Infection Trichuris Muris Failure to Expel in an Altered Th1/Th2

Absence of CC Chemokine Ligand 2 Results in an Altered Th1/Th2 Cytokine Balance and Failure to Expel Trichuris muris Infection

This information is current as Matthew L. deSchoolmeester, Matthew C. Little, Barrett J. of September 29, 2021. Rollins and Kathryn J. Else J Immunol 2003; 170:4693-4700; ; doi: 10.4049/jimmunol.170.9.4693 http://www.jimmunol.org/content/170/9/4693 Downloaded from

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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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Absence of CC Chemokine Ligand 2 Results in an Altered Th1/Th2 Cytokine Balance and Failure to Expel Trichuris muris Infection1

Matthew L. deSchoolmeester,2* Matthew C. Little,* Barrett J. Rollins,† and Kathryn J. Else*

Despite a growing understanding of the role of cytokines in immunity to intestinal helminth infections, the importance of chemokines has been neglected. As a chemokine with both chemoattractive properties and an ability to shape the quality of the adaptive immune response, CC chemokine ligand 2 (CCL2) was investigated as an attractive candidate for controlling resistance to these types of infection, which require highly polarized Th cell responses. We show here for the ﬁrst time that CCL2 plays an important role in the development of resistance to infection by the gastrointestinal nematode Trichuris muris. Thus, in the absence of CCL2, worm expulsion does not occur, and the lymph node draining the site of infection becomes a Th1-promoting environment. Elevated levels of IL-12 are produced by polarizing APCs, and the composition of the APC environment itself is perturbed, Downloaded from with reduced numbers of macrophages. The Journal of Immunology, 2003, 170: 4693–4700.

richuris muris is a natural mouse model of the gastrointes- moattractant protein-1, was originally described as being chemo- tinal nematode parasite Trichuris trichiura, one of the most tactic for monocytes (11–13), but is now known to also be che- T prevalent human helminth infections worldwide. Strain motactic for human NK cells (14, 15), memory T cells (16), and variation in the mouse gives a spectrum of phenotypes varying basophils (17). In the mouse, CCL2 is a functional monocyte che- http://www.jimmunol.org/ from resistant strains such as BALB/c and C57BL/6, in which the moattractant in vivo (18–22), and CCL2-deficient mice are unable parasite is quickly expelled, to a completely susceptible strain such to recruit monocytes to sites of inflammation in a number of mod- as AKR, in which fecund adult worms survive in the intestine for els, including to the peritoneum after thioglycolate administration many weeks or months (1). It is now well established that a Th2- (23). Importantly with regard to the development of a Th2-biased dominated immune response, characterized by the production of immune response, CCL2 causes a decrease in IL-12 production IL-4, IL-5, IL-9, and IL-13, is an absolute requirement for worm from activated macrophages and monocytes (24, 25). Furthermore, expulsion (2–5), and the development of a Th1 response associated the stimulation of naive T cells from OVA-specific TCR trans- with high levels of IFN-␥ and IL-12 leads to host susceptibility (6, genic mice in the presence of CCL2 induces IL-4 production and 7). For example, blocking the interaction of IL-4 with the IL-4R using the development of a Th2 phenotype (26, 27), and CCL2 stimu- by guest on September 29, 2021 an anti-IL-4R Ab induces susceptibility in normally resistant lates IL-4 production from Th2-differentiated cells, suggesting a BALB/K mice, whereas injection of an IL-4 complex into susceptible possible positive feedback mechanism for the maintenance or en- AKR mice causes worm expulsion (2). BALB/K mice also become hancement of Th2 responses (28). Analysis of the CCL2-deficient susceptible to infection following administration of IL-12 (7). Despite mouse revealed an inability to mount a Th2 response following this knowledge, the expulsion mechanism responsible for the elimi- OVA sensitization and challenge (29). Interestingly, this was not due nation of T. muris from its host is not yet understood, although the to abnormal T cell migration, suggesting a role for CCL2 in adaptive roles of mast cells, eosinophils, Ab, and CD4ϩ T cell-mediated cy- immunity through control of Th cell differentiation as well as in innate totoxicity are known not to be essential individually (8–10). immunity through effects on monocyte recruitment. Recently, an increasing number of chemokines have been shown With regard to models of infection, CCL2 is produced in re- to play important roles in shaping the adaptive immune response in sponse to the intestinal nematode, Trichinella spiralis, with ex- addition to their widely detailed chemotactic properties. The CC pression found in the serum and locally at the site of infection, in chemokine ligand (CCL)2,3 previously defined as monocyte che- the jejunum (30–32). CCL2-deficient mice are resistant to Leish- mania major infection, a model that requires a Th1 immune response for resolution (29), and the induction of lung granulomas *Department of Immunology, Microbiology, and Development, University of Manchester School of Biological Sciences, Manchester, United Kingdom; and †De- by Schistosoma mansonii egg Ag results in elevated levels of partment of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, CCL2 in whole lung tissue (33). A number of reports detail CCL2 Boston, MA 02115 being produced in human and mouse intestinal mucosa and asso- Received for publication November 11, 2002. Accepted for publication February ciated tissues (34, 35), leading to increased IL-4 and decreased 24, 2003. IL-12 production. Furthermore, human lamina propria macro- The costs of publication of this article were defrayed in part by the payment of page phages, endothelial cells, and intestinal epithelial cells (36) and mouse charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mesenteric lymph node (MLN) cells and Peyer’s patch cells (35) all 1 This work was supported by Wellcome Trust Grant 044494/Z (to M.L.D., M.C.L., have the potential to make CCL2. Therefore, it was decided to further and K.J.E.). investigate the role of this chemokine in intestinal infection. 2 Address correspondence and reprint requests to Dr. Matthew deSchoolmeester, De- It is reported here that mice naturally resistant to T. muris pro- partment of Immunology, Microbiology, and Development, University of Manchester duce higher levels of CCL2 than susceptible mice following in- School of Biological Sciences, 3.239 Stopford Building, Manchester, U.K. M13 9PT. E-mail address: [email protected] fection, and significantly, CCL2-deficient animals are totally un- 3 Abbreviations used in this paper: CCL, CC chemokine ligand; CCU, cecal crypt able to expel T. muris infection. This susceptible phenotype is units; E/S, excretory/secretory; MLN, mesenteric lymph node; p.i., postinfection. associated with a reduced Th2 response, characterized by low IL-4

