in gut washings of FcR γ -KO a nd W T mice challenged orally with 300 lung-recovered L 3 of S. venezuelensis and sacrificed on day 5 post challenge. Ch S- A :E = Chondroitin sulphate A-E; Us-Ch = Unsulphated chondroitin. (C) Chondroitin sulphate (ChS) concentrations ( µ g/m l) ( mean ± SD) in gut homogenates of FcR γ -KO and WT mice challenged orally with 300 lung-recovered L 3 of S. venezuelensis a nd s acrificed on day 8 post challenge. ChS-A:E = Chondroitin sulphate A-E; Us-Ch = Unsulphated chondroitin. 226 Denis Nnabuike Onah, Yukifumi Nawa and Professor Toshiyuki Takai and Dr. Masao Ono (Tohoku University, Sendai, Japan) for generously providing the mutant mice used in this study. DNO was a JSPS postdoctoral research fellow and funding for this work was provided by the Grant-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. 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(2004), / 5 (3), 221–226 Mucosal mast cell-derived chondroitin sulphate levels in and worm expulsion from FcR γ -knockout mice following oral challenge with Strongyloides venezuelensis Denis. proteoglycans (Chondroitin sulphates A, C, D, E, and total chondroitin sulphate) in gut washings of challenged KO and WT mice sacrificed on days 1 and 5 are presented in Figs. 3a and 3b respectively [16]. Proteoglycan assay in gut washings and homogenate The entire small intestine isolated from each of the Chondroitin sulphates and worm expulsion in FcR γ -knockout mice 223 sacrificed animal