CHITOSAN EFFECTS ON T(CD4+, CD8+) LYMPHOCYTES AND MACROPHAGES DURING EXPERIMENTALLY INDUCED TRICHINOSIS IN MICE

Jolanta Piekarska¹, Aleksandra Balicka-Ramisz², Alojzy Ramisz², Bogumiła Pilarczyk², Łukasz Laurans^1
1 Department of Internal and Parasitic Diseases, Agricultural University of Wrocław, Poland
² Department of Animal Hygiene and Prophylaxis, Agricultural University of Szczecin, Poland

ABSTRACT

The study involved mice of an inbred CFW line from the author’s own culture conducted according to the street light method (Lane Petter and Pearson 1971). All the animals, aged about 3 months and weighing 20 g each, were infected per os with 200 T. spiralis larvae per mouse. A total of 76 mice, divided into two groups, were used. Group I constituted the control and consisted of T. spiralis-infected mice, while Group II consisted of chitosan (chitosan adipinate)-treated mice receiving a dose of 0.4 mg per mouse, administered intra-peritoneally for 20 days (6 days prior to infection until day 13 post-infection). Four mice of each group were sacrificed by decapitation on invasion day 7, 14, 21, 28, 35, 42, and 60. Sections of the jejunum and mandibular muscle were used to prepare populations of cells involved in the inflammatory infiltration. The populations selected were the T (CD4⁺ and CD8⁺) lymphocytes and macrophages. The first were identified with immunofluorescence, using labelled monoclonal antibodies, while identification of the latter proceeded immunoenzymatically, with non-labelled monoclonal antibodies. In addition, the infected animals in each group were examined for the presence of parasites: on day 7, 10, 14, and 21 post-infection, the intestinal parasites were counted, while, the muscle-dwelling larvae being enumerated on day 60 post-infection. In this study, the macrophage count in the jejunum mucosa basement membrane of the chitosan-treated mice increased until day 21 post-infection and remained, until the observations were terminated, at a level higher than that in the control. On the other hand, the transversely striated muscles revealed, in addition to T CD4+ and T CD8+ lymphocytes, a stronger macrophage mobilisation throughout the period of observations. The chitosan-treated mouse jejunum were also a site of a faster removal (“expulsion”) of adult parasites than in the control, the muscle larval count in those mice being clearly lower than in the control.

Key words: Chitosan, mice, Trichinella spiralis, T(CD4⁺, CD8⁺) lymphocytes, macrophages.

INTRODUCTION

Cellular inflammatory infiltrations are observed both during intestinal and muscular stages of trichinosis. In both organs, the infiltrations involve the same populations of cells, but their respective contributions differ, depending on the organ and on duration of the invasion.

The study was aimed at elucidating the role of T (CD4⁺, CD8⁺) lymphocytes and macrophages in trichinosis. The lymphocyte and macrophage behaviour was observed in mice, experimentally infected with Trichinella spiralis, that were simultaneously treated with chitosan.

Chitosan is the simplest chitin derivative that, due to its high biological activity, the absence of any allergenic or irritating properties [4], and low toxicity [30] is used in prophylaxis and treating of both humans and animals. The most valuable chitosan properties include cytokine production stimulation, pathogenic bacteria immobilisation, binding of minerals indispensable in bacterial growth, immobilisation of yeasts and moulds, activation of macrophages, susceptibility to lysozymes, reduction of blood cholesterol level, and erythrocyte agglutination [29].

The present study was based on the assumption that comparison of data obtained from T.spiralis-infected animals (control) with data collected from experimentally infected mice that were treated simultaneously with chitosan, would result in a better understanding of the role T (CD4⁺, CD8⁺) lymphocytes and macrophages play during a T. spiralis infection and of their effects on the parasite.

MATERIAL AND METHODS

The study involved mice of an inbred CFW line from the author’s own culture conducted according to the street light method [15]. All the animals, aged about 3 months and weighing 20 g each, were infected per os with 200 T. spiralis larvae per mouse, as described by Kozar and Kozar [13]. A total of 76 mice, divided into two groups, were used. Group I constituted the control and consisted of T. spiralis-infected mice, while Group II consisted of chitosan (chitosan adipinate)-treated mice receiving a dose of 0.4 mg per mouse, administered intra-peritoneally for 20 days (6 days prior to infection until day 13 post-infection).