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 4694 CCL2-DEFICIENT MICE ARE SUSCEPTIBLE TO T. muris INFECTION and high IFN-␥ and IL-12. Macrophage recruitment to the large binding sites in the sections were blocked using 20% normal rat serum intestine and local draining node (MLN) was also decreased in the (Sigma-Aldrich) in PBS for1hatroom temperature. Endogenous avidin CCL2-deficient mouse, and relatively fewer CD4ϩ cells were and biotin binding sites were blocked using a commercial kit according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). present in the MLN. The sections were incubated with either 4 ␮g/ml biotinylated rat anti- mouse f4/80 mAb (Caltag Laboratories, Burlingame, CA) or 5 ␮g/ml bi- Materials and Methods otinylated rat anti-mouse CD4 mAb (BD Biosciences) in PBS for1hat Animals room temperature. Alternatively, a number of sections were incubated in parallel with the appropriate biotinylated isotype control Abs (BD Bio- Ϫ Ϫ Breeding pairs of SCYA2 / (CCL2-deficient) (23, 37) were provided by sciences). A Vectastain Elite avidin-biotin-peroxidase complex kit fol- B. Rollins (Dana-Farber Cancer Institute, Boston, MA) and bred in-house lowed by a 3,3Ј-diaminobenzidine chromagen kit were then used according at University of Manchester. The appropriate control mice, C57BL/6, as to the manufacturer’s instructions (Vector Laboratories). Sections were well as AKR and BALB/c mice were purchased from Harlan U.K. (Bic- counterstained in Harris’ hematoxylin solution and mounted in Aquamount ester, U.K.). Male mice were used in all experiments. Mice were infected aqueous mounting medium (BDH Laboratory Supplies). The number of with T. muris when 8–10 wk old. positively stained cells per 20 crypt units was assessed in triplicate by light microscopy after randomization and blinding. Parasite Flow cytometry The maintenance of T. muris, the method of infection, and the production of excretory/secretory (E/S) protein were previously described (38). Infec- MLN cell suspensions from infected or control mice were prepared as tions were designed to give each animal ϳ100–150 infective eggs. Mice described previously, except that washing steps were performed in PBS were sacrificed at various time points after infection, and the worm burden containing Dulbecco’s A and B salts, 0.1% sodium azide, and 2% FCS. in the large intestine was assessed as previously described (39). Cells were resuspended at a final concentration of 2 ϫ 106/ml, and 0.5 ml was used for each sample. Single staining was performed with anti-CD4- Downloaded from Culture of MLN cells for in vitro cytokine measurement FITC, anti-CD8-FITC, anti-B220-FITC (all from BD Biosciences), or anti- F4/80-FITC (Caltag). Double staining was performed with a combination MLN cell suspensions from infected or control mice were prepared as of anti-CD11b-FITC and anti-I-A/I-E-PE (BD Biosciences), anti-CD11c- described previously (6, 40). Briefly, 5 ϫ 106 total MLN cells were resus- FITC and anti-I-A/I-E-PE, anti-CD11b-FITC and anti-F4/80-PE (Caltag), pended in RPMI 1640 medium supplemented with 5% FCS, 2 mM L- or anti-B220-FITC and anti-I-A/I-E-PE. Appropriate isotype controls of glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin (all from Invitro- irrelevant specificity (rat IgG2b-PE (Caltag), rat IgG2b-FITC and hamster gen, Paisley, U.K.), and 60 ␮M monothioglycerol (Sigma-Aldrich, Poole,

