Interleukin-10-deficient mice develop chronic enterocolitis

Cell, Vol. 75, 263-274, October22, 1993,Copyright© 1993by Cell Press

Interleukin-lO-Deficient Mice Develop Chronic Enterocolitis Ralf K~Jhn,* J(irgen L6hler,l Donna Rennick,~ Klaus Rajewsky,* and Werner MOiler* *Institute for Genetics University of Cologne Weyertal 121 50931 Cologne Federal Republic of Germany tHeinrich Pette Institute for Experimental Virology and Immunology University of Hamburg Martinistrasse 52 20251 Hamburg Federal Republic of Germany $DNAX Research Institute of Molecular and Cellular Biology 901 California Avenue Palo Alto, California 94304

Summary Interleukin-10 (IL-10) affects the growth and differentiation of many hemopoietic cells in vitro; in particular, it is a potent suppressor of macrophage and T cell functions. In IL-10-deficient mice, generated by gene targeting, lymphocyte development and antibody responses are normal, but most animals are growth retarded and anemic and suffer from chronic enterocolitis. Alterations in intestine include extensive mucosal hyperplasia, inflammatory reactions, and aberrant expression of major histocompatibility complex class II molecules on epithelia. In contrast, mutants kept under specific pathogen-free conditions develop only a local inflammation limited to the proximal colon. These results indicate that the bowel inflammation in the mutants originates from uncontrolled immune responses stimulated by enteric antigens and that IL-10 is an essential immunoregulator in the intestinal tract. Introduction Cytokines are potent regulatory molecules secreted by cells of the immune system upon activation. Interleukin-10 (IL-10) was initially identified as an activity produced by T helper cell subset 2 (Th2) (see below), inhibiting the synthesis of cytokines by Th 1 cells (Fiorentino et al., 1989). It was subsequently found to be produced also by Ly-1 B (B-l) cells, macrophages, thymocytes, and keratinocytes upon activation (for review see Moore et al., 1993). IL-10 affects the growth and differentiation of various cell types of the immune system in vitro. Like IO4, it enhances the expression of major histocompatibility complex (MHC) class II molecules on resting murine B cells and increases their viability (Go et al., 1990), but, in contrast with IL-4, proliferation and immunoglobulin class switching of lipopolysaccharide (LPS)-stimulated B cells are not affected (de Waal Malefyt et al., 1992). The combination of IL-10

and anti-CD40 antibodies stimulates human B cells to proliferate and differentiate into plasma cells (Rousset et al., 1992). IL-10 is a potent suppressor of macrophage activation in vitro. It inhibits the production of inflammatory cytokines such as I1_-1,IL-6, and tumor necrosis factor ~ (TNFcz) by macrophages stimulated with LPS and interferon y (IFNy) (Fiorentino et al., 1991b). Accordingly, mice become more sensitive to LPS-induced shock by treatment with anti-lL-10 antibodies (Ishida et al., 1992), while lethal endotoxemia and elevated serum TNF~ levels are suppressed upon the administration of IL-10 (Howard et al., 1993). In addition, IL-10 prevents the IFNy-stimulated synthesis of nitric oxide, resulting in decreased resistance to intracellular parasites (Gazzinelli et al., 1992). It also suppresses the ability of macrophages to stimulate the production of IFNy and other cytokines by Thl cells (Fiorentino et al., 1991a) and inhibits the macrophage-dependent development of Thl cells (Hsieh et al., 1992, 1993). Since activated macrophages also produce IL-10, the production of inflammatory cytokines by macrophages could be controlled in an autoregulatory manner (Fiorentino et al., 1991b). It is thought that IL-10 may control the type of immune response that develops upon parasite infection (Sher et al., 1992; Mosmann and Moore, 1991). CD4+T cells, secreting IL-4, IL-5, IL-6, and IL-10 but not IFN7 (Th2 cells), are effective helper cells for B cell antibody production, whereas Thl cells, producing IL-2 and IFN7, mediate cellular immune reactions to parasites or protein antigens (for review see Mosmann and Coffman, 1989). Parasite infections often lead to polarized immune responses of the Thl or Th2 type, either of which can mediate protection or suspectibility, depending on the pathogen (Sher and Coffman, 1992; Urban et al., 1992). The production of IL-10 is strongly increased in mice that develop an immune response of the Th2 type upon infection with Leishmania major, Schistosoma mansoni, Trypanosoma cruzi, or Nippostrongylus brasiliensis (Heinzel et al., 1991; Sher et al., 1991; Silva et al., 1992; Mosmann et al., 1991). Since IL-10 inhibits the activity of Thl cells, 11_-10may represent an effector produced by Th2 cells to suppress the development of a Thl response, resu,lting in an appropriate defense in case a Th2 response mediates resistance, but an inappropriate defense when a Thl response is needed for resistance to a parasite. The recent advance of embryonic stem (ES) cell technology has made it possible to study the in vivo functions of cytokines through mouse mutants specificallydeficient for a given cytokine or cytokine receptor. Mice deficient for IL-2, IL-4, transforming growth factor 131 (TGFI~I), iFNy, or IFNy receptor provided insights into the essential functions of these cytokines for the immune system and provided a tool to investigate their role in the host defense to pathogens (Schorle et al., 1991; KQhn et al., 1991; Shull et al., 1992; Dalton et al., 1993; Huang et al., 1993). In the present paper, we describe the generation and phenotype of a mouse mutant in which the IL-10 gene is inacti-

