Trichuris Muris Nematode Parasite Intestine of Mice Infected with The

The Characterization of Intraepithelial Lymphocytes, Lamina Propria Leukocytes, and Isolated Lymphoid Follicles in the Large Intestine of Mice Infected with the Intestinal This information is current as Nematode Parasite Trichuris muris of September 28, 2021. Matthew C. Little, Louise V. Bell, Laura J. Cliffe and Kathryn J. Else J Immunol 2005; 175:6713-6722; ; doi: 10.4049/jimmunol.175.10.6713 Downloaded from http://www.jimmunol.org/content/175/10/6713

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Matthew C. Little,2 Louise V. Bell, Laura J. Cliffe, and Kathryn J. Else

Despite a growing understanding of the role of cytokines in immunity to the parasitic helminth Trichuris muris, the local effector mechanism culminating in the expulsion of worms from the large intestine is not known. We used flow cytometry and immunohistochemistry to characterize the phenotype of large intestinal intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) from resistant and susceptible strains of mouse infected with T. muris. Leukocytes accumulated in the epithelium and lamina propria after Downloaded from infection, revealing marked differences between the different strains of mouse. In resistant mice, which mount a Th2 response, the number of infiltrating CD4؉, CD8؉, B220؉, and F4/80؉ IEL and LPL was generally highest around the time of worm expulsion from the gut, at which point the inflammation was dominated by CD4؉ IEL and F4/80؉ LPL. In contrast, in susceptible mice, which mount a Th1 response, the number of IEL and LPL increased more gradually and was highest after a chronic infection had developed. At this point, CD8؉ IEL and F4/80؉ LPL were predominant. Therefore, this study reveals the local immune responses underlying the expulsion of worms or the persistence of a chronic infection in resistant and susceptible strains of mouse, respectively. In addition, for the first time, http://www.jimmunol.org/ we illustrate isolated lymphoid follicles in the large intestine, consisting of B cells interspersed with CD4؉ T cells and having a central zone of rapidly proliferating cells. Furthermore, we demonstrate the organogenesis of these structures in response to T. muris infection. The Journal of Immunology, 2005, 175: 6713–6722.

richuris muris is a natural mouse model of the nematode immunodeﬁcient SCID mice by the transfer of CD4ϩ donor cells parasite, Trichuris trichiura, one of the most prevalent hu- (9, 11). However, in this model, protective immunity can be ab- T man helminth infections worldwide. The range of protec- rogated (using a combination of Ab against the gut-homing adhe- ␤ tive immunity mounted against T. muris in the mouse infection sion molecules mucosal addressin cell adhesion molecule-1, 7 model varies depending upon the background genetics of the in- integrin, and CD103 (11)) by blocking T cell migration to the gut by guest on September 28, 2021 bred strain of mouse (1, 2) and parallels the range of responses (12). This supports the theory that locally acting T cell-dependant observed within an outbred human population exposed to T. tri- effector mechanisms are responsible for the expulsion of T. muris chiura. The majority of mouse strains, such as BALB/c, are resis- from the large intestine. tant to T. muris and quickly expel the parasite, whereas a few Lamina propria leukocytes (LPL)3 and intraepithelial lympho- strains, such as AKR, are susceptible, allowing the development of cytes (IEL) are the effector compartments of the gut mucosal im- fecund adult parasites, culminating in a chronic infection of the mune system (13). By virtue of their anatomical location, IEL have cecum and proximal colon. the closest direct contact with foreign Ags derived from the gut It is now well established that a Th2-dominated response, char- lumen and are thought to play a key role in the immune responses acterized by the production of IL-4, IL-5, IL-9, and IL-13, is an to these Ags and in the pathogenesis of a variety of disease states. absolute requirement for the expulsion of worms by resistant Small intestinal IEL have been extensively studied in the mouse. strains of mouse (3–6). Susceptible strains, rather than failing to Most are T cells, but compared with peripheral T cells found in respond to T. muris, instead mount an inappropriate Th1 response secondary lymphoid organs, a high proportion of IEL are CD8ϩ, associated with high levels of IFN-␥ and IL-12 (7, 8). Despite this express TCR␥␦, and develop independently of the thymus. Thy- knowledge, the effector mechanism ultimately responsible for the mus-independent IEL, which can be either TCR␣␤ϩ or TCR␥␦ϩ, expulsion of T. muris by the host is not understood. Many of the are relatively abundant and express CD8 in its ␣␣ homodimeric Th2 responses typically associated with helminth infection, such as form. Contrastingly, the thymus-dependent population expresses mastocytosis, eosinophilia, and strong parasite-speciﬁc Ab re- TCR␣␤ and bears either CD4 or CD8 in its more familiar ␣␤ sponses, are not essential (9, 10). Resistance can be conferred to heterodimeric form (14–19). However, IEL from the large intestine are seldom studied de-

