Abstract: The role of B cells in the pathogenesis of inflammatory bowel disease is not well understood. We utilized a pathogen-driven model to evaluate the function of B cells in the development of acute ileitis. B cell deficient mice (B -/-) exhibited a significant delay in the development of acute, lethal ileitis as well as the number of intracerebral cysts following oral infection with tissue cysts of Toxoplasma gondii. Oral infection in the B deficient mice with a sub-lethal dose of tissue cysts resulted in significantly reduced expression and as well as secretion of TNF-a , IFN-g , iNOS and IL-10 at day 3 and 7, but not day 30 after infection compared to the parental control mice. CD19+ cells isolated from the mesenteric lymph nodes, Peyer's patch or lamina propria of parasite infected C57BL/6 mice produced considerably greater quantities of TNF-a compared to uninfected control mice. In vitro, co-culture of antigen primed CD19+ B cells with CD4+ T cells increased the production of detectable IFN-g in the culture supernatant suggesting one potential mechanism for the enhanced Th1 cytokine profile in mice following oral infection with T. gondii.

Certain strains of inbred mice, in particular C57BL/6, develop an acute, lethal hyperinflammatory response of the small intestine following oral infection with tissue cysts of T.gondii (1). The hyperinflammation is characterized by the loss of normal cytologic architecture, infiltration of inflammatory cells and tissue necrosis with early mortality. Reversal of this inflammation may result in prolonged survival. IFN-g plays an essential role in the mediation of the hyperinflammatory bowel disease in these strains of mice (2). Mice with either genetic deficiencies of IFN-g (gene knockout) or depleted of this cytokine by mAb are rendered susceptible to oral acquisition of parasites and die from parasitemia. Thus IFN-g has a dichotomous role in the host: first as a protective cytokine and second as a mediator of severe hyperinflammation.

The functional immune role of B cells and antibody production in resistance against T. gondii remains uncertain (3). In a series of recent studies by Kang, H and co-workers, uMT mice were used to analyze the role of B cells in host resistance to this parasite (4).

These mice which are generated by disruption of one of the membrane exons of the u-chain gene have no detectable B cells or circulating Ab, yet display normal development of the T lymphocyte compartment (4). It is reported that uMT mice have normal Ag-presenting function for priming of CD4+ T cells to most soluble Ags as well as unimpaired CD8+ T lymphocytes response (4). More over, CD4 T cells deficient mice exhibited impaired resistance to a challenge infection with T. gondii, and administration immune sera partially restore this deficiency (5). This suggests that CD4 T cells can also participate to protection via their role as helper cells for protection of isotype-switched antibodies.

Recently we have reported that CD40/CD154 interaction is important in the development of the acute ileitis following oral infection. In the three intestinal compartments (lamina propria, Peyers patch and mesenteric lymph nodes) evaluated the increase in CD40 expression was found to be primarily within the B cell compartment. FACScan analysis indicated that a high frequency of the positive cells express both CD19 and CD40 (6). In this paper, the functional role for the increase in CD19+ B cells following oral infection with T. gondii is explored. Our data suggest that these cells maybe involved in the early production of several important Th1-type cytokines that are associated with both idiopathic and pathogen-driven inflammatory bowel disease.

C57BL/6 (B6) mice and B6.129S2-Igh-6 ^tm1Cgn Tnfrsf5^tm1Kik mice (B -/-) which are deprived of B cells, were obtained from Jackson Laboratories (Bar Harbor, ME). Each group was composed of six mice and each experiment was repeated at least twice.

The strain 76K of T. gondii was used. It produces large numbers of cysts containing bradyzoites in the brain of infected mice. Mice were infected orally by intragastric gavage of cysts collected from the brain of infected mice. Cysts were maintained by passage every 2 months in naïve mice. For this study, brain tissue containing cysts were suspended in saline buffer, and the mice were orally challenged with cysts.

Toxoplama lysate antigen (TLA) was prepared from tachzoites of the RH strain of T. gondii. The parasites were cultured in human fibroblasts, released by forced extrusion through a 27-gauge needle and separated from host cell debris on a Percoll gradient. The parasites were sonicated eight times (18,000 Hz, 4° C) at 10s intervals. The sonicate was centrifuged at 10,000 ´ g for 15 mins, soluble antigen were collected in the supernatant, and the protein concentration was determined by a BCA protein Assay Kit (Rockford, Illinois, USA). The antigen was aliquoted and stored at -20 ° C.