Throughout the period of the experiment, the mice were kept under identical conditions. Four mice of each group were sacrificed by decapitation on invasion day 7, 14, 21, 28, 35, 42, and 60. Sections of the jejunum and mandibular muscle were used to prepare cryostatic mounts in which, following appropriate staining, the selected populations of cells involved in the inflammatory infiltration were examined. The populations selected were the T (CD4⁺ and CD8⁺) lymphocytes and macrophages. The first were identified with immunofluorescence, using labelled monoclonal antibodies, while identification of the latter proceeded immunoenzymatically, with non-labelled monoclonal antibodies. In addition, the infected animals in each group were examined for the presence of parasites: on day 7, 10, 14, and 21 post-infection, the intestinal parasites were counted, while, the muscle-dwelling larvae being enumerated on day 60 post-infection.

RESULTS

The T CD4⁺ lymphocyte level in the jejunum mucosa basement membrane of the Trichinella spiralis-infected mice (control) increased as early as on day 7 post-infection (Table 1). The cell count continued to increase until day 21, to gradually decrease thereafter. T CD4⁺ lymphocytes in the mucosa of the chitosan-treated mice showed a similar dynamics until day 14 post-infection, the lymphocyte number, however, being lower than that in the mice not receiving chitosan. Those animals showed their T CD8⁺ lymphocytes to be activated on day 14 post-infection only (Table 2). In the jejunum mucosa basement membrane of the Trichinella spiralis-infected mice the macrophages attained peak mobilisation on day 7 post-infection, whereupon their activity was gradually slowing down until day 60 (Table 3). On the other hand, the chitosan-treated group showed the macrophage count to increase until day 21 post-infection (a=0.05), to subsequently decrease thereafter, but to a level still generally higher than that in the control.

The T CD4⁺ lymphocytes were spotted in the mandibular muscle of the T. spiralis-infected mice as early as on day 14 post-infection, their count increasing until day 28; on the other hand, although the chitosan-treated mice showed the peak activity on day 28 as well, it was higher and the subsequent decrease weaker than in the control (Table 4). Activation of the T CD8⁺ lymphocytes was observed in those animals (Table 5) on day 28 and 35 post-infection (a=0.01). The number of macrophages that appeared on day 14 post-infection in the infected mice was rapidly increasing up to day 21 post-infection (Table 6), to gradually decrease thereafter. A very similar dynamics was shown by those cells in the chitosan-treated mice. However, the level they were attaining throughout the period of observations was much higher, and on days 35, 42, and 60 post-infection, the difference with respect to the control group was statistically significant at a=0.01.

The mean jejunum count of T. spiralis on day 7 post-infection in the control was 65.0 (SE 7.0); it was 15.0 (SE 6.0) on day 10, 2.0 (SE 1.0) on day 14, and 1.0 (SE 1.0) on day 21. The chitosan-treated mice showed the mean count of parasites in the jejunum to change from 60.0 (SE 12.0) on day 10 post-infection to 1.0 (SE 1.0) on day 14, no parasites being found in the jejunum on day 21. The mean number of muscle-embedded larvae in the control on day 60 post-infection was 22350 (SD 4510.0), the chitosan-treated mice showing the mean count of 19105 (SD 6682).

DISCUSSION

Studies on Guinea pigs, carried out by Nishimura et al. [19, 20] and by Rodgers and Mims [28] showed chitosan to have high immunoadjuvant properties and to activate peritoneal macrophages and increase the antibody production. Application of chitosan to combat parasitic and bacterial diseases in pigs and sheep [2, 24, 25, 26, 27] demonstrated chitosan to be capable of significantly altering organismic responses. Chitosan salts administered intramuscularly turned out to protect lambs from respiratory diseases, while intraperitoneal application stimulated macrophage and neutrophilous granulocyte activity in mice [26]. Taking those results into consideration, it was decided to check, in this work, whether chitosan may also affect the immunological response during experimental trichinosis in mice.