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom spite the marked differences in both function and luminal environ- ment between the different regions of the intestine and the devel- Received for publication January 10, 2005. Accepted for publication July 22, 2005. opment of diseases speciﬁc to the large intestine, such as ulcerative The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance colitis and colon cancer. Accordingly, a few studies have shown with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Wellcome Trust Grant 044494/Z (to M.C.L., L.V.B., and K.J.E.). 2 Address correspondence and reprint requests to Dr. Matthew C. Little, Faculty of 3 Abbreviations used in this paper: LPL, lamina propria leukocyte; E/S, excretory/ Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, secretory; IEL, intraepithelial lymphocyte; ILF, isolated lymphoid follicle; MLN, Manchester, U.K. M13 9PT. E-mail address: [email protected] mesenteric lymph node; p.i., postinfection.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 6714 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION that IEL of the large intestine have a different phenotype and func- samples were quantified using recombinant murine cytokine standards tion than those of the small intestine (20–22). Within the large (R&D Systems). The plates were read at 405 nm. intestine, the proportion of CD8ϩ cells is lower; however, they still ϩ Parasite-specific Ab ELISA constitute a major subset of the T cell pool, with the ratio of CD4 to CD8ϩ being approximately equal. Principally, although thy- Serum was assayed by capture ELISA for T. muris-specific IgG1 and IgG2a as described previously (26). Briefly, Immulon 96-well plates mus-independent T cells, characterized by their expression of ␮ ␣␣ ␥␦ (Thermo Electron) were coated with 5 g/ml T. muris E/S Ag and incu- CD8 and TCR , predominate in the small intestine, they are bated with serum diluted through eight serial 2-fold dilutions from 1/20 to much less abundant in the large intestine (20–22). IEL from the 1/2560. Parasite-specific Ig was detected using either biotinylated anti- large intestine have much less cytolytic activity in vitro than IEL murine IgG1 (Serotec) or IgG2a (BD Biosciences). from the small intestine (20). Furthermore, although similar patterns of IFN-␥ production are seen, more of the type 2 cytokines, Isolation of IEL IL-4 and IL-5, are produced by IEL of the large intestine (21, 23). IEL were isolated by an accepted modification of the method described by Therefore, a pronounced regional specialization of epithelial T Davies and Parrott (27). Briefly, large intestines (cecum and ϳ6cmof cells is found in the gut. proximal colon) were removed, and macroscopically visible lymphoid ag- gregates on the cecum were cut off and dispensed with. Fat and connective Given that T. muris forms syncitial tunnels within the epithelium tissues were removed, and the large intestines were opened longitudinally, of the cecum and proximal colon (24), IEL are especially close to then washed twice, to remove the feces, in calcium- and magnesium-free the parasites and their Ag. Therefore, IEL may play a major role in HBSS containing 2% FCS (at 4°C). The intestinal tissue from 10 mice was the immune response to and, ultimately, the elimination of T. pooled and then cut into 1-cm pieces. This tissue was placed in 50-ml tubes and washed three times in HBSS containing 2% FCS at 4°C. The tissue was muris. Because Th cells are essential for the expulsion of T. muris, Downloaded from transferred to 25-cm3 tissue culture flasks and incubated at 37°C in HBSS we hypothesize that these cells migrate to the large intestine in containing 10% FCS, 0.2 mmol/l EDTA, 1 mmol/l DTT, 100 U/ml peni- resistant strains of mouse in temporal association with the expul- cillin, and 100 ␮g/ml streptomycin. After 20 min, the flasks were shaken sion of worms. In susceptible strains of mouse, the accumulation vigorously for 30 s, and the supernatant containing the IEL was separated of inappropriate subsets of leukocytes in the large intestine might from the tissue fragments using a stainless steel sieve. The supernatant was collected and put on ice, the tissue fragments were retuned to the flasks, underlie their inability to expel the parasite, leading to the devel- and the process was repeated. After this process, the tissue pieces were opment of a chronic infection. This study characterizes large in- examined microscopically to ensure that the epithelium had been removed http://www.jimmunol.org/ testinal IEL throughout the infection of resistant and susceptible and that the characteristic folds and ridges of the lamina propria were still mice with T. muris. intact. The epithelial cell suspensions from both incubations were pooled, washed, and suspended in RPMI 1640 at 4°C, then passed through nylon wool columns. The cell suspension was collected and suspended in 44% Materials and Methods Percoll, which was layered on top of 67.5% Percoll and centrifuged at Mice 600 ϫ g for 20 min at 4°C. The IEL were collected from the interface between the Percoll gradients and prepared for phenotypic analysis by flow Specific pathogen-free AKR and BALB/c mice were purchased from Har- cytometry. lan U.K. and were maintained in individually ventilated cages. In all experiments, male mice were infected with T. muris when they were 6–8 wk Flow cytometry old. SCID mice were used to investigate the influx of macrophages into the by guest on September 28, 2021 large intestine during T. muris infection in the absence of an adaptive IEL were washed in PBS containing Dulbecco’s A and B salts, 0.1% so- immune system and to examine whether isolated lymphoid follicles (ILF)- dium azide, and 2% FCS. Triple staining was performed on samples of 1 ϫ like structures could develop in the absence of lymphocytes. SCID mice 106 cells using a combination of the following Abs: anti-CD3-PE, biotin- were bred in isolators at the University of Manchester, and male mice used ylated anti-CD25 used in conjunction with streptavidin-TriColor (BD Bio- when they were 6–8 wk old. The animal studies were reviewed and ap- sciences), and one of the following FITC-conjugated Abs: anti-CD4, anti- proved by the Home Office and were performed under the legal require- B220, anti-CD30 (Serotec), anti-CD69, or anti-CD103. Alternatively, ments of the Animal (Scientific Procedures) Act (1986). triple staining was conducted using anti-CD8␣-PE or anti-TCR␤-PE, anti- CD8␤-FITC or anti-TCR␥␦-FITC, and biotinylated anti-CD25 used in con- Parasite junction with streptavidin-TriColor (BD Biosciences). Appropriate isotype controls of irrelevant specificity (rat IgG2a-PE, rat IgG2b-PE, rat IgG2a- T. muris was maintained as described previously (24). Mice were infected ϳ FITC, rat IgG2b-FITC, hamster IgG-FITC, and biotinylated rat IgG2a) orally with 150 infective eggs. Mice were killed at various time points were included. All Abs were obtained from BD Biosciences unless other- postinfection (p.i.), and the worm burdens in the large intestine were as- wise stated. All cells were stained for 30 min in the dark on ice and then sessed as described previously (1, 2). T. muris excretory/secretory (E/S) Ag fixed by the addition of 2% formaldehyde in PBS, 0.1% sodium azide, and was prepared from adult worms after a 4-h in vitro culture as described 2% FCS. The data were acquired on a FACSCalibur flow cytometer and previously (24). analyzed using CellQuest Pro software (both from BD Biosciences). Cell culture Immunohistochemistry Mesenteric lymph nodes (MLN) were removed, and single-cell suspensions were prepared. Total MLN cells were suspended in RPMI 1640 me- Mice were killed at various time points p.i. with T. muris. Age-matched, uninfected control mice were killed on day 21 p.i. Approximately 6 mm of dium supplemented with 5% FCS, 2 mmol/l L-glutamine, 100 U/ml peni- cillin, 100 ␮g/ml streptomycin (all from Invitrogen Life Technologies), the proximal colon (juxtaposed to the distal cecum) was removed, trisected, and 60 ␮mol/l monothioglycerol (Sigma-Aldrich). The cells were stimu- and carefully positioned in OCT embedding medium (R. A. Lamb). The ␮ ϫ 6 tissue was snap-frozen in liquid nitrogen-chilled isopentane (BDH-Merck), lated with 50 g/ml T. muris E/S Ag in 48-well plates (5 10 cells/well) ␮ at 37°C for 24 h. The cell supernatants were harvested and stored at Ϫ20°C and 6- m sections were cut using a cryomicrotome. The tissue was air- until they were assayed for cytokines. dried for1htomaximize its adhesion to gelatin-coated microscope slides, then fixed using 4% paraformaldehyde (Sigma-Aldrich) in PBS for 10 min Cytokine ELISA at 4°C. Slides were washed in PBS, and endogenous peroxidase activity was quenched using 0.064 mg/ml sodium azide, 1.5 U/ml glucose oxidase, Cytokines were analyzed by sandwich ELISA as described previously (25). and 1.8 mg/ml D-glucose (Sigma-Aldrich) in PBS for 20 min at 37°C. After The following mAb pairs were used: IFN-␥, R4-6A2, and XMG1.2; IL-4, another wash in PBS, nonspecific binding sites in the sections were blocked 11B11, and BVD-24G.2; IL-5, TRFK5, and TRFK4 (all from BD Bio- using 10% normal rat serum (Sigma-Aldrich) in PBS for1hatroom sciences); IL-9, 249.2 (E. Schmitt, University of Mainz, Mainz, Germany), temperature. Endogenous avidin and biotin binding sites were blocked us- and DC9302C12 (BD Biosciences); and IL-12 p40, C15.6 (G. Trinchieri, ing a commercial kit according to the manufacturer’s instructions (Vector Schering-Plough, Dardilly, France), and C17.8 (BD Biosciences). The de- Laboratories). The sections were incubated at room temperature for 1 h tection Ab were biotinylated, and a streptavidin-peroxidase (Roche) system with one of the following rat anti-mouse biotinylated mAb: anti-CD4 was used in conjunction with the substrate ABTS (Sigma-Aldrich). The (5 ␮g/ml; BD Biosciences), anti-CD8␣ (10 ␮g/ml; BD Biosciences), The Journal of Immunology 6715 anti-B220 (10 ␮g/ml; BD Biosciences), anti-CD11b (5 ␮g/ml; BD Bio- ␮ ␤ sciences), anti-F4/80 (5 g/ml; Caltag Laboratories), or anti- 7 integrin (10 ␮g/ml; BD Biosciences). Alternatively, a number of sections were incubated in parallel with the appropriate biotinylated isotype control Abs (BD Biosciences). A Vectastain Elite avidin-biotin-peroxidase complex kit, followed by a 3,3Ј-diaminobenzidine chromagen kit, were then used according to the manufacturer’s instructions (Vector Laboratories). The sections were counterstained in Harris’s hematoxylin solution and mounted in Aquamount aqueous mounting medium (BDH-Merck). The number of positively stained cells per 20 crypt units was assessed in triplicate by light microscopy after randomization and blinding. In vivo labeling and in situ immunohistochemical visualization of proliferating lymphocytes Mice were injected i.p. with 10 mg of BrdU, which is taken up by proliferating cells during the S phase of the cell cycle. After 40 min, the mice were killed, and the detection of nuclei that had incorporated BrdU was performed by immunohistochemistry using an anti-BrdU mAb (Mas 250b; Harlan Sera Laboratories) as described previously (28). Statistical analysis