At day 7 after parasite challenge, mice were sacrificed by CO₂ inhalation. The intestines were preserved in 10% buffered formalin. Fixed tissues were embedded in paraffin, serially sectioned at 5 µm and stained with hematoxylin and eosin.

Peyer’s patches (PP) and mesenteric lymph nodes (MLN) were excised from the wall of the small intestine, and minced in culture medium. The cell suspensions were passed through a cell strainer (Fisher Scientific, Chicago, IL), then a sterilized gauze band to remove cell debris and washed twice with cold RPMI 1640 medium. Mononuclear cells were obtained by centrifugation on a Ficoll layer (d=1.077).

Intestinal lamina propria mononuclear cells (LPMC) were isolated as described (7, 8). Briefly following dissection and removal of Peyer’s patches, the sectioned (0.3cm) intestines were incubated in RPMI containing 25 mM EDTA, 100 UI/ml penicillin-streptomycin, 1% fungizone, 50 UI/ml gentamicin (Sigma, St Louis, MO) under a magnetic stirring (300 rpm) at room temperature (six times, 20 min). The intestine pieces were then incubated at 37 ° C into RPMI-10%FCS with 125 UI/ml collagenase VIII (Sigma). After 3 hours, the digested suspension containing LPMC was filtered on cell strainer and the pellet resuspended in RPMI-10%FCS. Mononuclear cells were obtained by centrifugation on a Ficoll layer (d=1.077). CD4+ and CD19+ cells were purified by a standard positive selection procedure using microbeads and the MACS system (Miltenyi Biotec, Aubum, CA). This procedure routinely allow ³ 95% of cell purity.

Intracellular cytokines staining

Cell suspension of LPMC, MLN and PP containing 1¥106 cells were added to 96-well plates and washed twice in PBS.before stimulation with PMA and ionophore for 4 hours. Cells were then incubated (20 min, 4ƒC) with 100 ml of 1% Fc-block (CD32/CD16), to prevent nonspecific Ab staining After washing cells were incubated (1 h on ice) in the presence of 1% FITC conjugated surface marker antibody to CD19 (1D3) (PharMingen, San Diego, CA). Cells were then fixed 30 min in 2% paraformaldehyde, washed and then permeabilized (10 min) in 1%BSA/PBS containing 0.5% saponin. Finally, cells were washed and resuspended in 1 % BSA/PBS containing saponin and incubated with anti TNF-a antibodies for 30 min. After washing, the cells were fixed with 1% paraformaldehyde in PBS and analyzed by FACScan (Becton Dickinson, Mountain View, CA) the following day.

Primed or unprimed CD19+ and CD4+ T cells from the lamina propria were co-cultured together in various combination and were and were not restimulated with 10ug/ml TLA. Primed cells were isolated from 7 days earlier infected mice and unprimed cells were isolated from naïve mouse. The supernatants were collected after 72 hours of co-culture and frozen at -20° C before being examined for cytokine content by an ELISA assay.

Sera were collected at day 7 after infection, the concentration of INF-g, TNF-a and IL-10 were measured by ELISA following the manufacture's instructions (Biosouce, Camarillo, CA).

The tissues from intestine and spleen were frozen in liquid nitrogen and stored at —80ƒC until RNA preparation. The samples were homogenized in 2.0 ml Trizol reagent (Life Technology, Grand Island, NY) and incubated (5 min, 20 ° C) to allow the complete dissociation of nucleoprotein complexes. After adding 0.2 ml of chloroform per 1 ml of Trizol reagent and incubating them (3 min, 20 ° C), the samples were centrifuged at 12,000 x G (15 min, 4 ° C). RNA remains exclusively in the upper aqueous phase. RNA precipitation was done with isopropyl alcohol, and washed with 75% ethanol. The isolated mRNA was resuspended in RNase-free water. The concentration was determined by UV spectroscope at 260 nm.