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Results

in exon 3 deletes an EcoRI site, resulting in the recognition of a 2.9 kb band by probe B instead of the 1.1 kb wild-type fragment (Figure 1A, c; Figure 1B). Heterozygous offspring from two independent clones that transmitted the mutation through the germline were intercrossed separately to generate animals homozygous for the IL1OT mutation. For the experiments described below, age-matched homozygous offspring (denoted as IL10T mice below) from different litters were combined into groups, using wild-type littermates as controls. The animals were kept under conventional conditions unless otherwise stated. One of the strains was transferred into a specific pathogen-free (SPF) facility by embryo transfer. To confirm that the IL10T mutation inactivates the IL-10 gene, I L10T and control mice were infected with the nematode N. brasiliensis, which induces a strong Th2 immune response in normal mice. Highly elevated levels of IL-4, IL-5, and IL-10 can be measured in the supernatants of in vitro cultures of splenic T cells after stimulation with the mitogen concanavalin A (Con A) (Mosmann et al., 1991). As shown in Table 1, both IL10T and control mice responded to the infection with an increased production of IL-5, but levels of IL-10 detectable by a specific enzymelinked immunosorbent assay (ELISA) were found only in the supernatants derived from wild-type animals. The absence of IL-10 activity in the supernatants from IL10T mice was confirmed using a biological assay based on the suppression of IFNy production by IL-10 (Fiorentino et al., 1991a) (data not shown).

Generation of IL-10-Deficient Mice The IL-10 gene (Figure 1A, a) was disrupted in murme ES cells by replacement of a 500 bp fragment containing codons 5-55 of the first exon by a linker, providing a termination codon, and a n e o expression cassette (Figure 1A, b) and b y t h e introduction of a termination codon into exon 3. The structure of the mutated IL-10 gene in targeted ES cell clones and the progeny of germline chimeras was confirmed by Southern blot analysis (Figure 1B). Since a novel EcoRI site is introduced by the n e o gene, an EcoRI digest hybridized with probe A yields a 4.3 kb fragment for the mutated allele rather than the 5.7 kb wild-type band (Figure 1A, c; Figure 1B). The cointegration of the mutation

B and T Lymphocytes Develop Normally in IL10T Mice For the analysis of T and B cell development in IL-10deficient mice, we used animals at the age of 4 and 6 weeks to minimize side effects due to the disease developed by the mutants (see below), since the disease is less severe in young IL10T mice. At these time points, the T cell compartment in thymus and spleen was similar in IL10T and control mice (Table 2). IL-10 is therefore not essential for the generation of the CD4 ÷ and CD8 ÷ T cell subsets. B-1 cells (Kantor, 1991) are the main source of B cellderived IL-10, and it was suggested that IL-10 acts as an