Statistical analysis was performed by ANOVA and Tukey’s post-test (us- Downloaded from ing the statistical package GraphPad PRISM 3.0). Results BALB/c mice are resistant to T. muris and mount a Th2 response, whereas AKR mice are susceptible to infection and mount a Th1 response http://www.jimmunol.org/ After infection with T. muris, BALB/c mice expelled the majority of the worms from the large intestine before day 21 p.i. and were free of worms by day 35 p.i. In contrast, AKR mice failed to expel the worms and were chronically infected with T. muris (Fig. 1A). MLN cells from uninfected mice and mice infected with T. muris were stimulated in vitro with T. muris E/S Ag. The cells from infected BALB/c mice produced the Th2 cytokines IL-4, IL-5, and IL-9, whereas AKR mice displayed a Th1-skewed, Ag-specific cytokine response, characterized by higher levels of IFN-␥ and by guest on September 28, 2021 IL-12 p40 (Fig. 1B). Furthermore, the Ag-specific Ab produced by BALB/c mice in response to infection were predominantly IgG1, FIGURE 1. The immune response to infection with T. muris and the in contrast to AKR mice, in which high levels of IgG2a were isolation of IEL from the large intestine. Susceptible (AKR) and resistant detected (Fig. 1C). Taken together, these data confirmed that the (BALB/c) strains of mouse were infected with ϳ150 T. muris ova. Mice immune response to T. muris was Th2 and Th1 dominated in re- were killed at several time points p.i. (three mice per group), and the num- sistant BALB/c mice and susceptible AKR mice, respectively. ber of T. muris worms inhabiting the large intestine was counted and is expressed as the mean Ϯ SEM (A). MLN cells, isolated from uninfected Reduction in yield of IEL during the infection mice and from infected mice on day 21 p.i., were stimulated in vitro for 24 h with T. muris E/S Ag. The supernatant was analyzed for cytokines by Using accepted standard methods, the number of IEL extracted ELISA. The data are expressed as the mean Ϯ SEM of five mice (B). from the large intestine of one uninfected mouse was typically in Ag-specific IgG was analyzed in the serum of uninfected (naive) mice and 6 6 the range of 1 ϫ 10 to 1.5 ϫ 10 (Fig. 1D). However, p.i., the infected mice 35 days p.i. The serum was serially diluted, and only data for yield of IEL decreased (Fig. 1D) in temporal association with the the 1/80 dilution (within the linear range) are expressed as the mean Ϯ expulsion of worms from the large intestine (Fig. 1A). The lowest SEM of five mice (C). IEL were isolated from the large intestine of unin- yield of IEL from BALB/c mice occurred on days 14 and 21 p.i., fected (naive) mice and from mice infected with T. muris at various time when the worms were being actively expelled. The IEL yield grad- points p.i., as indicated. A representative example of the yield of IEL ually returned to normal as the mice became free of infection. In during the infection is shown in D. AKR mice, a progressive reduction in the yield of IEL was noted as the chronic infection developed. The expulsion of worms or the development of a chronic infection is associated with enteropathy of CD3ϩCD4ϩ IEL occurred that differed between the two strains in the large intestine, including crypt hyperplasia, goblet cell hy- of mouse. In BALB/c mice, the percentage of CD3ϩCD4ϩ IEL perplasia, and the hypersecretion of mucus (24). This appears to increased gradually, and at its peak (21 days p.i.) had risen by interfere with our method of IEL extraction from the large intes- Ͼ2-fold (Fig. 2A). There followed a decline in the percentage of tine, leading to an artificially low yield p.i. The percentage of IEL CD3ϩCD4ϩ IEL in BALB/c mice, approaching preinfection levels ␣ ␤ ϳ expressing CD103 ( E 7 integrin) was 85%, and this was un- by 35 days p.i. (Fig. 2A). In contrast, the percentage of affected by infection (data not shown). CD3ϩCD4ϩ IEL in AKR mice continued to rise throughout the