IgG-FITC (BD Biosciences)) were included. Cells undergoing double http://www.jimmunol.org/ U.K.). MLN cells were stimulated with 50 ␮g/ml T. muris E/S Ag in staining were preincubated with 0.03 mg/ml rat IgG for 10 min on ice 48-well plates (Helena Biosciences, Sunderland, U.K.), with supernatants before staining to reduce nonspecific Ab binding. All cells were stained for harvested after either 24 or 48 h, as described in the figure legends, and 30 min on ice in the dark and then fixed by the addition of 2% formal- stored at Ϫ20°C until use. dehyde in Dulbecco’s A and B salts, 0.1% sodium azide, and 2% FCS Cytokine and chemokine ELISAs and stored at 4°C in the dark until analyzed. Results were acquired on a FACSCalibur flow cytometer and analyzed using CellQuest software (both Cytokines were analyzed by sandwich ELISA as previously described (40). from BD Biosciences). The mAb pairs used were: IFN-␥, R4-6A2 and XMG1.2; IL-4, 11B11 and BVD-24G.2; IL-5, TRFK5 and TRFK4 (all from BD Biosciences, Oxford, Statistical analysis U.K.); IL-9, 249.2 (E. Schmidt and J. van Snick) and DC9302C12 (BD Statistical analysis was performed using either Student’s t test or one-way by guest on September 29, 2021 Biosciences); and IL-12, C15.6 (G. Trinchieri, Schering-Plough, Dardilly, ANOVA with the statistical package GraphPad PRISM (GraphPad Soft- France) and C17.8 (BD Biosciences). All detection Abs were biotinylated ware, San Diego, CA). A value of p Ͻ 0.05 was considered significant. and used in conjunction with streptavidin-peroxidase (Roche, Sussex, U.K.). ABTS (Sigma-Aldrich) was used as a substrate, and plates were Results read at 405 nm. IL-13 was assayed using the Quantikine M ELISA kit (R&D Systems, Abingdon, U.K.) following the manufacturer’s instruc- Resistant (Th2-dominated) and susceptible (Th1-dominated) tions. Measurements of macrophage inflammatory protein-1␣ (CCL3) and strains of mouse also differ in their chemokine responses CCL2 were performed using the DuoSet ELISA development system following T. muris infection (R&D Systems) and the OptEIA mouse CCL2 set (BD Biosciences), respectively. To assess whether CCL2 and CCL3 were produced in response to infection with T. muris, in vitro MLN cell production of these Cincinnati cytokine capture assay chemokines was measured in BALB/c (resistant) and AKR (sus- The reagents and protocol for performing the in vivo cytokine capture ceptible) mice. Following infection mice were sacrificed on days 0, assay were provided by F. Finkelman (University of Cincinnati, Cincinnati, 3, 7, 10, 14, 21, 28, and 35 p.i. As expected, BALB/c mice ex- OH). The method used was previously described (41). Briefly, naive or T. pelled the majority of worms before day 21 p.i., whereas AKR muris-infected mice were injected with biotinylated anti-IL-4 (clone BVD- 1D11) 24 h before sacrifice. Serum was collected and diluted 1/2 for mea- mice maintained worms through to day 35 p.i. (Fig. 1A). MLN surement of IL-4 by ELISA. ELISA plates were coated with anti-IL-4 cells were cultured with E/S Ag, and the levels of cytokine pro- capture Ab (clone BVD6-24G2.3) overnight and blocked with 10% FCS in duction were assessed by ELISA. AKR mice produced a Th1- PBS. A standard was prepared by mixing IL-4 with biotinylated BVD- skewed cytokine response, as demonstrated by high levels of 1D11 for 3 min before diluting to 100 ng/ml. Samples and standards were then IFN-␥ and negligible IL-9, whereas BALB/c mice exhibited the assayed in duplicate using buffers and reagents described previously (40). opposite profile (Fig. 1, B and C). This pattern was reflected in Immunohistochemistry other Th1- and Th2-associated cytokines, as MLN cells from Mice were killed on days 13, 21, and 35 postinfection (p.i.) with T. muris. BALB/c mice produced less IL-12 and more IL-4, IL-5, and IL-13 Age-matched naive controls were killed on day 21. Approximately 8 mm than AKR mice (data not shown). of the large intestine, juxtaposed to the distal cecum, was removed, tri- These results confirmed that the immune response of T. muris- sected, and carefully positioned in optimal cutting temperature (OCT) com- infected AKR and BALB/c mice had become either Th1 or Th2 pound embedding medium (R. A. Lamb, Eastbourne, U.K.). The tissue was snap-frozen in liquid nitrogen-chilled iso-pentane (BDH Laboratory Sup- dominated, respectively. The production of CCL2 and CCL3 was plies, Poole, U.K.), and 7-␮m sections were cut using a cryomicrotome. assessed in the same MLN cell supernatants to determine whether The tissue was air-dried for1htomaximize its adhesion to gelatin-coated these chemokines were associated with either of these immune microscope slides and then fixed using 4% paraformaldehyde (Sigma-Al- states. No CCL2 was detected in MLN cell supernatants in either drich) in PBS for 10 min at 4°C. Slides were washed in PBS, and endogenous peroxidase activity was quenched using 0.064 mg/ml sodium azide, mouse strain. However, CCL2 was detectable in the sera of AKR 1.54 U/ml glucose oxidase, and 1.8 mg/ml D-glucose (Sigma-Aldrich) in and BALB/c mice, with T. muris-infected BALB/c mice producing PBS for 20 min at 37°C. Following a further wash in PBS, nonspecific almost 5-fold more CCL2 than naive animals on day 21 p.i. (372.9 The Journal of Immunology 4695 Downloaded from

FIGURE 2. CCL2 and CCL3 levels in BALB/c and AKR mice following infection with T. muris. Mice (three per group) were infected on day 0 with ϳ100 embryonated eggs and were sacriﬁced on days 0, 3, 7,10, 14, 21, 28, and 35 p.i. A, Serum collected from clotted whole blood was used to measure CCL2 by ELISA. B, Single-cell suspensions of MLN cells were cultured for 24 h, and the supernatant was recovered for measurement of http://www.jimmunol.org/ CCL3 by ELISA. The limits of detection of the assays were: CCL2, 10.4 pg/ml; and CCL3, 189.4 pg/ml. Results are expressed as the mean Ϯ 1 SD. .p Ͻ 0.05 compared with AKR mice on the same day p.i ,ء

