Studies On The Destructive Effect On Structure And Lymphocytes Of Spleens In Mice Infected With Schistosoma Japonicum

Schistosomiasis is still a neglected tropical disease hazardous to the health of human, it is mainly caused by Schistosoma japonicum in China. Tremendous progress in the control of schistosomiasis has been achieved over the past50years, however, it is still one of the most important infectious disease in China. Splenohepatomegalia is a notable character of schistosomiasis. Earlier studies mainly focus on the hepatic pathology, portal hypertension caused by trapped eggs was generally accepted as the most conceivable reason of splenomegaly in schistosomiasis, but no deepgoing investigation about the destructive effect on structure and lymphocytes of spleens was found. One of our recent studies showed that S. japonicum infection destroyed the structure of the lymphoid follicles through apoptosis of spenic lymphocytes, in this study, we observed the dynamic changes in structure of the lymphoid follicles in infected C57BL/6mice and splenic cells apoptosis caused by antigen from S. japonicum in vivo. In addition, we also attempted to investigate the relationship between the splenic granulomas and the reestablishment of the lymphoid follicles in the spleen of mice in advanced stage schistosomiasis.Material and methods1. Destructive effect of eggs of S. japonicum on splenic structure in C57BL/6mice1.1Dynamic observation of eggs deposited in the spleen of infected mice: Thirty-three male C57BL/6mice were infected percutaneously with20cercariae of S. japonicum and sacrificed at5^7^9..16weeks after infection. Spleen were embedded in paraffin and stained with hematoxylin and eosin (HE) prior to microscopic examination. Remanent spleen samples were weighed and ground into homogenate, all eggs released was quantified by light microscopy. Liver samples as control were digested in4%potassium hydroxide and for each mouse, total count of eggs was calculated through examination of three same volume digestion. 1.2Observation of destructive effect of eggs on splenic structure:Twelve male mice were divided randomly into4groups:mice injected intravenously with eggs、mice injected intravenously with soluble eggs antigen (SEA) or mice injected intravenously with soluble worms antigen (SWA) and mice injected surgically into the spleens. Eggs used for injection were obtained from mice then adjusted to2500-3000eggs/ml and used immediately, mice were injected intravenously0.1ml each time and lasted for4weeks; each mice injected SEA or SWA50μg every2days and lasted for4weeks;0.1ml suspension of nearly250eggs were injected surgically into into the lower pole of the spleen. Four weeks later, all groups of mice were sacrificed, the splenic paraffin sections were stained with H E and the splenic structures were observed. Twenty mice were divided into3groups:mice infected with single-sex of schistosome cercariae、mice infected with both sexes of schistosome cercariae and normal mice as control.9mice were convinced as single-sex of schistosome cercariae infected by checking the sex of adult worms and liver of mice after they were sacrificed. Each mice of the first2groups was infected percutaneously with20cercariae of S. japonicum, all mice were sacrificed at9weeks post infection, the weight of mice were compared among the groups and splenic structure was observed.2. Analyzation of protein fractions in SEA to identify the pro-apoptotic molecules to T cells2.1Apoptosis of splenic cells from mice infected with S. japonicum in vivo and ex vivo:Ten male mice were infected percutaneously with20cercariae of S. japonicum and sacrificed at5and8weeks post infection, single cell suspensions were prepared from spleens, splenic cells from naive or infected mice(five mice for each group) were stained with anti-B220and anti-CD3in combination with Annexin V to show total T or B cells and apoptosis cells of them. Apoptotic cells around the eggs in the spleen of infected or egg-injection mice were evaluated by TUNEL. Imuno histochemistry staining was used to show T cells localized in the granulomas of spleen and liver from mice infected for18weeks.2.2Apoptosis of splenic lymphocytes from mice infected for different times in vitro stimulated by SEA:Mice were infected percutaneously with20cercariae of S. japonicum at various intervals and sacrificed at same time, splenic cells of infected mice were pooled from three to four animals at each time point(because of limited number of cells left after TUNEL staining). Cells were cultured in the presence of different concentrations of SEA (15,30,60,120μg/ml). apoptotic cells labled by FITC-deoxynucleotides through TUNEL staining were analyzed by flow cytometry.2.3Apoptosis of splenic lymphocytes from mice of naive, immunized and8week after infected in vitro stimulated by SEA:Four male mice were infected with20cercariae of S. japonicum for8weeks,4mice were immunized through subcutaneously injection with50μg SEA in CFA for14days, another4normal mice were used as control. All mice were sacrificed at the same time, Splenic lymphocytes from mice of this3groups were cultured in the presence of SEA (40μg/ml,80μg/ml,120μg/ml), apoptotic B220+or CD3+cells labled with Annexin V were analyzed by flow cytometry.2.4Observation of T lymphocytes localized in the splenic egg granulomas: Mice infected with S. japonicum for18weeks were sacrificed, then spleens and livers were removed and embedded in paraffin, immunohistochemistry staining method were used to detect CD3+cells in the sections of spleen and liver.2.5Pro-apoptotic activity of separated SEA fractions molecule to splenic T cells:Soluble Egg Antigen (SEA) was prepared, proteins in the SEA were size-separated initially into3different fractions by superdex200gel filtration. They were concentrated by ultrafiltration using5kDa centricon concentrator. Molecules<5kDa was concentrated by lyophilization as fraction4. Each fraction (100μg/ml) was then tested for the presence of pro-apoptotic activity through annexin V and propidium iodide staining、DNA fragmentation and RT-PCR that aimed to determine the mRNA