Cytokine mRNA expression in the intestine and spleen were detected as described (8), 10 mg of mRNA were used and radioactively labeled for RPA analysis. Cytokine mRNA expressions in the cells were detected with RiboQuant multi-Probe RNase Protection Assay (RPA) System Kit (PharMingen, San Diego, CA) as described by the manufacturer. Briefly, the probe either for cytokine detection iNOS, TNF-a, IFN-g, and TGF-b1 and TGF-b2 were labeled with ³²P. Then, the hybridization procedure between RNA samples and the labeled probe were performed. RNA samples were digested by RNase treatment 45 min at 30°C. The RNase digests were extracted carefully. Samples were loaded in 5% acrylamide gel. For quantification, autoradiographs were scanned and band densities were analyzed by NIH Image 1.61/ppc software in Macintosh computer. The housekeeping gene probes GAPDH, allows for normalizing sampling and technique errors to permit comparison of individual mRNA species between samples. Results were expressed as the % of the intensity of the band corresponding to the studied cytokines relative to the intensity of the housekeeping RNA.

Quantification of the parasite burden was carried out by PCR. Intestine and spleen were recovered from infected mice at day 7 post infection. Genomic DNA was extracted from tissue using the Qiamp tissue kit (Qiagen, Valencia, CA), and 400 ng of each samples were analyzed. Amplification of parasite DNA was performed using primers specific for the Toxoplasma B1 gene (5' -GGAACTGCATCCGTTCATGAG-3' AND 5' -TCTTTAAAGCTTCGTGTCT-3'), a 35-fold sequence found in all known parasite strains (9, 10). A134 bp competitive standard containing the primer template sequences as the 194 bp B1 PCR fragment was generated and used as an internal standard for competitive PCR. Amplification of the 194 base pairs segment of the B1 gene and the 134 base pairs segment of the internal standard was performed in a 50 ml reaction mix containing 1.25 units of Amplitaq DNA polymerase, 1¥ buffer (Perkin Elmer), 0.2mM each of dGTP, dATP, dTTP, dCTP and 0.4mM (each) B1 primers. To quantify the number of parasites from the tissue, T gondii DNA was isolated from a known number of cell culture-derived extracellular tachyzoites. The toxoplasma B1 sequence was used as a probe to quantify tissue parasite load and run in parallel with the infected tissues, a known number of parasites were used as a standard to quantitate the number of parasites/microgram of tissue DNA (9,10).

Data were expressed as means and standard deviations and plotted in corresponding figures. The differences between experimental groups and comparison between groups were examined with Student t-test.

B -/- mice exhibited an increased survival rate after oral infection.

B -/- mice were infected per os with a lethal dose of T. gondii tissue cysts containing bradyzoites. We observed a statistically significant increase in delay to death compared to B6 wild type mice (Fig. 1). Histological analysis performed at different times after infection demonstrated considerable inflammation in the small intestine of the control B6 mice compared to the B -/- animals (Fig. 2). At day 7 post infection the intestinal architecture is markedly altered in which the villi are shortened and the lamina propria is swollen and packed with inflammatory cells. There is considerable necrosis and hemorrhage evident at the tip of the villi. In contrast, at day 7 post infection, the B -/- intestinal mucosal surface appeared normal with no definitive evidence of either inflammation or necrosis. However, at day 9 after infection when all of the control mice had succumbed, histologic changes consistent with early inflammation were evident in the small intestine of the B -/- mice (Fig. 2). The degree of intestinal inflammation increased over the next 48 hours at which time the B -/- mice succumbed. The delayed time to death in the B -/- mice suggested that B cells may participate to the early development of the acute inflammatory response.

Overproduction of the Th1-type cytokines IFN-g and TNF-a with subsequent induction of nitric oxide has been associated with intestinal necrosis in parasite infected B6 mice (11). The expression of these pro-inflammatory cytokines was assessed in B -/- mice post oral infection with a lethal dose of T. gondii tissue cysts. Compared to the wild type susceptible B6 mice, B -/- mice at day 7 PI produced significantly less mRNA for iNOS, IFN-g and TNF-a (Fig. 3A). At day 9 PI all of the control mice had succumbed to infection. Analysis of the intestinal tissue for cytokine production in the B -/- mice demonstrates a marked increase in the production of iNOS consistent with that observed at an earlier time point in the control mice (Fig. 3A). The pattern and kinetics of cytokine mRNA production in the spleen is similar to that observed in the intestinal samples (Fig. 3B). A sera ELISA was performed in an attempt to quantify the production of IFN-g and TNF-a protein secretion in the B -/- mice. At day 7 PI, B-/- mice produced significantly less TNF-a and IFN-g than the control wild type group (Fig. 3C). To identify if the lack of IFN-g, TNF-a and subsequently NO production was due to a lack of stimulation or to an overproduction of regulatory cytokines, the production of TGF-b and IL-10 was estimated in the two groups. Independent of the technique, both RPA and ELISA, demonstrated that TGF-b and IL-10 production remained low in B -/- mice post infection. This would suggest that the delay in the inflammatory process was not due to an overproduction of these downregulatory cytokines (data not shown).