ϩ infection, reaching a 3-fold increase by 35 days p.i. (Fig. 2A). The number of CD4 IEL increased p.i. CD4 detection by immunohistochemistry allowed the numerical Using flow cytometry, the percentage of IEL exhibiting a Th cell quantification of CD4ϩ IEL and LPL within the large intestine. phenotype (CD3ϩCD4ϩ) was ϳ5% in both BALB/c and AKR Dynamic changes in the number of CD4ϩ IEL (Fig. 2, B and C) mice (Fig. 2A). However, p.i., dynamic changes in the percentage and LPL (Fig. 2, B and D) occurred p.i. with T. muris, revealing 6716 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION Downloaded from http://www.jimmunol.org/ FIGURE 2. Analysis of Th cells in the large intestine of susceptible (AKR) and resistant (BALB/c) strains of mouse infected with T. muris. IEL were isolated from the large intestine of uninfected (naive) mice and from mice infected with T. muris at various time points p.i., as indicated. CD3ϩCD4ϩ IEL were analyzed by flow cytometry. The data are presented graphically in A as the mean Ϯ SD of four separate experiments. Immu- nohistochemical staining of CD4ϩ cells in the proximal colon was conducted at all time points p.i. A representative photographic example is shown for BALB/c mice 21 days p.i., where positively stained cells are brown. Arrows show examples of IEL and LPL. Scale bar ϭ 50 ␮m(B). by guest on September 28, 2021 Quantitative analysis of the immunohistochemistry is illustrated in C (CD4ϩ IEL) and D (CD4ϩ LPL). The values represent the mean Ϯ SEM of five mice at each time point, and the results are representative of three FIGURE 3. Analysis of CTLs in the large intestine of susceptible p Ͻ 0.001. (AKR) and resistant (BALB/c) strains of mouse infected with T. muris. IEL ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .separate experiments were isolated from the large intestine of uninfected (naive) mice and from mice infected with T. muris at several time points p.i., as indicated. CD8␣- differences between the strains of mouse. Initially, in BALB/c ϩ and CD8␤-expressing IEL were analyzed by flow cytometry. A represen- mice, the number of CD4 IEL increased, reaching a peak 21 days tative example for naive mice is shown in A, where the percentage of gated p.i., then declined at later time points. In contrast, the number of ϩ cells in each quadrant of the scatter plots is denoted. The flow cytometry CD4 IEL in AKR mice continued to increase throughout the data are presented graphically (as the mean Ϯ SD of three separate exper- infection (Fig. 2, B and C). These strain-specific patterns of change iments) in B only for CD8␣ϩ IEL. Immunohistochemical staining of ϩ in CD4 IEL during infection are essentially similar to those dem- CD8␣ϩ cells in the proximal colon was conducted at all time points p.i. A onstrated by flow cytometry (Fig. 2A). The number of CD4ϩ LPL representative photographic example is shown for BALB/c mice 21 days increased in both strains of mouse p.i. (Fig. 2, B and D). There p.i., where positively stained cells are brown. Arrows show examples of ϩ ϭ ␮ were roughly 15 times more CD4 cells in the lamina propria than IEL and LPL. Scale bar 50 m(C). Quantitative analysis of the immu- ␣ϩ ␣ϩ in the epithelium regardless of infection with T. muris (Fig. 2, C nohistochemistry is illustrated in D (CD8 IEL) and E (CD8 LPL). The values represent the mean Ϯ SEM of five mice at each time point, and and D). ;p Ͻ 0.01 ,ءء .the results are representative of two separate experiments Ͻ ءءء The number of CD8ϩ IEL increased p.i. , p 0.001. The expression by IEL of both isoforms of CD8 was investigated using flow cytometry. This revealed major differences between the within the large intestine to be quantified. Paradoxically, although strains of mouse in the relative abundance of both CD8␣␣ϩ and CD8ϩ IEL were found in BALB/c mice by flow cytometry (Fig. 3, CD8␣␤ϩ IEL in the large intestine. Twice the percentage of A and B), virtually no CD8ϩ IEL or LPL were detected by immu- CD8␣␣ϩ IEL were detected in AKR mice compared with BALB/c nohistochemistry in uninfected BALB/c mice (Fig. 3D). After in- mice before infection (Fig. 3A). Conversely, although a significant fection, there was a limited influx of CD8ϩ IEL and LPL into the proportion of IEL expressed CD8␣␤ in BALB/c mice, the per- large intestine in BALB/c mice (Fig. 3, C–E). Compared with centage of these cells was negligible in AKR mice (Fig. 3A). After BALB/c mice, CD8ϩ IEL and LPL were relatively abundant in infection, CD8␣ϩ IEL were more abundant in AKR mice than in uninfected AKR mice, and the number of these cells was consid- BALB/c mice (Fig. 3B). erably greater p.i. (Fig. 3, C–E). Approximately 20 times more Immunohistochemical detection of the CD8 ␣-chain (which is CD8ϩ leukocytes were found in the lamina propria than in the expressed by all CD8ϩ cells) enabled all CD8ϩ IEL and LPL epithelium in uninfected AKR mice (Fig. 3, D and E). The Journal of Immunology 6717

Analysis of TCR subunits to normal levels, whereas in AKR mice the highest levels of B There were ϳ4 times more TCR␣␤ϩ IEL than TCR␥␦ϩ IEL in cells occurred at later time points (Fig. 4, C and D). However, uninfected mice, as determined by flow cytometry (data not there was also some disparity in the data between the two methods ␣␤ϩ of B cell analysis. Most strikingly, by immunohistochemical anal- shown). In BALB/c mice, the percentages of TCR and ϩ TCR␥␦ϩ IEL were 59 and 15%, respectively. No clear pattern of ysis, there were significantly more B220 IEL in AKR mice than change over time p.i. to TCR␣␤ϩ or TCR␥␦ϩ IEL was evident in in BALB/c mice 21 days p.i. (Fig. 4, B and C), although by flow either strain of mouse (data not shown). cytometry the converse was found (Fig. 4A).

ϩ Large influx of macrophages into the large intestine p.i. The number of B220 IEL increased p.i. ϩ Ϫ Because no macrophage markers are entirely specific, two such The percentages of T cells (CD3 B220 ) and B cells ϩ Ϫ markers (F4/80 and CD11b) were used to investigate more clearly (B220 CD3 ) in the IEL compartment were evaluated by flow the influx of macrophages into the large intestine by immunohis- cytometry. T cells were always more abundant than B cells (data ϩ Ϫ tochemistry. In practice, there was little difference between the two not shown). In BALB/c mice, the percentage of B220 CD3 IEL methods of analysis. In uninfected mice, F4/80ϩ and CD11bϩ IEL increased 21 days p.i. (Fig. 4A). An increase in the percentage of ϩ ϩ ϩ Ϫ were scarce (Fig. 5, A, B, D, and E), whereas F4/80 and CD11b B220 CD3 IEL was associated with a decrease in the percentage ϩ Ϫ LPL were relatively abundant (Fig. 5, A, C, D, and F). After in- of CD3 B220 IEL (data not shown). fection, there was a significant increase in the number of F4/80ϩ B cells in the large intestine were also examined by immuno- ϩ ϩ and CD11b IEL (Fig. 5, A, D, B, and E). There was a more histochemistry. There were ϳ4 times more B220 IEL than LPL ϩ ϩ striking increase in the number of F4/80 and CD11b cells in the Downloaded from in uninfected mice (Fig. 4, C and D). After infection, there were ϩ lamina propria p.i., uncovering major differences between the two greater numbers of B220 IEL and LPL (Fig. 4, B–D). The pat- strains of mouse. In BALB/c mice, the number of F4/80ϩ and terns of this change over time broadly mirrored those found by CD11bϩ LPL reached a peak 21 days p.i., declining at later time flow cytometry (Fig. 4A). That is to say, the number of B cells points. In contrast, the numbers of F4/80ϩ and CD11bϩ LPL in initially increased p.i. in BALB/c mice and subsequently returned AKR mice continued to increase throughout the infection (Fig. 5, ϩ