expression level of Fas, FasL. These studies narrowed the activity to fraction1. Subsequently, bands in fraction1were isolated by SDS-PAGE, then divided into3sub-fractions and electroeluted from the gel slices by electroelution tube, pooled fractions were concentrated and used for next stimulation (60μg/ml), the pro-apoptotic activity of the sub-fractions was tested again.3. Primary study on the reestablished lymphoid follicles in advancing infected miceFifteen male mice were infected with20cercariae of S. japonicum for18weeks, they were divided into2groups according to whether they have splenic granulomas or not, the structure of spleens were observed, serum antibodies、cytokines secreted by splenic lymphocytes stimulated in vitro and the ratio of follicular helper T Cells between the2groups were also examined. Results1. Destructive effect of eggs of S. japonicum on splenic structure in C57BL/6mice1.1Dynamic observation of eggs deposited in the spleen of infected mice:It is very common phenomenon that eggs deposited in the spleen of C57mice as infection progressed, at5weeks post infection, when eggs startting deposit in the liver, no eggs were found in the spleen, eggs were first seen in spleen at7weeks post infection, nearly75%(6/8) mice have eggs deposited in their spleens after9weeks post infection. Deposited eggs in the spleens forming granulomas or not, egg granulomas formed in the cortex frequently but not in the medulla in spleen while eggs deposited in the medulla did not induce granulomas even at16weeks post infection.1.2Observation of destructive effect of eggs on splenic structure:Four weeks after injection, the splenic structure of mice injected intravenously with eggs were destroyed, and characterized with decreased number of lymphoid follicles and blurred marginal zone. Lymphoid follicles around the eggs injected into the spleen were also seriously depauperated. The splenic weight of the mice infected with infected with both sexes cercariae (n=7,0.41g±0.03g) significantly more than that single-sex cercariae (n=9,0.15g±0.01g)(F=60.914, P<0.001) and normal mice (n=4,0.07g±0.01g)(P<0.01). However, the splenic structure of the mice infected with single-sex cercariae kept integrated.2. Analyzation of protein fractions in SEA to identify the pro-apoptotic molecules to T cells2.1Splenic cells apoptotic in vivo and ex vivo:TUNEL staining demonstrated apoptotic cells in the granulomas in situ, apoptotic cells were also seen around the eggs injected into the spleen, Flow cytometry of the stained cells revealed a sharp reduction in splenic B (F=33.789, P<0.001) and T (F=11.143, P<0.01) cells and a significantly increase of Annexin V positive T cells (F=12.436, P<0.01) at8weeks post infection in comparison to naive mice, but there was no significant changes in the number of apoptotic B cells (P>0.05).2.2Apoptosis of splenic lymphocytes from mice infected for different times in vitro stimulated by SEA:More cells underwent apoptosis at antigen concentration of120μg/ml than at60μg/ml at all time post infection while apoptosis of cells at concentrations below60μg/ml was not obvious, Cells from8weeks post infection seemed more susceptible to the SEA induced apoptosis.2.3Apoptosis of splenic lymphocytes from mice of naive, immunized and8week after infected in vitro stimulated by SEA:As concentration of SEA increases, T cells but not B cells underwent apoptosis, especially in cells from mice of immunized and infected. In this2groups, more T cells underwent apoptosis at antigen concentration of120μg/ml than at80μg/ml (F=0.139, P<0.05; F=47.608, P<0.001) while apoptosis of B cells at concentration of120μg/ml was not obvious (P>0.05).2.4Observation of T lymphocytes localized in the splenic egg granulomas: The result of immunohistochemistry staining showed that more CD3positive signals around the eggs in splenic granulomas than in liver granulomas.2.5Pro-apoptotic activity of separated SEA fractions molecule to splenic T cells:SEA were separated into4fractions, their pro-apoptotic activity to T cells were measured by DNA fragments ladder and Annexin V staining, mRNA expression level of Fas, FasL of T cells were also detected by RT-PCR, the result showed that significant pro-apoptotic activity was associated with fraction1(>55kDa). Fraction1was then divided into sub-fractions by molecule mass, final comparison showed that the pro-apoptotic activity was correlated with the fraction of molecules between55to72KDa. It induced more apoptosis in T cells in vitro (P<0.001).3. Primary study on the reestablished lymphoid follicles in advancing infected miceEgg granulomas were first seen in spleen of mice at7weeks post infection, lymphoid follicles reestablished or lymphoid follicles-like structures were seen in the spleen with egg granulomas. Mice infected with S. japonicum for18weeks were divided into2groups:group of mice with egg granuloma and group of mice without egg granuloma. mice with egg granuloma has higher serum antibodies, especially significant higher IgG2a (t=4.956, p<0.05). Under the stimulation with SEA or ConA, splenic lymphocytes from mice with egg granuloma secreted higher level of IL-4(F=25.119, P<0.001) and IL-5(F=14.234, P<0.001). The follicles reestablished composed mainly by B lymphocytes, PNA positive germinal center (GC) can not be detected in reestablished lymphoid follicles although mice with egg granuloma were found having higher ratio of follicular helper T (Tfh) Cells in their CD4+cells. Conclusions1. It is common phenomenon that eggs deposited in the spleen of C57BL/6mice as infection progressed, eggs of S. japonicum had a destructive effect on the splenic structure in C57BL/6mice.2. SEA induce apoptosis of splenic lymphocytes, higher concentrations of SEA have induced more apoptosis T cells, molecules between55and72kDa has more significant pro-apoptotic activity.3. Mice in advanced stage of schistosomiasis may have egg granulomas in their spleens and granulomas may change the local immune environment, characterized with prominent Th2response which may benefit for the reestablishment of the destructive lymphoid follicles in the spleen.