To investigate the direct role of the B cell population in the gut inflammatory process, a cytokine analysis of the CD19+ cells isolated from the small intestine was performed. Recent studies have suggested that B cells can be subclassed into a Th1 -type profile and have the capacity to synthesize IFN-g in response to infection with T. gondii (12). TNF-a is an essential molecule involved in the gut inflammatory process. In order to assess the potential role for TNF-a in this experimental model of acute ileitis, a FACScan analysis was performed at day 7 PI. The lymphocyte population was isolated from the lamina propria. Approximately half of the lymphocytes isolated from the intestine of control mice expressed a profile consistent with the production of TNF-a. Of this population, CD19+ cells (B cells) account for 20% of those TNF-a producing cells. In the B -/- (CD19 negative) mice, a significantly lower percentage (20%) of the lamina propria lymphocytes produced TNF-a in response to infection (Fig. 4A). In both groups of mice, the overall proportion of TNF-a producing cells increased over time post infection, although production in the B -/- mice remained below that observed in the controls. A similar distribution in percentage of lymphocytes expressing CD19 and TNF-a was observed in other tissues associated with the intestinal compartment. At day 7 PI 48% and 20% of the purified lymphocytes from the Peyer’s patches and mesenteric lymph nodes respectively produced TNF-a and among them 16% and 9% (PP, MLN respectively) were CD19+ (Fig. 4B, C). In both Peyer’s patches and mesenteric lymph nodes, the proportion of lymphocytes producing TNF-a remain below 5%, and none of them were CD19+. These data suggested that CD19+ B cells may be directly involved in the inflammatory response via secretion of the pro-inflammatory molecule, TNF-a.

B cells enhancement IFN-g production by CD4 T cells.

In addition to their direct inflammatory activity, B cells may syngergize with T cell to amplify this process by the increased expression of IFN-g (12). To address this question, CD19+ cells isolated from the lamina propria of infected (primed) or non infected (unprimed) mice were co-cultured with T cells isolated from the same primed or unprimed mice. Unprimed mice produced negligible quantities of IFN-g. A low level of IFN-g production was noted in the culture supernatant of primed CD19+. Following parasite restimulation, the quantity of IFN-g produced rose significantly (244pg/ml to 550pg/ml) as shown in Table 1. Unprimed splenic CD4 T cell produced negligible quantities of IFN-g whereas primed CD4 cells in the presence or absence of parasite antigen produced significant quantities of IFN-g. Co-culture of primed CD4 T cells together with unprimed CD19+ cells nominally increased the production of IFN-g. However, there was a considerable increase in cytokine production when primed B cells were co-cultured with CD4 T cells (Table 1). Addition of parasite antigen further enhanced the production of this cytokine. These observations would suggest that CD19+ B cells may have the capacity to synergize with CD4+ T cells in order to enhance the inflammatory response to oral infection with this parasite.

B cells are required for the clearance of the parasite.

Previous observations from our lab have indicated that the intestinal inflammatory response although detrimental to the host also protects against parasite infection. In order to assess the role of B cells in parasite clearance from the gut, B -/- mice were infected per os and the parasite burden evaluated in their intestine at day 7 post infection. The B -/- mice exhibited a significantly higher parasite burden in their intestine compared to B6 control mice (Fig. 5A). In order to assess the long term consequences of B -/- on chronic parasite burden, B -/- mice were infected with a sub-lethal dose of tissues cysts. One month post infection, the cyst burden in the brain of B -/- and control B6 mice was enumerated. Brain cyst enumeration was consistent with the quantitative PCR for parasite burden performed at day 7 PI. B-/- mice harbored significantly more brain cysts than the wild type mice (Fig. 5B).