FIGURE 1. Worm burden and cytokine responses in BALB/c and AKR larvae at days 12 and 21 p.i. but had completely cleared the infec- mice following infection with T. muris. Mice (three per group) were in- tion by day 35 p.i. (Fig. 3). CCL2-deficient mice, however, main- fected on day 0 with ϳ100 embryonated eggs and were sacrificed on days tained a parasite burden on day 35 p.i. with the presence of fecund, 0, 3, 7,10, 14, 21, 28, and 35 p.i. A, The number of T. muris inhabiting the adult worms indicating complete susceptibility to infection. by guest on September 29, 2021 large intestine was assessed from day 14 p.i. Single-cell suspensions of To investigate the Th1/Th2 status of CCL2-deficient mice, MLN MLN cells from all time points were cultured for 24 h, and the supernatant cells were recovered from naive and infected animals and stimu- was recovered for analysis of cytokines by ELISA. B, IFN-␥; C, IL-9. The lated with E/S Ag, and the production of IFN-␥, IL-4, IL-5, IL-9, limits of detection of the assays were: IFN-␥, 0.4 ng/ml; and IL-9, 1.6 U/ml. Results are expressed as the mean Ϯ 1 SD. IL-12, and IL-13 was assessed by ELISA. The Th2 cytokines IL-4, IL-5, IL-9, and IL-13 were all present at higher levels in C57BL/6 wild-type mice than in CCL2-deficient animals. Although these differences did not reach statistical significance, after in vitro re- Ͻ vs 75.4 pg/ml; Fig. 2A; p 0.05). The peak of CCL2 production stimulation with E/S Ag, strong consistent trends were observed in in AKR mice was later, on day 28 p.i., and was less than that two separate experiments, with reduced Th2 cytokine levels in measured in the BALB/c mice on day 21 p.i. (118.1 vs 372.9 CCL2-deficient mice. Also, restimulation of cells with Con A (a pg/ml). In contrast, CCL3 was present at significantly higher levels more potent method of restimulating cells that have been primed in E/S-stimulated MLN supernatants from Th1-dominated AKR mice than in those from Th2-dominated BALB/c mice from day 21 p.i. and remained higher through the remainder of the time course (Fig. 2B; p Ͻ 0.05). Levels of CCL3 increased only slightly in BALB/c mice during the course of infection. The time course of production of CCL2 and CCL3 was identical with that for the cytokines shown in Fig. 1, A and B, i.e., the first measurable increase over background was about day 21 p.i. It appears, therefore, that following infection with T. muris, CCL2 and CCL3 are associated with Th2- and Th1-dominated immune responses respectively.

CCL2-deficient mice fail to expel T. muris and exhibit diminished Th2 cytokine responses FIGURE 3. Worm burden in C57BL/6 and CCL2-deficient mice fol- To determine the importance of CCL2 in the Th2-driven protective lowing infection with T. muris. Mice (four or five per group) were infected immunity in response to T. muris infection, mice of the normally on day 0 with ϳ100 embryonated eggs and were sacrificed on days 12, 21, resistant C57BL/6 strain in which the gene for CCL2 (SCYA2) has and 35 p.i. The number of larvae (days 12 and 21 p.i.) or adult worms (day been disrupted, were utilized. There was a highly significant dif- 35 p.i.) in the large intestine was determined. Results are expressed as the ference in the ability of these mice to expel T. muris compared mean Ϯ 1 SD and are representative of five independent experiments. ND, .p Ͻ 0.05 compared with C57BL/6 mice ,ء .with the C57BL/6 wild type controls. Wild type mice harbored none detected 4696 CCL2-DEFICIENT MICE ARE SUSCEPTIBLE TO T. muris INFECTION by parasite Ag in vivo (40)) confirmed these trends, with cells from but slow expelling, C57BL/6 mouse strain (3, 8) and different from CCL2-deficient MLN producing significantly lower levels of Th2 the faster expelling BALB/c mice in which no increase in IFN-␥ cytokines (IL-4: CCL2-deficient, 37.5 Ϯ 22.6 pg/ml; C57BL/6, production occurs (Fig. 1B). However, differences in production of 307.2 Ϯ 205 pg/ml; IL-5: CCL2-deficient, 10 Ϯ 4.7 U/ml; the Th1-associated cytokine IL-12 were seen, with CCL2-deficient C57BL/6, 69.1 Ϯ 55.1 U/ml; IL-9: CCL2-deficient, 0.3 Ϯ 0.6 mice producing 686.3 pg/ml compared with 277.4 pg/ml for wild- U/ml; C57BL/6, 10.0 Ϯ 10.0 U/ml; p Ͻ 0.05 for each cytokine). type MLN cells (Fig. 4C; p Ͻ 0.05). To confirm the decreased ability of MLN cells from CCL2-defi- The production of CCL2 and CCL3 in T. muris-infected AKR cient mice to produce Th2 cytokines following T. muris infection, and BALB/c mice was associated with Th2 and Th1 immune re- the sensitive CCCA method (41) was used to measure levels of sponses, respectively (Fig. 2, A and B). As CCL2-deficient IL-4 in vivo on day 21 p.i. Fig. 4A shows that following infection, C57BL/6 mice showed a Th1-biased response (decreased IL-4, the amount of IL-4 in the serum of wild-type mice rose dramati- increased IL-12), the levels of these chemokines in MLN cell su- cally from 77.2 to 268.7 pg/ml, significantly greater than the pernatants of CCL2-deficient and wild-type animals were mea- amount detected in infected CCL2-deficient mice (57.8 pg/ml; p Ͻ sured. CCL2 was significantly up-regulated in C57BL/6 mice on 0.01). In addition, levels of IL-4 in CCL2-deficient animals did not day 21 p.i. compared with that in naive mice (Fig. 4D; p Ͻ 0.001). rise significantly after infection (naı¨ve, 11.6 pg/ml; infected, 57.8 Although CCL3 was strongly associated with a Th1 response in pg/ml). No IL-4 was detectable in control animals injected with susceptible AKR mice, there was no difference in the production of PBS. Importantly, even postinfection, IL-4 levels in CCL2-defi- this chemokine between CCL2-deficient and wild-type C57BL/6 cient mice did not reach naive wild-type levels. MLN cells after restimulation with E/S Ag (Fig. 4E).