--57 -43 - 1,1 probe A

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Figure 1. Disruption of the IL-10 Gene (A) Strategy for the mactivatnonof the IL-10 gene by homologous recombination. Abbreviations: E, EcoRI; Bg, Bglll; H, Hindlll. (a) Genomlc structure and partial restriction map of the IL-10 gene. Exons are represented by closed boxes and numbered. The lengths of diagnostic restriction fragments and probes used for Southern blot analysis are shown. (b) The targeting vector with homology regions of 0 9 kb on the 5' side and 4.2 kb on the 3' side of the nee gene. As indicated by the dotted lines, a 0.5 kb fragment wtth codons 5-55 of the first exon was replaced by a linker, providing a termination codon, and the neo gene. The termination codon in exon 3 (TAA) was generated by the mutagenesls of an EcoRI stte. An HSV TK expression cassette (HSVtk) and plasmid sequences were attached to the long arm of homology (c) The predicted structure of the IL10T locus. Recombinant ES cell clones were ndentifiedby polymerase chain reaction; triangles indicate the position of pnmers used. (B) Southern blot analysns of tail DNA from intercrosses of heterozygous IL10T mice. (a) Probe A was hybridized to EcoRI-digested DNA from a wild-type animal (wt/wt), a heterozygous mouse (IL10T/wt), and a homozygous mouse (IL10T/IL10T). (b) EcoRI-digested DNA from the same animals as in (a) was hybridized to probe B.

vated by targeted mutation. The mutant animals develop a disease that appears largely to depend upon an inappropriate immune response to intestinal antigens.

IL-10-Deficient Mice Develop Chronic Enterocohtls 265

Table 2. Analysis of T and B Cell Subsets in IL10T and Control Mice Thymus

Spleen

Mouse Mouse Number Strain

Body Weight (g)

Cell Number (x 10 6)

CD4+ (%)

CD8÷ (%)

CD4/CD8 ÷ (%)

Cell Number Lymphocytes CD4÷ (x 10-') (%) (%)

CD8(%)

B Cells (%)

1 2

IL10T ND Wild type ND

85 24

8 13

2 3

82 74

35 36

95 93

21 14

13 7

54 49

3 4 5 6

IL10T IL10T Wild type Wild type

40 39 70 70

15 6 9 7

4 2 2 2

77 87 87 89

21 37 50 32

72 82 87 91

22 19 13 13

8 5 6 4

51 39 49 29

13 12 25 25

Bone Marrow Mouse Mouse Number Strain

Peritoneum Cell Pre-B Number Lymphocytes Cells ( x l 0 -6) (O/o) (%)

Newly Generated BCells(O/o)

B Cells (%)

Cell Number Lymphocytes CD5 Conventional (x 10=6) (%) BCells B Cells

1 2

IL10T 14 Wild type 14

57 32

33 31

15 13

6 5

1 2

45 45

54 31

14 35

3 4 5 6

IL10T IL10T Wild type Wild type

15 31 46 41

38 23 35 40

12 10 16 14

8 6 8 4

1 2 1 1

28 20 34 37

55 32 29 40

22 26 32 30

14 20 19 15

Mice 1 and 2 were 4 weeks old, and mice 3-6 were 6 weeks old. Single cell suspensions were prepared from thymus, spleen, and bone marrow, and cell numbers were determined in a hemocytometer. Cells were stained with labeled antibodies and analyzed by flow cytometry as described in Experimental Procedures. The percentages of cells in the lymphocyte gate are shown. T cell subsets were analyzed by staining with anti-CD4 and anti-CD8 antibodies. B cell subsets were analyzed by staining with anti-CD45R/B220, anti-lgM, and anti-lgD antibodies. B cells in spleen were ~dentified as CD45R/B220 and surface IgM-positive cells. Pre-B cells in bone marrow were CD45R/B220 du"and surface immunoglobulin negative, newly generated B cells were CD45R/B220 bng"tand surface IgM positive but negative for surface IgD. B cells were CD45R/B220 br'Qhtand IgM and IgD posJhve CD5 B cells and conventional B cells m the peritoneum were identified as CD5*,CD45R/B220 du" or CD5 ,CD45R/B220 br'ghtcells, respectively. ND, not determined.

autocrine growth factor for this B cell subset and is essential for its self-renewing capacity (O'Garra et al., 1990, 1992). The flow cytometric analysis of B cell subsets in bone marrow, spleen, and peritoneum of IL10T and normal mice did not reveal any significant difference between mutants and the controls (Table 2). In particular, the B-1 cell subset in the peritoneum is present in IL10T mice (Table 2). Accordingly, the antibody response of mutants to (~(1-3)-dextrane, restricted to the B-1 subset, was normal (data not shown). IL-10 is therefore essential neither for the generation of conventional B cells in bone marrow, spleen, and peritoneum nor for the generation and maintenance of the B-1 cells in the peritoneum.