C and F). Interestingly, there were twice as many F4/80 and http://www.jimmunol.org/ CD11bϩ cells in the lamina propria of BALB/c mice than in AKR mice 21 days p.i. (Fig. 5, A, C, D, and F). In uninfected SCID mice there were 52 Ϯ 11 F4/80ϩ LPL/20 crypts (data not shown). After infection, the number of F4/80ϩ LPL continued to increase in SCID mice (139 Ϯ 12 F4/80ϩ LPL/20 crypts after 21 days and 192 Ϯ 17 F4/80ϩ LPL/20 crypts after 35 days; data not shown), resembling that found in AKR mice (Fig. 5C).

Expression of activation markers by IEL does not change p.i. by guest on September 28, 2021 There were more CD25ϩ IEL in AKR mice than in BALB/c mice (Fig. 6, A–C). Only a small proportion of CD3ϩ IEL expressed CD25 in either strain of mouse (Fig. 6A); accordingly, few CD4ϩ IEL expressed CD25 (Fig. 6B). The majority of CD25ϩ IEL in AKR mice were B cells (Fig. 6C). In contrast to BALB/c mice, in which few B220ϩ IEL expressed CD25, in AKR mice most B220ϩ IEL expressed CD25 (Fig. 6C). A high proportion of CD3ϩ IEL expressed the activation marker CD69 (Fig. 6D). The infection of mice by T. muris caused no discernable difference in the percentage of CD25ϩ or CD69ϩ IEL (Fig. 6). Less than 0.5% of the IEL expressed CD30 (an early marker of activation) in uninfected mice or at any time point p.i. (data not shown). Lymphoid follicles filled mainly with B cells are present in large intestine Numerous pronounced follicular structures were discovered in the large intestine of both AKR and BALB/c mice. These follicles FIGURE 4. Analysis of B cells in the large intestine of susceptible were comprised primarily of closely packed B cells interspersed by ϩ ϩ (AKR) and resistant (BALB/c) strains of mouse infected with T. muris. IEL small clusters of CD4 T cells. CD8 T cells were much less were isolated from the large intestine of uninfected (naive) mice and from common, but could occasionally be found at the edge of the fol- mice infected with T. muris at several time points p.i., as indicated. licles. Macrophages were found at the marginal zone of the folli- ϩ Ϫ B220 CD3 IEL were analyzed by flow cytometry, and the data are pre- cles, and occasionally, individual macrophages could also be de- sented in A as the mean Ϯ SD of three separate experiments. Immunohis- ϩ tected more centrally. Some cells around the outside of the follicles tochemical staining of B220 cells in the proximal colon was conducted at expressed ␣ integrin (Fig. 7A). BrdU was incorporated by leuko- all time points p.i. A representative photographic example is shown for 4 cytes in the core of the follicles, suggesting a central zone of pro- BALB/c mice 21 days p.i., where positively stained cells are brown. Ar- rows show examples of IEL and LPL. Scale bar ϭ 50 ␮m(B). Quantitative liferating B cells (Fig. 7B). Infrequently, structures resembling fol- analysis of the immunohistochemistry is illustrated in C (B220ϩ IEL) and licles were also found in the large intestine of infected SCID mice, D (B220ϩ LPL). The values represent the mean Ϯ SEM of five mice at although they consisted of neither B cells nor macrophages (Fig. each time point, and the results are representative of two separate experi- 7C). In some mice, multiple follicles were found in the gut sections p Ͻ 0.001. (Fig. 7D). In AKR mice, but not in BALB/c mice, there was a ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .ments 6718 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION

BALB/c and AKR strains of mouse. CD8ϩ IEL were more abundant in AKR mice than in BALB/c mice. Furthermore, although CD8␣␣ϩ IEL were found in both strains of mouse, CD8␣␤ϩ cells were found only in BALB/c mice. Indeed, there are numerous examples in the literature of phenotypic differences between strains (20–22). Regardless of interstrain differences, we were able to compare the present study with previous investigations because BALB/c mice are routinely used. Consistent with previous descriptions of IEL isolated from the large intestine (20, 21), ϳ76% were CD3ϩ T cells, of which 80% were TCR␣␤ϩ and 20% were TCR␥␦ϩ. However, a discrepancy with previous reports was evident when the T cell subsets were subjected to a more detailed analysis. In the present study the proportions of CD3ϩ IEL expressing CD4 and CD8 were 7 and 51%, respectively, and by deduction, the remain- ing T cells (ϳ42%) were double negative (CD4ϪCD8Ϫ). Others estimate a higher proportion of CD4ϩ T cells (between 32 and 72%), with the ratio of CD4ϩ to CD8ϩ being approximately equal ϩ (20–22). Indeed, the relative abundance of CD4 T cells from the Downloaded from large intestine is thought to distinguish them from T cells of the small intestine, where CD8ϩ cells predominate (20–22). Further- more, contrary to previous reports, CD8␣␣ϩ cells were found to be more plentiful than CD8␣␤ϩ, again resembling the phenotype commonly associated with the small intestine (17, 21, 22). Al- Ϫ Ϫ