IFN-␥ was undetectable in naive mice, but was strongly ex- Downloaded from pressed in both wild-type and CCL2-deﬁcient mice following in- CCL2-deﬁcient mice differ in the cellular composition of the fection (Fig. 4B). Elevated IFN-␥ levels are typical of the resistant, large intestine and MLN both before and after infection As CCL2 is a chemoattractant for various cell types, including activated T cells, NK T cells, and basophils, and may be the main chemoattractant for monocyte/macrophages under some condi-

tions, the cellular composition of the large intestine was assessed http://www.jimmunol.org/ by histological and immunohistochemical methods, and the composition of the MLN was determined by flow cytometry in naive and T. muris-infected WT and CCL2-deficient mice. Infection with T. muris caused an expansion of crypt goblet cells and an influx of eosinophils and mast cells to the large intestine. There were no differences in goblet cell hyperplasia, eosinophilia, or mastocytosis between wild-type and CCL2-deficient mice after infection (data not shown). Immunohistochemical staining for the macrophage- specific Ag, F4/80, revealed a significant reduction in the infiltra- by guest on September 29, 2021 tion of macrophages to the large intestine following infection of CCL2-deficient mice. Fig. 5A shows intense F4/80 staining in wild-type mice on day 20 p.i., whereas CCL2-deficient mice exhibit much weaker staining (Fig. 5B). Very few macrophages are present in naive tissue (Fig. 5C). To quantify the differences in staining, sections from four animals on days 0 (naive), 13, 20, and 35 p.i. were stained, and the number of macrophages in 20 cecal crypt units (CCU) of three serial sections for each animal was assessed. There were no differences in the number of macrophages in naive animals or on day 13 p.i. (Fig. 6A). However, there were significantly fewer macrophages in the large intestine of CCL2- deficient animals on day 20 p.i. ( p Ͻ 0.01). This trend was also present on day 35 p.i., but was not statistically significant due to large variation in macrophage number in the wild-type animals. It is also clear from these data that macrophages do migrate into the large intestine under resting and inflammatory conditions, albeit in reduced numbers, in the absence of CCL2 (Figs. 5B and 6A). Im- munohistochemical staining of CD4ϩ T cells showed that there was no difference in the number of cells infiltrating the large in- FIGURE 4. Cytokine and chemokine responses in C57BL/6 and CCL2- testine of wild-type or CCL2-deficient mice in naive animals or at deficient mice following infection with T. muris. Mice (four or five per any time point following infection with T. muris (Fig. 6B). group) were infected on day 0 with ϳ100 embryonated eggs and were In the MLN, macrophages (CD11bϩF4/80ϩ cells) and dendritic sacrificed on day 21 p.i. A, Some animals were injected with biotinylated cells (CD11cϩMHC IIϩ cells) made up only a small fraction of the anti-IL-4 Ab 24 h before sacrifice for the measurement of IL-4 in serum by total lymphocyte population (Fig. 7A, R1) when assessed by flow ELISA. Single-cell suspensions of MLN cells from mice not injected with cytometry (0.1–2%; data not shown). However, the application of biotinylated anti-IL-4 were cultured for 48 h, and the supernatant was recovered for the measurement of IFN-␥ (B), IL-12 (C), CCL2 (D), and a separate region (Fig. 7A, R2) revealed that the majority of these CCL3 (E) by ELISA. The limits of detection of the assays were: IL-4, 4.29 APCs resided among the larger, more granular cell population, pg/ml; IFN-␥, 4.5 ng/ml; IL-12, 240.5 pg/ml; CCL2, 80.1 pg/ml; and forming between 1 and 10% of this population. Consequently, CCL3, 6.6 pg/ml. Results are expressed as the mean Ϯ 1 SD. ND, none quantification of macrophage and dendritic cell numbers was per- p Ͻ 0.001. formed on this subpopulation. The proportions of macrophages in ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .detected The Journal of Immunology 4697

FIGURE 5. Immunohistochemical detection of macrophages in the large intestine of C57BL/6 and CCL2-deficient mice. Mice (four per group) were infected on day 0 with ϳ150 embryonated eggs and were sacrificed on day 20 p.i. Naive mice were sacrificed 20 days after the start of the experiment. A section of the large intestine was recovered and processed for immunohistochemistry. Sections were stained with F4/80 for identification of macrophages. A, Wild-type mice, day 20 p.i.; B, CCL2-deficient mice, day 20 p.i.; C, wild-type naı¨ve mice; D; isotype control (performed on wild-type tissue). Original magnifica- tion of the sections, ϫ200. Arrows indicate the

anterior portion of T. muris embedded within gut Downloaded from epithelium. http://www.jimmunol.org/ the MLN of wild-type and CCL2-deficient mice both before and from MLN of wild-type and CCL2-deficient mice 20 days after T. during infection are shown in Figs. 7 and 8. Fig. 7, BÐD, shows muris infection. At this time point there were more macrophages in isotype control staining and staining for macrophages isolated the MLN of wild-type mice (9.92%) than in CCL2-deficient mice by guest on September 29, 2021

FIGURE 7. Identification of macrophages in single-cell suspensions of MLN cells from T. muris-infected C57BL/6 and CCL2-deficient mice. Mice ϳ ϩ (four to six per group) were infected on day 0 with 150 embryonated eggs FIGURE 6. Quantification of macrophage and CD4 T cell infiltration and were sacrificed on day 20 p.i. Single-cell suspensions of MLN cells were of the large intestine following T. muris infection. Mice (four per group) prepared and stained with anti-mouse CD11b-FITC and F4/80-PE for macro- were infected on day 0 with ϳ150 embryonated eggs and were sacrificed phages or the appropriate control Abs. A, A typical forward vs side scatter plot on days 13, 20, and 35 p.i. Naive mice were sacrificed 20 days after the for MLN cells indicating the main lymphocyte population (R1) and the area start of the experiment. A section of the large intestine was recovered and richest in macrophages (R2). B, Isotype control staining; C, wild-type MLN processed for immunohistochemistry. Sections were stained with F4/80 for cells, day 20 p.i.; D, CCL2-deficient MLN cells, day 20 p.i. The number in the ϩ identification of macrophages (A) or with CD4 for CD4 T cells (B), and upper right quadrant indicates the percentage of double-positive cells present the number of positive cells in 20 CCU was assessed. Results are expressed in area R2 of the scatter plot for that cell population. Plots are representative as the mean Ϯ 1 SD. **, p Ͻ 0.01 compared with CCL2-deficient mice. of two independent experiments. 4698 CCL2-DEFICIENT MICE ARE SUSCEPTIBLE TO T. muris INFECTION