A pivotal role for cell mediated immunity and the induction of the Th1-type cytokines, IFN-g and TNF-a, has been well established for both experimental and human infection with Toxoplasma gondii. The role for the B cells in the immune response to this parasite is less well appreciated. Although antibody production has long been recognized as a hallmark of both acute and chronic infection with this parasite very little is understood regarding the role of B cells in the induction or persistence of the host response. Our observations suggest that one function of B cells may be the associated with the innate response to oral infection with this parasite.

Our observations indicate that the intestinal B cell compartment following oral infection with T. gondii is involved in the early immune response. B -/- mice exhibit a delay in time to death compared to the control parental strain of mice following oral infection with a lethal dose of T. gondii tissue cysts. This difference in time to death was associated with a reduction in several Th-1 inflammatory cytokines including TNF-a, IFN-g and NO. Our data further indicate that antigen primed CD19+ B cells account for a considerable amount of the TNF-a production in the parasite infected intestine. In vitro, intestinally derived B cells enhance the overall production of IFN-g when co-cultured with CD4+ T cells. The presence of intestinal B cells results in the reduction of parasite burden as determined by semi-quantitative PCR.

Taken together these data illustrate that B cell which are numerous in the intestine, especially in the Peyer’s patches where they outnumber all other lymphoid cells may

serve as a specialized population of APC. Several co-stimulatory molecules have been identified as participating to the interaction of the APC and effector lymphocytes. Among these co-stimulatory molecules, CD40 expressed on a variety of APC including B cells is involved in this response. A recent study from our laboratory has demonstrated that CD40-CD154 interaction is essential for the development of acute, lethal ileitis in certain strains of susceptible mice following oral infection with T. gondii. Although expressed by macrophages and dendritic cells, B cell from the lamina propria macrophages and dendritic cells, B cells isolated from the lamina propria, Peyer’s patches and mesenteric lymph nodes account for the major expression of CD40. The lack of CD40 expression correlated with a lack of intestinal pathology and as well as reduced expression of inflammatory cytokine and chemokine production. These data are consistent with the present observations and suggest that B cells are perhaps involved in the activation of IFN-g producing T cells via CD40-CD154.

Our data demonstrate that B cells produce TNF-a, a function well associated with B cell activation (13). Several recent trials of intravenously administered antitumour necrosis factor-alpha (TNF-a) monoclonal antibody have shown dramatic responses among patients with Crohn's disease (14-16).

These results indicate a primary role for TNF-a in the mediation of altered mucosal immune function in this disease. Moreover in a murine models of IBD in which there is an over-expression of TNF-a, (TNFDARE) the pivotal role of this cytokine is appreciated (17). We demonstrated that a lack of B cells led to a decrease in TNF-a production that might account for the postponed initiation of the immune response. Antigen activated B cells and antigen primed CD4+ T cells synergized when co-cultured to over-produce IFN-g. IFN-g and TNF-a maybe involved in the activation of other cells, including macrophages, dendritic cells and enterocytes to produce NO. Nitric oxide has a dichotomous role during T.gondii infection that is both microbicidal as well as pathologic (11). The low production of iNOS mRNA in the B -/- mice may further explain the delayed immune response in these mice.

Although the regulatory role of B cells in immunological response has received limited attention, it is speculated that B cells are involved in ulcerative colitis (18). However some reports the regulatory role of mature B cells in a murine model of inflammatory bowel disease. TCRa-/- mice spontaneously developed chronic colitis that is mediated by CD4TCRa-/b+ cells, and it is more severe in the absence of mature B cells. B cells were reported to play an important role in the development of chronic colitis in TCRa-/- mice by directly regulating the pathogenic T cells (19). This regulatory mechanisms is perhaps dependent upon a CD40-CD154 interaction and may involve IL-10 or TGF-b secretion In contrast to those observations and in agreement with our current observations when infected with Cryptosopridium parvum, TCRa-/- X JH-/- (B deficient mice) did not develop inflammatory or hyperplastic intestinal lesions as detected in C.parvum-infected TCRa-/- mice (20).