Normal Immune Response of IL10T Mice to T Cell-Dependent Immunization A n t i b o d y responses to protein antigens require the cooperation of helper T cells and B cells. We measured the "antibody response of IL10T and control mice upon immunization with h a p t e n a t e d chicken "y-globulin (NP-CG; NP is 4-hydroxy-3-nitrophenyl-acetyl) to determine whether IL-10 is required for this cellular interaction in vivo. The helper cells that d e v e l o p in normal mice in response to NP-CG have the typical features of Th2 cells in that they induce i m m u n o g l o b u l i n G1 (IgG1) and immunoglobulin E (IgE) (KQhn et al., 1991; unpublished data). As shown in Figure 2, the concentrations of NP-specific antibodies of all immunoglobulin isotypes were not significantly different in the sera of IL10T and control mice after primary or sec-

o n d a r y immunization (Figure 2), except that the primary IgA levels were higher in s o m e of the mutants. Thus, IL-10 has no decisive function for the induction of antibody production and d e v e l o p m e n t of B cell m e m o r y during the interaction of Th2 and B cells. In addition, we measured the concentrations of antibodies of the various isotypes in the sera of n o n i m m u n i z e d IL10T and control mice (Figure 3). The levels of most isotypes were not significantly different between the two groups, indicating also that the antibody response of IL10T mice to environmental antigens is not fundamentally disturbed. The elevated levels of IgG1 and especially of IgA found in the majority of the IL10T mice probably reflect the increased numbers of IgA and IgG 1 plasma cells associated with the d e v e l o p m e n t of chronic enterocolitis in these animals (see below).

IL10T Mice Develop a Thl Response upon Nematode Infection Infection of mice with the n e m a t o d e N. brasiliensis induces the d e v e l o p m e n t of a Th2 response, with increased production of IL-4, IL-5, and IL-10. IL-10 might act as an effector molecule through which Th2 cells inhibit the develo p m e n t of a Tht response to this parasite (Mosmann et al., 1991). We c o m p a r e d the pattern of cytokines produced by Con A - s t i m u l a t e d spleen cells from Nippostrongylusinfected IL10T and wild-type mice to determine w h e t h e r IL-10-deficient animals would d e v e l o p a T h l response in addition to a nematode-induced Th2 response. The levels

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F~gure3. Serum Concentrationsof ImmunoglobulinIsotypes from IL10T and Control M~ce IL10T mice (8 weeks old) (closedcircles) and control mice (opencircles) were bled from the tail vein, and the serum concentrationsof the indicatedisotypesweredeterminedby ELISA.Eachsymbolrepresents the value obtainedfrom one animal.

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Figure 2. T Cell-DependentAntibodyResponseof ILl 0T and Control Mice IL10T mice (8 weeks old) (closedcircles) and control mice (opencircles) were immunizedw~th NP-CG, and the concentrationsof NPspecificantibodiesof the indicatedisotypesweredeterminedby ELISA in sera taken 14 days after primary(A) or 6 days after secondary(B) ~mmunization,exceptfor IgM, which was measuredin sera collected at day 6 of the primary response.Concentrationsof NP-bmdingantibodies in preimmunesera were below0.5 I~g/mlin all animals(data not shown).The valuesgiven for the secondaryresponserepresent the increaseof the concentrationsof NP-bmdingantibodiesin sera within 6 days after secondaryimmumzation.Eachsymbolrepresents the value obtainedfrom one animal.

of IL-4 and IL-5 were increased to a similar extent in cultures from infected mutants and controls (Figure 4), indicating that IL-10 is not necessary for the development of Th2 cells, in accordance with the results obtained for the anti-NP-CG response. The levels of IFN7 were 5-fold higher in spleen cell cultures from infected IL10T mice compared with infected controls. A similar difference in the production of IFN~, was found in cultures of anti-CD3stimulated splenic CD4 ÷T cells from infected animals (Figure 4). Using immunostaining for IFN~, and flow cytometry (Schmitz et al., 1993), we found a 3-fold increase in the frequency of IFN~,-producing T cells (data not shown) in CD4 ÷ T cell cultures from IL10T mice compared with controls. Taken together, these data provide evidence that IL10T mice develop a Thl response that is suppressed in normal mice by the production of IL-10 by Th2 cells upon nematode infection. In conclusion, IL-10 is not needed for the development of Th2 cells, but limits the development of Thl cells during a parasite-induced Th2 response in vivo. IL10T Mice Are Growth Retarded and Develop Anemia Among the offspring from matings of heterozygous mutants, it was apparent that the size and body weight of most animals homozygous for the IL10T mutation was reduced compared with their normal littermates (Figure 5a). At the age of 7-11 weeks, approximately 75% of the IL10T mice