MLN lymphocyte population among the cell types that make up the majority of the node population, namely T cells (CD4ϩ or CD8ϩ cells) and B cells (B220ϩ cells). When expressed as a percentage of the total cells in the MLN, CD4ϩ cells comprise 32.3 and 28.4% of cells in the MLN of naive wild-type and CCL2- deficient mice, respectively, a small, but statistically significant, difference ( p Ͻ 0.05) that is maintained postinfection (Fig. 8C and Table I). Thus, following infection the relative percentage of CD4ϩ T cells typically decreases in both wild-type and CCL2- deficient mice p.i. (40) (our unpublished observations), only re- covering if worms are expelled (i.e., after day 20 p.i. for wild-type animals). In contrast, the relative percentage of CD8ϩ cells did not differ between wild-type and CCL2-deficient mice before or postinfection (data not shown). In both strains the percentage of B cells in the MLN is increased over naive levels on days 20 and 34 p.i., with the peak percentage again corresponding to the presence of worms, occurring on day 20 p.i. in wild-type mice and day 34 p.i. in CCL2-deficient mice (Fig. 8D and Table I). This difference may be due to the persistence of worms in CCL2-deficient Downloaded from mice maintaining B cell proliferation in the MLN and is consistent with data comparing other T. muris-resistant and susceptible mouse strains (our unpublished observations).

FIGURE 8. The cellular compositions of MLNs from infected and naive http://www.jimmunol.org/ C57BL/6 and CCL2-deficient mice. Mice (four to six per group) were infected Discussion on day 0 with ϳ150 embryonated eggs and were sacrificed on days 12, 20, and That a Th2-dominated immune response, characterized by high 34 p.i. Naive mice were sacrificed after the final group of infected mice. Sin- levels of IL-4, IL-9, and IL-13, is required for the successful clear- gle-cell suspensions of MLN cells were prepared and stained with anti-mouse ance of a T. muris infection in mice is well documented (2–5). CD11b-FITC and F4/80-PE (A), anti-mouse CD11c-FITC and MHC II-PE However, there has been little investigation into the importance of (B), anti-mouse CD4-FITC (C), or anti-mouse B220-FITC (D) Results are chemokines in the development of immunity to T. muris and to expressed as the mean relative percentage of total cells staining positively Ϯ p Ͻ 0.05; intestinal nematode parasites in general. It is shown here that the ,ء .SD and are representative of two independent experiments 1 p Ͻ 0.01 (compared with C57BL/6 mice). levels of two chemokines, CCL2 and CCL3, differ in AKR and ,ءء

BALB/c strains of mice during the course of T. muris infection. by guest on September 29, 2021 These strains, respectively, exhibit susceptibility (Th1 dominated) (3.2%). Analysis of individual groups (n ϭ 4–6 mice/group) con- and resistance (Th2 dominated) to infection. As such they offer firmed that significantly more macrophages were present in the oppositely polarized immune responses, allowing study of the MLN of wild-type mice than in CCL2-deficient mice by day 20 p.i. roles of various mediators under such conditions. The levels of ( p Ͻ 0.01), a difference that was maintained on day 34 p.i. (Fig. CCL2 and CCL3 were assessed at several time points during the 8A; p Ͻ 0.01). These results were confirmed in a second identical course of infection, and we found increased production of CCL2 experiment (Table I). Unlike in the large intestine, however, there associated with resistance in BALB/c mice, while levels remained were significantly fewer macrophages in the MLN of naive CCL2- low in AKR mice. In contrast, a dramatic and sustained increase in deficient mice than in wild-type mice. In comparison, no differ- CCL3 was seen in susceptible AKR mice from day 21 p.i., whereas ences in the numbers of dendritic cells (CD11cϩMHC IIϩ) were CCL3 levels did not increase in BALB/c mice over the time course observed in naive mice or at any time point following infection studied. This suggests that CCL2 is associated with a Th2-domi- (Fig. 8B). Interestingly, differences were also found in the total nated response (high IL-4, IL-5, IL-9, and IL-13 and low IFN-␥),

Table I. Flow cytometry data showing the proportions and CD4ϩ T cells, B220ϩ B cells, and CD11bF4/ 80ϩ macrophages in the MLN of naive (day 0 p.i.) and T. muris-infected wild-type and CCL2-deﬁcient micea