In our model of pathogen driven ileitis, B cells appear to play a direct role in the induction of the T cell response although the specific role for antibody function remains elusive. As anticipated, B -/- mice infected with T. gondii are devoid of antibody (data not shown) further confounding the functional role of the humoral response to this parasite as well as inflammation. In some IBD patients, in particular those with ulcerative colitis, a predominant colonic mucosal B cell response against human tropomyosin

isoform 5 was described (21). The median value of percentage of total immunoglobulin, IgA, G, M in those patients with ulcerative colitis or Crohn’s colitis was significantly higher than in non IBD patients; moreover in some murine model of IBD, the pathology is associated with an inappropriate B-cell response with autoantibody production (22-23). However it is unlikely that ileitis observed after oral infection with T. gondii is related to autologous antibody production because the effect produced by the lack of B cells is only transitory. The observed delay in the development of the hyperinflammatory ileitis would indicate that although B cells contribute to the lethal process there are other immune cell compartments involved in the initiation of this deleterious immune response.

The inflammatory process aims at the elimination of the parasites and a lack of IFN-g, iNOS and/or TNF-a has been shown to impair the host resistance against parasite invasiveness. In the current work, the delayed immune response induced a less efficient clearance of the parasite which is consistent with all other observations (11, 24-25).

1. Liesenfeld O, Kosek J, Remington JS, Suzuki Y. Association of CD4+ T cell-dependent, interferon-gamma-mediated necrosis of the small intestine with genetic susceptibility of mice to peroral infection with Toxoplasma gondii. Journal of Experimental Medicine 1996; 184: 597-607.

2. Suzuki Y, Orellana MA, Schreiber RD, Remington JS. Apr 1988. Interferon-gamma: the major mediator of resistance against Toxoplasma gondii. Science. 22;240(4851):516-518.

3. Sayles PC, Gibson GW, Johnson LL. B cells are essential for vaccination-induced resistance to virulent Toxoplasma gondii. Infect Immun. 2000 Mar;68(3):1026-33.)

4. Kang H, Remington JS, Suzuki Y.Decreased resistance of B cell-deficient mice to infection with Toxoplasma gondii despite unimpaired expression of IFN-gamma, TNF-alpha, and inducible nitric oxide synthase.J Immunol. 2000 Mar 1;164(5):2629-34.

5. Johnson LL, Sayles PC. Deficient humoral responses underlie susceptibility to Toxoplasma gondii in CD4-deficient mice. Infect Immun. 2002 Jan;70(1):185-91.

6. Li W, Buzoni-Gatel D, Debbabi H, Hu MS, Mennechet FJD, Durell BG, Noelle RJ, Kasper LH. CD40/CD154 ligation is required for the development of acute ileitis following oral infection with an intracellular pathogen in mice. Gastroenrology 2002; Mar.

7. Buzoni-gatel D, Debbabi H, Mennechet FJD, Martin V, Lepage AC, Schwartzman JD, Kasper LK. Acute inflammatory ileitis after intracellular parasite infection is controlled by TGF-b producing intraepithelial lymphocytes. Gastroenterology 2001; 120: 914-924.

8. Davies MD, Parrott DM. Preparation and purification of lymphocytes from the epithelium and lamina propria of murine small intestine. Gut 1981; 22: 481-488.

9 Burg JL, Grover CM, Pouletty P, Boothroyd JC. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. Journal of Clinical Microbiology 1989; 27: 1787-1792.

10 Khan IA, MacLean JA, Lee FS, Casciotti L, DeHaan E, Schwartzman JD, Luster AD. IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection. Immunity 2000; 12: 483-494.

11. Proc Natl Acad Sci U S A 1997 Dec 9;94(25):13955-60) A dichotomous role for nitric oxide during acute Toxoplasma gondii infection in mice. Khan IA, Schwartzman JD, Matsuura T, Kasper ).

12 Harris DP, Haynes L, Sayles PC, Duso DK, Eaton SM, Lepak NM, Johnson LL, Swain SL, Lund FE. Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat Immunol. 2000 Dec;1(6):475-82.

13. O'Garra A, Stapleton G, Dhar V, Pearce M, Schumacher J, Rugo H, Barbis D, Stall A, Cupp J, Moore K. Production of cytokines by mouse B cells: B lymphomas and normal B cells produce interleukin 10. Int Immunol. 1990;2(9):821-32.

14. Blam ME, Stein RB, Lichtenstein GR. Integrating anti-tumor necrosis factor therapy in inflammatory bowel disease: current and future perspectives. Am J Gastroenterol. 2001 Jul;96(7):1977-97. Review.