were affected, with the majority of animals exhibiting a weight reduction of about 30%. The weight difference is established postnatally, and growth retardation becomes evident between the age of 3 and 4 weeks (Figure 5e). For some mutant mice, the body weight was normal up to the age of 12 weeks. In these animals (e.g., group 2 in Figure 5a), disease progression is slower, but finally all mutants are affected by weight loss. Both sexes are equally affected. About 90% of the IL10T mice are anemic at the age of 7-11 weeks, as shown by decreased numbers of erythrocytes and hemoglobin concentration in the blood (Figures 5b and 5c). The numbers of leukocytes (Figure 5c) and platelets (data not shown) in the blood of IL10T mice were within the normal range or were increased up to 2-fold. Differential counts on blood films showed that the elevated leukocyte values were caused by an increase in granulocytes. The anemia could be defined in most cases as microcytic or normocytic and hypochromic. Because iron levels in sera of IL10T mice were reduced by 50% compared with normal animals and iron stores were found depleted using the Prussian blue reaction on sections of bone marrow and spleen (data not shown), iron deficiency most likely contributes to the anemia. Colony assays revealed no reduction in the frequency of stem cells or myeloid and erythroid progenitors in bone marrow of mutants with milder manifestations of anemia (data not shown). As shown by the mortality diagram in Figure 5d, the disease developed by the IL10T mutants is lethal for about 30% of the animals up to the age of 3 months. Death was correlated with severe anemia and a gradual weight loss. At later time points, the mortality increased but did not reach 100%. Anemia and weight loss appeared among mutants derived from two independent ES cell clones and were also observed in homozygous mutants after six backcrosses of the IL10T mutation to the C57BL/6 background. Since the phenotype of the latter animals was less variable and more distinct, the variability in onset of the disease observed among the mice shown in Figure 5 is probably partly due to genetic heterogeneity. To define the origin

IL-10-Deficient Mice Develop Chronic Enterocolitis 267

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Figure 4. Cytokine Production of Stimulated T Cells from Nippostrongylus-lnfected IL10T and Control Mice IL10T and control mice (9 weeks old) were infected with N. brasthensis larvae, and spleen cells were isolated after 8 days. Shown are the concentrations (mean _ SD) of the indtcated cytokines in 48 hr culture supernatants of Con A-stimulated spleen cells from infected IL10T (closed bars) or control (hatched bars) ammals (plus Nippostrongylus, eight ammals per group) in comparison to parallel cultures from noninfected mice (minus Nippostrongylus; five animals per group). The cytokine levels were determmed by ELISA. IL-4, IL-5, and IFN~/were measured from cultures containing the anti-lL-10 antibody SXC1 to exclude inhibitory effects of IL-10 produced in cultures from normal animals. The relattve numbers of splenic CD4+ and CD8+ T cells were not significantly different between the IL10T mice and controls (data not shown). In parallel, splenic CD4+ T cells from three infected IL10T and three control mice were isolated by fluorescence-activated cell sorting and stimulated with anti-CD3 antibodies for 48 hr (CD4÷; plus Nippostrongylus). ND, not detectable. The detection limit of the IL-10 ELISA was 2 U of IL-10 per milliliter of supernatant.

of weight reduction and a n e m i a in IL10T mice, animals kept under conventional or SPF conditions were sacrified and their organs e x a m i n e d histologically.