though double-negative (CD4 CD8 ) cells constituted a major T http://www.jimmunol.org/ cell subset in the present study, previous reports suggest they are less prevalent (from 1 to 27%) (20–22). Therefore, in this study we reproducibly define a large intestinal T cell phenotype that con- trasts with previous descriptions. It was vital in the present study to use only mice that were free of gastrointestinal infections before infection with the cecum-dwelling nematode T. muris. However, laboratory mice are often chronically infected with the gut-dwelling nematodes Aspiculuris tetraptera and Syphacia obvelata (29), and as we discuss later, infection does alter the balance of different by guest on September 28, 2021 IEL subsets. Thus, our results may differ from those of previous reports in part due to the use of specific pathogen-free laboratory mice housed in individually ventilated cages. Hence, this study challenges previous descriptions of IEL isolated from the large FIGURE 5. Analysis of macrophages in the large intestine of suscepti- intestine (suggesting that they are phenotypically similar to IEL ble (AKR) and resistant (BALB/c) strains of mouse infected with T. muris. from the small intestine), and therefore, fundamentally, the poten- Staining for either F4/80 or CD11b, macrophages were detected by immu- tial for CD8-mediated cytotoxicity in the large intestine is greater nohistochemistry in the proximal colon of uninfected (naive) mice and than described previously. from mice infected with T. muris. Representative photographic examples The number of IEL extracted from the large intestine p.i. ap- of F4/80 (A) or CD11b (D) staining are shown for BALB/c mice 21 days p.i. Positively stained cells are brown. Arrows show examples of IEL and peared to be affected by infection-associated gut enteropathy. It is LPL. Scale bars ϭ 50 ␮m(A and D). Quantitative analysis of the immu- therefore misleading and does not reflect the actual number of IEL nohistochemistry is illustrated in B (F4/80ϩ IEL), C (F4/80ϩ LPL), E in the large intestine p.i. However, a reliable account of IEL num- (CD11bϩ IEL), and F (CD11bϩ LPL). The values represent the mean Ϯ bers is given by histological examination, demonstrating the SEM of five mice at each time point. The results are representative of three marked accumulation of IEL in the large intestine p.i., uncovering -p Ͻ 0.001. differences between the contrasting stains of mouse and reinforc ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .separate experiments ing the value of using both flow cytometry and immunohistochemistry. In BALB/c mice, the number of IEL increased (peaking at significant increase, per mouse in the number of follicles at later ϳ21 days p.i.), then reverted toward normal levels, corresponding time points p.i. (Fig. 7E). However, the follicles tended to be larger with the kinetics of worm expulsion. In contrast, the number of in BALB/c mice than in AKR mice, particularly p.i. (Fig. 7F). IEL in AKR mice increased and remained high as the infection progressed. Discussion Th cells are known to play a pivotal role in the mechanism of T. Typical of previous investigations, only ϳ1 ϫ 106 IEL were ob- muris expulsion, because the depletion of CD4ϩ cells confers a tained from the large intestine of an individual uninfected mouse. susceptible phenotype to resistant strains of mouse (30). As we Consequently, to analyze their phenotype comprehensively, it is confirm, the generation of a Th2 response is essential for the ex- common practice to pool IEL from several individuals (20–22). A pulsion of worms (3–6). A locally acting mechanism for Th2 cells considerable percentage of IEL extracted from the large intestine has been postulated, yet no previous studies have shown the mi- ␣ ␤ ϩ expressed the classical IEL marker CD103 ( E 7 integrin), con- gration of CD4 cells into the large intestine. Importantly, the firming the reliability of our preparation technique. Some differ- present study demonstrates for the first time that in resistant mice ences in the phenotype of IEL were apparent between uninfected exhibiting a Th2 response, CD4ϩ Th cells do indeed accumulate in The Journal of Immunology 6719

FIGURE 6. The activation state of IEL in the large intestine of susceptible (AKR) and resistant (BALB/c) strains of mouse infected with T. muris. The expression of the lymphocyte activation markers CD25 and CD69 was analyzed by ﬂow cytometry in uninfected (naive) mice and in infected mice 21 days p.i. A, CD25 expression by T cells. B, CD25 expression by Th cells. C, CD25 expression by B cells. D, CD69 expression by T cells. The results are representative of two separate experiments. Downloaded from http://www.jimmunol.org/

the epithelium of the large intestine around the time of worm ex- intestine, the contamination of IEL by B cells from ILF is inevi- pulsion. In susceptible mice that mount a Th1 response, the num- table, because these follicular structures are intimately associated ber of CD4ϩ Th cells increases more gradually and is greatest with the epithelium. Furthermore, we suggest (with relevance to during the chronic phase of infection. Recently, several studies all previous studies of isolated IEL) that although LPL can be have suggested various potential effector mechanisms by which T. excluded from the preparations, a degree of contamination from by guest on September 28, 2021 muris may be expelled from the gut. These theories include an ILF is to be expected. Because ILF tended to be larger in increased rate of epithelial cell turnover (31) and the release of BALB/c mice than in AKR mice (particularly p.i.), this is the factors by goblet cells that may impair chemotaxis of the parasite most likely explanation for the abundance of B cells in IEL (32). Nevertheless, both these potential mechanisms depend upon preparations from resistant mice. Recently, other authors have the secretion of Th2 cytokines in the large intestine. Because the considered it inevitable that B cells from germinal centers con- present study suggests that Th2 cells migrate to the large intestine taminate IEL suspensions (13). Therefore, the present study at the time of worm expulsion, this bridges the gap in our knowl- highlights the importance of immunohistochemistry as a vital edge between the well-characterized afferent immune responses to tool to address this problem. the putative efferent immune effector mechanisms of worm It is difficult to describe the activation state of IEL, because expulsion. some markers of activation were widely expressed by IEL, and In contrast to resistant mice, a large population of CD8ϩ cells others were expressed by only a small minority of cells; the ma- infiltrated the mucosa of the large intestine in susceptible mice p.i. jority of CD3ϩ IEL expressed CD69, whereas CD25, in accor- However, a recent study in our laboratory shows that the depletion dance with other studies (20), was expressed by a very small per- of CD8ϩ cells in susceptible mice fails to influence the develop- centage of T cells. Because CD3ϩ IEL isolated from the large ment of a chronic infection (33). Therefore, although they are not intestine have been shown to express CD25 after TCR stimulation essential for the development or maintenance of a chronic in- in vitro (20), it is perhaps surprising that there was no change in the fection, the sheer magnitude of CD8ϩ cell migration into the frequency of these cells p.i. with T. muris. Interestingly, CD25 was gut underlines the inability of susceptible mice to mount an expressed most notably by B cells in AKR mice. A distinct pop- appropriate protective immune response to the parasite. A much ulation of CD25ϩCD4ϩ cells, namely, regulatory T cells, is lower number of CD8ϩ IEL was found in resistant BALB/c thought to play a role in the persistence of infection to the parasite mice p.i. These cells were detected less frequently by immu- Leishmania major (34). However, a role for regulatory T cells in nohistochemistry than by flow cytometric analysis of isolated the immune response to T. muris seems unlikely, because Ͻ1% of IEL, perhaps indicating a difference in the sensitivity of the the IEL were CD25ϩCD4ϩ. contrasting methods. There was a sizeable influx of leukocytes into the lamina propria The accumulation of B cells in the gut was also noted p.i. B cells of the large intestine p.i. The phenotype of LPL differed markedly were shown by immunohistochemistry to be more numerous in from that of IEL. CD4ϩ cells were up to 10 times more abundant susceptible mice. However, in resistant mice, especially 21 days in the lamina propria than in the epithelium. Conversely, CD8ϩ p.i., the percentage of B cells in the isolated IEL (as shown by flow and B220ϩ LPL were less numerous than CD8ϩ and B220ϩ IEL. cytometry) was considerable, somewhat contradicting the immu- Macrophages made up a large fraction of the LPL p.i., whereas nohistochemical findings. During their extraction from the large they were rarely encountered in the IEL population. This work 6720 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION

FIGURE 7. Identiﬁcation and characterization of ILF in the proximal colon of mice. Staining for a range of leukocyte markers, ILF were characterized by immunohistochemistry in the proximal colon of uninfected mice and from mice infected with T. muris. Representative photographic examples are shown for uninfected BALB/c mice (except for F4/80 staining, which is from an uninfected AKR mouse), where positively stained cells are brown (A). Immuno-

staining revealed the incorporation of Downloaded from BrdU into proliferative cells in the proximal colon. A representative photographic example of BrdU staining (where BrdU-containing cells are brown) is shown for a particularly large ILF from an AKR mouse 21 days p.i.