15. Bell S, Kamm MA. Antibodies to tumour necrosis factor alpha as treatment for Crohn's disease. Lancet. 2000 Mar 11;355(9207):858-60.

16. Kollias G, Douni E, Kassiotis G, Kontoyiannis D. On the role of tumor necrosis factor and receptors in models of multiorgan failure, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. Immunol Rev. 1999 Jun;169:175-94. Review.

17. Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F, Kollias G. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity. 1999 Mar;10(3):387-98.

18. Dunn-Walters DK, Boursier L, Hackett M, Spencer J. Biased JH usage in plasma cell immunoglobulin gene sequences from colonic mucosa in ulcerative colitis but not in Crohn's disease. Gut. 1999 Mar;44(3):382-6.

19. Mizoguchi E, Mizoguchi A, Preffer FI, Bhan AK. Regulatory role of mature B cells in a murine model of inflammatory bowel disease. Int Immunol. 2000 May;12(5):597-605.

20. Waters WR, Palmer MV, Wannemuehler MJ, Sacco RE, Harp JA. B cells are required for the induction of intestinal inflammatory lesions in TCRalpha-deficient mice persistently infected with Cryptosporidium parvum. J Parasitol. 2000 Oct;86(5):1073-7.

21. Onuma EK, Amenta PS, Ramaswamy K, Lin JJ, Das KM. Autoimmunity in ulcerative colitis (UC): a predominant colonic mucosal B cell response against human tropomyosin isoform 5. Clin Exp Immunol. 2000 Sep;121(3):466-71.

22. Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, Horak I. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell. 1993 Oct 22;75(2):253-61.

23. Davidson NJ, Leach MW, Fort MM, Thompson-Snipes L, Kuhn R, Muller W, Berg DJ, Rennick DM. T helper cell 1-type CD4+ T cells, but not B cells, mediate colitis in interleukin 10-deficient mice. J Exp Med. 1996 Jul 1;184(1):241-51.

24. Yap GS, Scharton-Kersten T, Charest H, Sher A. Decreased resistance of TNF receptor p55- and p75-deficient mice to chronic toxoplasmosis despite normal activation of inducible nitric oxide synthase in vivo. J Immunol. 1998 Feb 1;160(3):1340-5.

25. Suzuki Y, Joh K. Effect of the strain of Toxoplasma gondii on the development of toxoplasmic encephalitis in mice treated with antibody to interferon-gamma. Parasitol Res. 1994;80(2):125-30.

Fig. 1: Survival rate in B -/- mice after oral T. gondii infection. B -/- mice (-D -) has increased survival time after oral infection with T. gondii compared to B6 mice (- l -).

Fig. 2: Histological analysis. At day 7 after infection, B6 mice exhibited an acute ileitis (B) compared to naïve control (A). B -/- intestinal mucosal surface was observed to be almost normal at day 7 post infection (C) but signs of acute inflammation were observed at day 9 post infection (D).

Fig. 3: Cytokine production. B -/- mice produced less inflammatory cytokines at day 7 post infection compared to B6 mice. B6 mice expressed significantly (P < 0.01) more iNOS, IFN-g and TNF-a mRNA in the gut (A), spleen (B) and produced more of the corresponding protein in their sera (C). The difference between the two groups progressively decreased within time post infection. At day 9, the difference was less important for each of the studied cytokines.

Fig. 4: Phenotypic analysis of TNF- a producing cells. A phenotypic analysis was performed to identify CD19+ cells (B cells) that produced TNF- a among the lymphocytes isolated from the lamina propria (A), Peyer's patches (B) and mesenteric lymph nodes (C) at different times after infection.

Table 1: IFN-g production was increased in the co-culture of CD4+ T cells within CD19+ cells. Either primed (P) or unprimed (U) CD4+ T cells were co-cultured for 72 h within primed or unprimed CD19+ cells isolated from the lamina propria, results were expressed as the quantities of IFN- g (pg/ml) produced in the supernatant of the co-culture either with or without antigen restimulation (+ Tx).

Fig. 5: Parasite load was increased in the intestine and brain of B -/- mice compared to B6 mice. The B -/- mice exhibited a significant higher parasite burden in their intestine at day 7 and day 9 after infection as attested by PCR determination (A), B -/- mice harbored significantly more brain cysts than the B6 mice one month after infection (B).