Histopathology of IL10T Mice from Conventional Breeding Major organs such as brain, heart, lungs, liver, pancreas, and kidneys from I L10T mice kept in a conventional animal facility were e x a m i n e d histologically and appeared normal. In contrast, profound alterations were present in the intestinal tract and hemopoietic tissues. The principal histopathologic finding in anemic and underweight animals was a chronic enterocolitis that involved the entire intestinal tract, the d u o d e n u m , proximal jejunum, and proximal colon being most severely affected. The intestinal pathology was characterized by a regionally variable pattern of mucosal inflammation associated with either hyperregenerative or d e g e n e r a t i v e lesions of the intestinal epithelia. In the d u o d e n u m and adjoining jejunum, the chronic inf l a m m a t o r y process caused excessive regenerative hyperplasia of the mucosa leading to a marked thickening of the intestinal wall. The typical architecture of the mucosa was disturbed by the formation of abnormal crypt and villus structures consisting of branched and fused villi, enlarged and branched crypts, and labyrinthine sheets of enterocytes on the surface of the m u c o s a (Figures 6A and

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Ftgure 5. Body Weight, Hemoglobin Concentrations, and Mortality of IL10T Mice (a and b) The body weights (a) and hemoglobin concentrattons in the blood (b) of several groups of IL10T (closed circles) and littermate control (open circles) mice at the indicated age are shown. Each symbol represents the value obtained from one animal. Males and females are not distinguished. (c) Correlation of the body weight with the concentration of hemoglobin, the number of erythrocytes, and the number of leukocytes m the blood of the individual antmals of group 1. (d) Mortality diagram of IL10T and control animals of group 3 from birth to the age of 3 months (e) Postnatal growth of the individuals of a group of IL10T mice and httermate controls between day 20 and day 44.

6B). In some aspects these lesions were reminiscent of inflammatory pseudopolyps. The typical quantitative relation between the duodenal villi and crypts was changed in the IL10T mice by e n l a r g e m e n t of the crypt compartment, leading to a complete loss of villi in some regions. Further, a marked desquamation of apical epithelia, multiple small superficial erosions of the mucosa covered by inflammatory exudate, mucus and cellular debris (Figure 6C), and a thickening of subepithelial b a s e m e n t m e m b r a n e s were detected. The abnormal architecture of the m u c o s a was associated with an extensive l y m p h o p l a s m o c y t i c and histiocytic infiltration of the lamina propria and tela submucosa. Apart from lymphocytes and plasma cells, macrophages, neutrophils, and occasional multinucleated giant cells and eosinophils contributed to the infiltrates. Along the jejunum these alterations gradually decreased, and in the ileum a milder inflammatory reaction was associated with a general atrophy of the m u c o s a equally involving crypts and villi and circumscribed epithelial destructions (Figures 6D and 6E). The gut-associated lymphatic tissue was diminished. Changes of the s a m e kind were present in the large bowel of most mutant mice (Figure 6F).

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Figure 6. Intestinal Histopathology of IL10T Mice from Conventional Breeding (A) Duodenal mucosa of an IL10T mouse showing abnormal architecture due to pathologic growth of the crypt compartment (compare with [B]). The depth of the branched crypts is increased. At the luminal surface (L), epithelial cells are desquamated from rudimentary villi. Magnification, 68.25 x. (B) Duodenal mucosa of a wild-type mouse. Magnification, 68.25 x. (C) Abnormal superficial epithelium of the duodenum disclosing circumscribed epithelial erosions (arrows) covered by an inflammatory exudate (asterisks). Magnification, 68.25 x. (D) The ileal mucosa of an ILl 0T mutant shows atrophic crypts and villi (compare with [E]). The arrow points to a deformed ileal villus. Magnification, 107.25 ×. (E) Ileum of a wild-type mouse. Magnification, 107.25 x. (F) Colon transversum from an IL10T mouse showing the atrophy of the mucosa with reduced numbers of goblet cells and degenerationof superficial epithelial cells. Magnification, 65 x. (A-E) Hematoxylin-eosin staining. (F) Periodic acid-Schiff reaction.