(B). ILF-like structures were character- http://www.jimmunol.org/ ized by immunohistochemistry in the proximal colon of SCID mice 35 days p.i. (C). A typical example of immunohistochemical staining of B220ϩ cells in AKR mice 28 days p.i., showing multiple ILF (D). The number of ILF per 6-mm section of proximal colon was determined histologically. A representative example is shown in E, where ﬁve mice by guest on September 28, 2021 per group were used (except for AKR mice 35 days p.i., where six mice were used). Each dot on the graph denotes the number of ILF found per 6-mm section of proximal colon in an individual mouse. To represent the relative size of ILF between AKR and BALB/c mice 21 days p.i., examples (ILF of average size stained for B220) are shown in F. All scale bars ϭ 100 ␮m. The results are representative of three separate experi- .p Ͻ 0.05 ,ء .ments

confirms that IEL and LPL are distinct components of the GALT. kine balance (35). Taken together, this suggests a potential role for Intriguingly, the migration of macrophages into the lamina propria macrophages in the mechanism of worm expulsion from the gut. reached a peak around the time of worm expulsion, at which point Future work will investigate the phenotype and role of lamina there were approximately twice as many macrophages in resistant propria macrophages in this context. mice than in susceptible mice. Greater numbers of macrophages Lymphoid structures known as ILF have been identified in both were observed p.i. in the lamina propria of SCID mice, suggesting small and large intestines of humans (36, 37); more recently, they that the accumulation of macrophages in the large intestine is in have been discovered and characterized, in some detail, in the part an innate immune response to the parasite. A recent study in small intestine of mice (38, 39). They are composed of a large B our laboratory shows that mice devoid of the macrophage chemo- cell area, including a germinal center, and like Peyer’s patches, the kine CCL2 fail to expel T. muris, and this is associated with fewer epithelium overlying the follicles contains M cells, suggesting that macrophages in the lamina propria and an altered Th1/Th2 cyto- they are inductive sites for local IgA responses. Although Hamada The Journal of Immunology 6721 et al. (38) and Dohi et al. (40) discovered ϳ50 ILF in the large 5. Faulkner, H., J. C. Renauld, J. Van Snick, and R. K. Grencis. 1998. Interleukin-9 intestine of normal mice in addition to ϳ10 colonic patches, they enhances resistance to the intestinal nematode Trichuris muris. Infect. Immun. 66: 3832–3840. neither described in detail nor showed any photographic examples 6. Richard, M., R. K. Grencis, N. E. Humphreys, J. C. Renauld, and J. Van Snick. of these structures (38, 40). We found numerous pronounced fol- 2000. Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in licular structures, invisible from the serosal or mucosal surface, in Trichuris muris-infected mice. Proc. Natl. Acad. Sci. USA 97: 767–772. 7. Else, K. J., L. Hultner, and R. K. Grencis. 1992. Cellular immune responses to the the large intestine of both AKR and BALB/c mice. Consisting of murine nematode parasite Trichuris muris. II. Differential induction of Th-cell B cells interspersed with CD4ϩ T cells and having a central zone subsets in resistant versus susceptible mice. Immunology 75: 232–237. 8. Bancroft, A. J., K. J. Else, J. P. Sypek, and R. K. Grencis. 1997. Interleukin-12 of rapidly proliferating cells, these lymphoid aggregations are promotes a chronic intestinal nematode infection. Eur. J. Immunol. 27: 866–870. analogous to ILF. Therefore, the present study extends our knowl- 9. Else, K. J., and R. K. Grencis. 1996. Antibody-independent effector mechanisms edge of the GALT, illustrating ILF in the large intestine of mice for in resistance to the intestinal nematode parasite Trichuris muris. Infect. Immun. 64: 2950–2954. the first time. While this manuscript was in revision, it was shown 10. Betts, C. J., and K. J. Else. 1999. Mast cells, eosinophils and antibody-mediated that although colonic patches and ILF of the large intestine both cellular cytotoxicity are not critical in resistance to Trichuris muris. Parasite contain M cells, they have a distinct structure and organogenesis Immunol. 21: 45–52. 11. Betts, C. J., M. L. deSchoolmeester, and K. J. Else. 2000. Trichuris muris: CD4ϩ (41). Lymphoid aggregations, equivalent in size to ILF, but devoid T cell-mediated protection in reconstituted SCID mice. Parasitology 121: of B cells, were found in the large intestine of SCID mice. This 631–637. suggests that the organogenesis of ILF depends on neither B cells 12. Picarella, D., P. Hurlbut, J. Rottman, X. Shi, E. Butcher, and D. J. Ringler. 1997. ␤ Monoclonal antibodies specific for 7 integrin and mucosal addressin cell adhe- nor T cells. Indeed, similar structures have been identified in the sion molecule-1 (MadCAM-1) reduce inflammation in the colon of scid mice small intestine of other immunodeficient mouse models, such as reconstituted with CD45Rbhigh CD4ϩ T cells. J. Immunol. 158: 2099–2106. athymic nude mice (nu/nu) and RAG-2 knockout mice (38). It has 13. Brandtzaeg, P., and R. Pabst. 2004. Let’s go mucosal: communication on slippery Downloaded from ground. Trends Immunol. 25: 570–577. been shown that ILF are formed in the small intestine in response 14. Bonneville, M., C. A. Janeway, Jr., K. Ito, W. Haas, I. Ishida, N. Nakanishi, and to normal gut flora in the cecum (39). In the present study there S. Tonegawa. 1988. Intestinal intraepithelial lymphocytes are a distinct set of ␥␦ was a significant increase p.i. with T. muris in the number of large T cells. Nature 336: 479–481. 15. Goodman, T., and L. Lefrancois. 1988. Expression of the ␥-␦ T-cell receptor on intestinal ILF in AKR mice. These findings provide evidence for intestinal CD8ϩ intraepithelial lymphocytes. Nature 333: 855–858. the organogenesis of ILF in response to luminal stimuli. In our 16. Bandeira, A., S. Itohara, M. Bonneville, D. O. Burlen, S. T. Mota, A. Coutinho, and S. Tonegawa. 1991. Extrathymic origin of intestinal epithelial lymphocytes model of T. muris infection, it is intriguing that ILF formation http://www.jimmunol.org/ bearing T-cell antigen receptor ␥␦. Proc. Natl. Acad. Sci. USA 88: 43–47. occurs specifically in susceptible AKR mice. It may arise due to 17. Guy-Grand, D., N. Cerf-Bensussan, B. Malissen, M. Malassis-Seris, C. Briottet, the chronic infection of these mice, where the epithelium is ex- and P. Vassalli. 1991. Two gut intraepithelial CD8ϩ lymphocyte populations posed for a longer duration to T. muris Ag in the gut. This may with different T cell receptors: a role for the gut epithelium in T cell differentiation. J. Exp. Med. 173: 471–481. represent a diversion of the local immune response away from an 18. Lefrancois, L. 1991. Phenotypic complexity of intraepithelial lymphocytes of the effective Th cell-dominated mechanism toward an inappropriate small intestine. J. Immunol. 147: 1746–1751. 19. Rocha, B., P. Vassalli, and G. D. Guy. 1991. The V␤ repertoire of mouse gut response dominated by B cells. The role of ILF in the pathogenesis ϩ ϩ homodimeric ␣ CD8 intraepithelial T cell receptor ␣/␤ lymphocytes reveals a of gastrointestinal infections and diseases should be investigated in major extrathymic pathway of differentiation. J. Exp. Med. 173: 483–486. the future. 20. Camerini, V., C. Panwala, and M. Kronenberg. 1993. Regional specialization of