Days p.i. Genotype CD4ϩ B220ϩ CD11bF4/80ϩ

0 WT 35.0 Ϯ 1.5 31.2 Ϯ 2.5 12.47 Ϯ 0.64 KO 26.3 Ϯ 2.1b 40.2 Ϯ 3.6b 9.07 Ϯ 1.37b 12 WT 34.2 Ϯ 2.8 32.6 Ϯ 4.7 6.72 Ϯ 1.75 KO 28.5 Ϯ 1.1b 39.3 Ϯ 4.5 8.02 Ϯ 4.21 21 WT 30.3 Ϯ 1.0 43.4 Ϯ 0.7 8.39 Ϯ 1.43 KO 25.8 Ϯ 4.4 47.0 Ϯ 5.6 5.99 Ϯ 1.74c 35 WT 32.1 Ϯ 3.2 35.2 Ϯ 3.7 19.69 Ϯ 1.96 KO 19.7 Ϯ 1.9b 49.9 Ϯ 3.4b 14.98 Ϯ 2.62b

a The data represents a repeat of the experiment presented in Fig. 8. There were no significant differences in the percentage of CD11cMHCIIϩ dendritic cells at any point in the experiment. Results are expressed as the mean relative percentage of total cells staining positively Ϯ 1SD(n ϭ 5 mice/group). b p Ͻ 0.01 vs wild-type mice. WT, wild-type; KO, knockout. c p Ͻ 0.05 vs wild-type mice. The Journal of Immunology 4699 and CCL3 is associated with a Th1 response (high IFN-␥ and low more chronic inflammatory response. This in combination with the IL-4, IL-5, IL-9, and IL-13) following infection with T. muris. increased levels of Th1-related cytokines might prolong the sur- This reciprocal relationship between CCL2 and CCL3 has been vival of the worm in the host. Indeed, two bacterial models of previously described with regard to T cell cytokine production. inflammation have shown that a lack of monocyte recruitment to Stimulation of OVA-transgenic T cells in the presence of CCL3 the site of infection is responsible for a more chronic pathology leads to IFN-␥ production, whereas the addition of CCL2 induces developing, although neither of these studies assessed cytokine IL-4 production (26, 27). Also, CCL2 increases and CCL3 inhibits status following infection (43, 44). Alternatively, the lack of mac- the production of IL-4 from differentiated Th2 lymphocytes (28). rophage recruitment to the gut could be seen as an indication of an The granulomatous lung inflammation model is also well charac- inadequate immune response being generated in the CCL2-defi- terized. A type 1 (Th1) granuloma can be induced by mycobacte- cient mice, leading to the failure of the mice to expel the parasite. rium purified protein derivative, whereas schistosomal egg Ag in- However, the equivalent production of IFN-␥ by MLN cells and duces a type 2 (Th2) granuloma. Under such conditions, CCL2 the influx of CD4ϩ T cells to the gut suggest that CCL2-deficient levels are higher in the local draining lymph nodes of mice with mice mount an immune response of equal magnitude to that of type 2 granulomas, and CCL2 inhibits IL-12 production by acti- wild-type mice in response to T. muris infection. vated macrophages isolated from these mice (24). Conversely, The cellular composition of the MLN, the lymph node draining CCL3 is produced following the induction of a type 1 granuloma the large intestine, was also analyzed during the course of T. muris (33). Previous work on the roles of chemokines in parasitic infec- infection. At this site there were significantly fewer macrophages, tions has associated CCL2 with two other intestinal parasites that but normal numbers of dendritic cells in the absence of CCL2 in naive provoke a strong Th2 response within the host; T. spiralis infection mice and on days 20 and 34 p.i. This may therefore result in quanti- Downloaded from causes an increase in the level of CCL2 in serum (31), and Nip- tative or qualitative differences in T and B cell activation in the MLN, postrongylus brasiliensis infection leads to increased CCL2 pro- producing an immune response biased toward Th1. Indeed, a macro- duction by murine intestinal epithelial cells on days 7 and 14 p.i. phage trafficking defect in CCR2 knockout mice (the receptor for (42). These data together with the evidence generated from our CCL2) has been shown to be responsible for a reduction in Ag-spe- work with AKR and BALB/c mice (Figs. 1 and 2) led us to ques- cific IFN-␥ production in lymph nodes (45). Although this study re- tion whether CCL2 was involved in generation of the Th2-asso- sults in mice exhibiting Th2-dominated, rather than Th1-dominated, http://www.jimmunol.org/ ciated protective immune response to T. muris. As shown in Fig. immune responses, it does set a precedent for the importance of the 3, CCL2-deficient mice on a C57BL/6 background were com- macrophage in stimulating Ag-specific T cells in the draining lymph pletely susceptible to infection, whereas wild-type mice expelled node. Interestingly, CCL2 decreases IL-12 production from both the parasite between days 21 and 35 p.i. The susceptibility of monocytes (25) and inflammatory macrophages (24), and also in- CCL2-deficient mice was associated with decreased production of creases IL-4 and decreases IL-12 production by mucosal tissue (35). Th2 cytokines (Fig. 4A and data not shown). With regard to Th1 Consequently, in the absence of CCL2, fewer macrophages are cytokine production, there was no difference in the levels of IFN-␥, present in the draining lymph node and the site of inflammation, and but there was increased IL-12 in the CCL2-deficient mice. Despite