Immunostaining revealed that most of the numerous plasma cells in the infiltrate produced IgA (Figure 7A). IgA immunolabeling was e n h a n c e d in d e g e n e r a t e d enterocytes of IL10T mutants (Figures 7A and 7B), and foci of

extracellularly deposited IgA were present in the connective tissue of the lamina propria (Figure 7A). As shown by immunostaining, the expression of M H C class II molecules in the gut of IL-10-deficient mice was altered. All of the

IL-10-Deficient Mice Develop Chronic Enterocoht~s 269

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Figure 7. Immunostaming for IgA and MHC Class II Antigen in the Intestinal Tract of IL10T Mice (A) IgA immunostaining of the duodenal mucosa of an IL10T mouse showing the infiltration of the lamina propna w~th IgA plasma cells. The arrow points to interstitial IgA deposits beneath altered apical enterocytes containing IgA• Magnification, 136.5 x. (B) Enterocytes of the ~rregular and atrophic colonic mucosa of an IL10T mouse d~splay strong cytoplasmic IgA ~mmunostaining Magnification, 1365x (C) Immunolabehng of MHC class II molecules in the duodenum of an IL10T mouse revealing class II expression on epithehal cells of duodenal villi and crypts (arrows) Magnification, 136.5 x . (D) Duodenal mucosa of a wild-type mouse Only mldvillus enterocytes are stained for MHC class II antigen• Magmficat~on, 136.5x. (E) Immunolabeling of MHC class II molecules m the colon of an IL10T mouse demonstrating class II expression on the irregular colonic ep~thehum. Magnification, 136.5 x . (F) Colomc epfthehum of a wild-type mouse, being MHC class II antigen negative Magnification, 136.5 x For methods see Experimental Procedures•

epithelium of the small intestine w a s labeled in I L10T mice, while in control a n i m a l s only midvillus e n t e r o c y t e s s h o w e d a faint staining signal (Figures 7C and 7D). In the large intestine of IL10T mice, we found e x p r e s s i o n of M H C class II m o l e c u l e s on the colonic epithelium, which is negative

for class II e x p r e s s i o n in wild-type animals (Figures 7E and 7F). T h e histopathological c h a n g e s in h e m o p o i e t i c tissues of IL10T mice w e r e variable, d e p e n d i n g on the severity of anemia. In s e v e r e l y a n e m i c animals, the b o n e m a r r o w

Cell 270

Figure 8. Histopathologyof IL10T Mice from SPF Conditions (A) Proximal colonic mucosa of an IL10T mouse, displaying hyperregenerativehyperplasia with enlargementof branched and blzzarely shaped crypts. The lamina propria is densely infiltrated by inflammatory cells. Beneath the superficial epithelium,an interstitial edema is present (arrowhead).Magnification, 65.1 x. (B) Colon of an IL10T mouse. Arrows point to a small erosionof the crypt epitheliumcovered by an exudate of inflammatorycells. The inflammatory reaction in the lamina propria (P) is predominantly histiocytic. Magnification, 130.2 x. (C) Bone marrowof an IL10T mouse. Myeloid cells prevail,and only a few erythroidcellswith dense dark nuclei are present. Magnification, 204.6 x. (D) Bone marrow of a wild-type mouse. Cells of the erythroldlineageare morefrequentthan myeloid cells. Magnification,204.6x. (A-D) Hematoxylin-eosinstaining. Animals were analyzedat the age of 8 weeks.

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was completely depleted of erythroid cells, while the myeloid compartment was hyperproliferative, and the red pulp of the spleen showed a hypoplasia.

Attenuation of the Disease under SPF Conditions IL10T mutants kept under SPF conditions lacked the alterations seen in the small intestine of mutants from conventional breeding, but exhibited lesions in the proximal colon similar to those in the duodenum of the latter animals (Figures 8A and 8B). The more caudal parts of the large intestine were normal. Thus, IL10T mice from SPF conditions suffer only from a local colitis and not a general enterocolitis as observed in mutants from conventional breeding, even when animals with a similar degree of anemia are compared• The alterations in hemopoietic tissues of mutants from SPF conditions were similar to those of IL10T mice from conventional breeding. While erythropoiesis in bone marrow was reduced, myelopoiesis was enhanced (Figures 8C and 8D).

Discussion IL-10 Is Not Required for Development and Function of T and B Cells In IL-10-deficient mice, T and B cell subsets in thymus, spleen, bone marrow, and peritoneum appeared normal by flow cytometric analysis. In particular, we show that IL-10, which is produced by activated B-1 cells, has no importance for the development of these ceils and their response to antigen, in contrast with the earlier finding that treatment of mice with anti-lL-10 antibodies leads to the depletion of the B-1 cell subset (Ishida et al., 1992). Since the serum levels of IFNy are elevated in anti-lL-10treated animals but not in IL10T mutants (data not shown) and since combined anti-lL-10 and anti-IFN'y treatment does not result in loss of B-1 cells (Ishida et al., 1992), the effect of anti-lL-10 antibodies is presumably indirect and not indicative of an autostimulatory role of IL-lO in B-1 cell development.