the mucosal immune system: intraepithelial lymphocytes of the large intestine by guest on September 28, 2021 In conclusion, in this study we characterize for the first time the have a different phenotype and function than those of the small intestine. J. Im- accumulation of cells into the epithelium and lamina propria of the munol. 151: 1765–1776. large intestine during infection of mice with T. muris. There were 21. Beagley, K. W., K. Fujihashi, A. S. Lagoo, S. Lagoo-Deenadaylan, C. A. Black, A. M. Murray, A. T. Sharmanov, M. Yamamoto, J. R. McGhee, C. O. Elson, et marked differences in this respect between resistant and suscepti- al. 1995. Differences in intraepithelial lymphocyte T cell subsets isolated from ble strains of mouse. In resistant BALB/c mice, the local inflam- murine small versus large intestine. J. Immunol. 154: 5611–5619. mation was dominated by CD4ϩ IEL and F4/80ϩ LPL at the time 22. Boll, G., A. Rudolphi, S. Spiess, and J. Reimann. 1995. Regional specialization of intraepithelial T cells in the murine small and large intestine. Scand. J. Im- of worm expulsion, in contrast to susceptible AKR mice, where munol. 41: 103–113. ϩ ϩ CD8 IEL and F4/80 LPL were predominant during the 23. Yamamoto, M., K. Fujihashi, K. W. Beagley, J. R. McGhee, and H. Kiyono. chronic phase of infection. Therefore, this study reveals the 1993. Cytokine synthesis by intestinal epithelial lymphocytes. J. Immunol. 150: 106–114. local immune responses underlying the expulsion of worms or 24. Wakelin, D. 1967. Acquired immunity to Trichuris muris in the albino laboratory the persistence of chronic infection, respectively. Furthermore, mouse. Parasitology 57: 515–524. we describe and illustrate ILF in the large intestine of mice and 25. Else, K. J., and R. K. Grensis. 1991. Cellular immune responses to the murine nematode parasite Trichuris muris. I. Differential cytokine production during demonstrate the organogenesis of these structures in response to acute or chronic infection. Immunology 72: 508–513. T. muris infection. 26. Else, K. J., G. M. Entwistle, and R. K. Grensis. 1993. Correlations between worm burden and markers of Th1 and Th2 cell subset induction in an inbred strain of mouse infected with Trichuris muris. Parasite Immunol. 15: 595–600. Acknowledgments 27. Davies, M. D., and D. M. Parrott. 1981. Preparation and purification of lympho- We are grateful to Neil E. Humphreys for helpful discussions and advice. cytes from the epithelium and lamina propria of murine small intestine. Gut 22: 481–488. 28. Potten, C. S., D. Booth, N. J. Cragg, G. L. Tudor, J. A. O’Shea, C. Booth, Disclosures F. A. Meineke, D. Barthel, and M. Loeffler. 2002. Cell kinetic studies in the murine ventral tongue epithelium: mucositis induced by radiation and its protec- The authors have no financial conflict of interest. tion by pre-treatment with keratinocyte growth factor (KGF). Cell Prolif. 35(Suppl. 1): 32–47. 29. Sueta, T., I. Miyoshi, T. Okamura, and N. Kasai. 2002. Experimental eradication References of pinworms (Syphacia obvelata and Aspiculuris tetraptera) from mice colonies 1. Else, K. J., D. Wakelin, D. L. Wassom, and K. M. Hauda. 1990. MHC-restricted using ivermectin. Exp. Anim. 51: 367–373. antibody responses to Trichuris muris excretory/secretory (E/S) antigen. Parasite 30. Koyama, K., H. Tamauchi, and Y. Ito. 1995. The role of CD4ϩ and CD8ϩ T cells Immunol. 12: 509–527. in protective immunity to the murine nematode parasite Trichuris muris. Parasite 2. Else, K. J., D. Wakelin, D. L. Wassom, and K. M. Hauda. 1990. The influence of Immunol. 17: 161–165. genes mapping within the major histocompatibility complex on resistance to Tri- 31. Cliffe, L. J., N. E. Humphreys, T. E. Lane, C. S. Potten, C. Booth, and churis muris infections in mice. Parasitology 101: 61–67. R. K. Grencis. 2005. Accelerated intestinal cell turnover: a new mechanism of 3. Else, K. J., F. D. Finkleman, C. R. Maliszewski, and R. K. Grencis. 1994. Cy- parasite expulsion. Science 308: 1463–1465. tokine-mediated regulation of chronic intestinal helminth infection. J. Exp. Med. 32. Artis, D., M. L. Wang, S. A. Keilbaugh, W. He, M. Brenes, G. P. Swain, 179: 347–351. P. A. Knight, D. D. Donaldson, M. A. Lazar, H. R. P. Miller, et al. 2004. RELM␤/ 4. Bancroft, A. J., A. N. McKenzie, and R. K. Grencis. 1998. A critical role for FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal IL-13 in resistance to intestinal nematode infection. J. Immunol. 160: 3453–3461. tract. Proc. Natl. Acad. Sci. USA 101: 13596–13600. 6722 LARGE INTESTINAL LEUKOCYTES AND T. muris INFECTION

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