the macrophages that are present lack a control mechanism for IL-12 by guest on September 29, 2021 the susceptible phenotype and enhanced Th1 response generated production. They may therefore express increased amounts of this by CCL2-deficient mice there was no increase in CCL3 above the Th1-inducing cytokine, and indeed, our data reveal significantly ele- level seen in wild-type mice. Thus, the relationships between vated IL-12 levels in the MLN in the absence of CCL2. In our model CCL2 and resistance (Th2 response) and between susceptibility and CCL3 (Th1 response) seen in BALB/c vs AKR mice (Fig. 2B) system, therefore, the absence of CCL2 may help promote a Th1 were not apparent. This may be due to the higher levels of IFN-␥ response through elevated IL-12 production from APCs, such as the macrophage or dendritic cell. found in C57BL/6 compared with BALB/c mice, leading to ele- ϩ vated levels of CCL3 in the wild-type mice. Fewer CD4 T cells were found in the MLN, but not the large Leukocyte recruitment was investigated in CCL2-deficient mice intestine, of both naive and infected CCL2-deficient mice. This infected with T. muris, as previous reports have described a defi- suggests that there is a population of T cells directly or indirectly ciency in macrophage recruitment in the absence of CCL2 (23, dependent on the presence of CCL2 for their migration into and/or 37). Using immunohistochemistry a decreased accumulation of maintenance in the MLN. CCL2 has been shown to be a chemoat- macrophages was found in the large intestine following T. muris tractant for T cells in vitro (16), and several studies have shown a role in vivo. Firstly, overexpression of human CCL2 in mouse type infection. Levels in naive animals were equivalent in wild-type and ϩ CCL2-deficient mice, revealing that other chemoattractants are II alveolar epithelial cells leads to an increase in CD3 T cells sufficient for the infiltration of resting numbers of macrophages to accumulating in the lung (21). Secondly, CCL2-deficient mice the large intestine (for example, other members of the monocyte have decreased lymphocyte infiltration in a model of inflammation chemoattractant protein family, CCL7, CCL8, and CCL12). The (46). Thirdly, treatment with a CCL2-neutralizing Ab decreases T number of macrophages in the large intestine of CCL2-deficient cell recruitment to the lung (47). Although Gu et al. (29) found no mice increased during the course of infection, indicating that difference in the migration of naive T cells to secondary lymphoid CCL2 is not essential for macrophage infiltration. However, as the organs, our study allows for differences in chemokine receptor number of macrophages infiltrating the large intestine of wild-type expression on naive, activated, and memory T cells also effecting mice was significantly higher on day 21 p.i. than in mice deficient T cell trafficking. Also, three independent in vitro experiments in CCL2, there is a subpopulation of F4/80ϩ cells that do require were performed in which MLN cells isolated from T. muris-in- CCL2 for influx to the gut postinfection. The primary role of the fected wild-type and CCL2-deficient mice were stimulated with macrophage at the site of infection and inflammation is probably E/S Ag in the presence or the absence of CCL2. There were no that of phagocytosis of dead or dying cells and foreign material to differences in the production of IL-4, IFN-␥, or IL-12 by MLN help resolve the inflammatory response and allow repair of dam- cells in vitro (data not shown), suggesting that the effect of CCL2 aged tissue. Therefore, it is possible that the reduced number of on cytokine production in MLN is indirect. Thus, in our model system macrophages found in the large intestine of CCL2-deficient mice the altered MLN environment of CCL2-deficient mice, containing following T. muris infection leads to decreased phagocytosis and a proportionally fewer CD4ϩ T cells and macrophages, may underlie 4700 CCL2-DEFICIENT MICE ARE SUSCEPTIBLE TO T. muris INFECTION the failure of CCL2-deficient mice to fully develop a Th2-dominated 22. Nakamura, K., I. R. Williams, and T. S. Kupper. 1995. Keratinocyte-derived immune response, resulting in long term persistent infection. monocyte chemoattractant protein 1 (MCP-1): analysis in a transgenic model demonstrates MCP-1 can recruit dendritic and Langerhans cells to skin. J. Invest. Here we show for the first time a requirement for the chemokine Dermatol. 105:635. CCL2 in protective immunity to an intestinal helminth. CCL2 de- 23. Lu, B., B. J. Rutledge, L. Gu, J. Fiorillo, N. W. Lukacs, S. L. Kunkel, R. North, C. Gerard, and B. J. Rollins. 1998. Abnormalities in monocyte recruitment and ficiency is linked to a decreased recruitment of macrophages to cytokine expression in monocyte chemoattractant protein 1-deficient mice. both the draining lymph node and the large intestine (the site of J. Exp. Med. 187:601. infection). In addition, it is shown that the absence of CCL2 leads 24. Chensue, S. W., K. S. Warmington, J. H. Ruth, P. S. Sanghi, P. Lincoln, and S. L. Kunkel. 1996. Role of monocyte chemoattractant protein-1 (MCP-1) in Th1 to significantly elevated levels of IL-12, reduced Th2 cytokine (mycobacterial) and Th2 (schistosomal) antigen-induced granuloma formation: levels, and failure to expel T. muris. relationship to local inflammation, Th cell expression, and IL-12 production. J. Immunol. 157:4602. 25. Braun, M. C., E. Lahey, and B. L. Kelsall. 2000. Selective suppression of IL-12 Acknowledgments production by chemoattractants. J. Immunol. 164:3009. We gratefully acknowledge the help of Louise Bell with the collection and 26. Karpus, W. J., N. W. Lukacs, K. J. Kennedy, W. S. Smith, S. D. Hurst, and analysis of data. We are also indebted to Fred Finkelman for kindly pro- T. A. Barrett. 1997. Differential CC chemokine-induced enhancement of T helper viding the reagents for the Cincinnati cytokine capture assay, and all the cell cytokine production. J. Immunol. 158:4129. 27. Karpus, W. J., and K. J. Kennedy. 1997. MIP-1␣ and MCP-1 differentially reg- staff at the Biomedical Services Unit at University of Manchester. ulate acute and relapsing autoimmune encephalomyelitis as well as Th1/Th2 lymphocyte differentiation. J. Leukocyte Biol. 62:681. References 28. Lukacs, N. W., S. W. Chensue, W. J. Karpus, P. Lincoln, C. Keefer, 1. Else, K., and D. Wakelin. 1988. The effects of H-2 and non-H-2 genes on the R. M. Strieter, and S. L. Kunkel. 1997. C-C chemokines differentially alter in- expulsion of the nematode Trichuris muris from inbred and congenic mice. Par- terleukin-4 production from lymphocytes. Am. J. Pathol. 150:1861. 29. Gu, L., S. Tseng, R. M. Horner, C. Tam, M. Loda, and B. J. Rollins. 2000.

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