Initial studies on the T lymphocyte behaviour in the course of trichinosis, carried out by Karmańska and Michalska [10], revealed a strong activation of those cells on about day 20 of the invasion, the stimulation occurring almost simultaneously in the lymphatic system organs and in inflammatory infiltrations on the small intestinal mucosa basement membrane and in muscles. Because numerous publications on other helminthoses had indicated that it was exclusively the CD4+ cells that were involved in host’s protection, this line of study was pursued also with respect to trichinosis, by comparing the behaviour of both the Th1 and Th2 cells in mice lines sensitive (B10.BR) and resistant (AKR) to T. spiralis infection [3, 21]. The importance of the Th population in the "expulsion” process was studied as well [6, 11]. In the intestinal phase of trichinosis, stimulation of the Th2 cells only was observed, the Th1 cells producing no response. It was also demonstrated that removal of adult Trichinella from the intestine involves cooperation between the Th cells and the antibodies [1]. When determining levels of cytokines Il -4 and Il-5 in the lymph, it was observed that cytokine increase was accompanied by Trichinella removal from the intestine, while IFN- showed a number of cycles of increased production during infection [23]. Other studies also confirmed the fact that the removal of adult T. spiralis depends on the activity of the CD4+cells, and specifically the Th2 ones and is mediated by the cytokines they release [5, 34]. The authors observed that both the "expulsion", and the accompanying contractions of the intestinal muscle layer are T lymphocyte-dependent, because both processes are slowed down in athymic animals. Not all authors, however, are unanimous in thinking that the CD8+ cells are not involved in defence processes during trichinosis. Yu and Lian [35] demonstrated, using the Mueller method, that the number of T lymphocytes increased between day 3 and day 14 post-infection, the elevated level being maintained for as long as until day 60. At that time, the number of the Th cells decreased, and the number of Ts cells increased. The suppression in T.spiralis infection, described earlier, would then be associated with a reduced Th/Ts ratio. Ivanoska et al. [7], too, were in favour of participation of both populations because they observed elevated levels of the CD4+ and CD8+ cells in the peripheral blood of T.spiralis-infected animals. On the other hand, Ramaswany et al. [22] concluded that, during rat tichinosis, the CD8+ lymphocytes are not involved in immunological processes. Karmańska et al. [8] disagreed with that conclusion following their studies on monoclonal antibodies-related behaviours of the CD4+ and CD8+ lymphocytes in sections of spleen, jejunum, and mandibular muscle of T. spiralis-infested mice. They found instead that the two lymphocyte populations were involved both in the intestinal and in the muscle phases of the disease. Following the T cell kinetics in Trichinella-infected mice, Lan Yan et al. [14] showed the Il -2 level to dominate at the early stage of the disease, the Il-6 being dominant at the late stage. Between days 14 and 25, the number of the CD4+ cells decreased to increase later on, while the CD8+ cell count was observed to gradually increase. Recently, Shen et al. [32] who used flow cytometry, determined the T lymphocyte count on day 7, 14, 21, 28, and 35 post-infection in experimentally infected mice. The results they obtained showed the levels of the CD4+ T cells to decrease initially, those of the CD8+ cells increasing. A reduction was also seen in the CD4+/CD8+ ratio that reached its minimum on day 14 post-infection; although it did gradually increase thereafter, as late as on day 35 post-infection it was lower than that in the healthy mice. The authors quoted concluded that, during an acute T. spiralis invasion, the host’s immunological function becomes inhibited.

The presence of macrophages among cells of an inflammatory infiltration during a T. spiralis infection was demonstrated as early as in the 1950s [16, 17]. Karmańska and Michalska [10] observed macrophages in the intestinal mucosa basement membrane and in transversely striated muscles of the infected mice. The role of macrophages in trichinosis was studied by Kozar et al. [12] who treated the T. spiralis infected mice with antimacrophage serum. Immunoenzymatic techniques and monoclonal antibodies used by Karmańska et al. [9] made it possible to elucidate behaviour of those cells in experimentally induced trichinosis in mice. The highest and fastest macrophage mobilisation was observed on day 7 post-infection in the intestinal mucosa basement membrane. The highest macrophage count in muscular inflammatory infiltrations was recorded on day 21 post-infection, and on day 28 inside larval cysts. Szkudliński [33], who studied peritoneal macrophage phagocytosis during trichinosis in mice showed invasions of T. pseudospiralis and T. spiralis to result in a decrease of the percentage of active macrophages and in reduction of their phagocytal ability. That was a consequence of the activity of prostaglandins which are important inflammatory response inducers. Having treated the mice with prostaglandin synthesis inhibitors, the author quoted found inhibition of the process and activation of phagocytosis. Mohda et al. [18] pointed out to the role of macrophages in initiating Th1 cells’ response during a T. spiralis infection. Those authors showed the mouse macrophages activated by larval antigens to be able to secrete IL-12 that induces activity and proliferation of Th1 lymphocytes.

In this study, the macrophage count in the jejunum mucosa basement membrane of the chitosan-treated mice increased until day 21 post-infection and remained, until the observations were terminated, at a level higher than that in the control. On the other hand, the transversely striated muscles revealed, in addition to T CD4+ and T CD8+ lymphocytes, a stronger macrophage mobilisation throughout the period of observations. The chitosan-treated mouse jejunum were also a site of a faster removal (“expulsion”) of adult parasites than in the control, the muscle larval count in those mice being clearly lower than in the control.

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