IL-10-DeficlentMice DevelopChronicEnterocohtis 271

Based on the pattern of cytokine production upon nematode infection of IL10T and control mice, we concluded that IL-10 is not needed for the differentiation of Th2 cells, but that the production of IL-10 inhibits the development of Thl cells during a Th2 response in vivo. An IL-10-mediated inhibition of Thl cell activity during nematode infection could be important for the host to suppress an inappropriate (immunopathologic) reaction to the parasite. In fact, it has been shown that the protective immunity of Nippostrongylus-infected normal mice is inhibited by the administration of IFN7 or the induction of an additional Thl response (Urban et al., 1992). Thus, IL-10 produced by Th2 cells during nematode infections might be of equal importance as the Th2 cytokine IL-4, which supports the development of Th2 cells (LeGros et al., 1990), induces IgE (Finkelman et al., 1986), and is needed for immunity to at least certain nematodes (Urban et al., 1992). However, IL-10 plays no role in the in vivo cooperation of Th2 cells and B cells during antibody responses since we found that the levels and distribution of immunoglobulin isotypes produced upon immunization of IL10T mice with a protein antigen were normal. The normal distribution of isotypes in this response suggests that, in contrast with the nematode-induced immune response, Thl development is not enhanced in IL10T mutants, since Thl cells induce IgG2adominated antibody responses (Mosmann and Coffman, 1989; Rizzo et al., 1992) and the levels of IgG2a anti-NP antibodies were not increased in the mutants compared with the controls. This discrepancy could result from an inherent difference of the two stimuli in terms of antigen presentation, since it is known that the nature of the antigen-presenting cell may affect T helper subset differentiation (Hsieh et al., 1993; Schmitz et al., 1993).

IL10T Mice Develop Chronic Enterocolitis IL-10-deficient mice raised under conventional conditions develop a disease that becomes apparent by weight loss and anemia at the age of 4-8 weeks and that is lethal for most animals. The mutants suffer from chronic enterocolitis predominantly affecting the duodenum, adjoining jejunum, and colon. Some histopathological features of the IL10T mutants are reminiscent of human intestinal disorders with an assumed immunopathogenesis, e.g., ulcerative colitis and celiac disease, but the enterocolitis developed by IL10T mice cannot directly be equated with any human disease. However, the presence of inflammatory infiltrates in the mucosa of the gut, the deposits of fibrinoid material and IgA, and the aberrant expression of MHC class II molecules in intestinal epithelia suggest a primary role for the immune system in the pathogenesis of the disease of IL10T mice. How can the bowel inflammation in IL10T mice be related to the IL-10 deficiency? The mucosal surface of the gut is exposed to high concentrations of antigens derived from food and microorganisms, leading to a continuous stimulation of the intestinal immune system. Since IL-10 is a potent suppressor of cytokine synthesis by macrophages, natural killer cells, and T cells, we suggest that the primary defect in IL-10-deficient mice is a failure to

control normal intestinal immune responses against enteric antigens, leading to chronic inflammation via continuous overproduction of cytokines such as TNFot, IL-1, or IFN~,. In a second step, enhanced epithelial MHC class II expression and antigen presentation and epithelial lesions may lead to a more massive exposure of lymphoid cells in the mucosa to luminal antigens and bacterial cell wall components (e.g., LPS), reinforcing the inflammatory process. The disease of IL10T mice is probably perpetuated by chronic, but readily reversible, overexpression of cytokines, because repeated administration of IL-10 into mutants can transiently cure their disease (D. R., unpublished data). Under normal conditions, macrophage activation is probably tightly controlled by IL-10 produced by T cells and, perhaps, systemically by intestinal epithelial cells. Uncontrolled macrophage activation in IL-10-deficient mice may cause an enhanced stimulation of Thl cells and natural killer cells, leading to an overproduction of IFN~, and other cytokines since IL-10 is known to suppress the macrophage-dependent activation of these cell types (Fiorentino et al., 1991a; Moore et al., 1993). IFN? could cause the aberrant expression of MHC class II molecules found on intestinal epithelia of IL10T mice, which in turn could further stimulate immune responses. Since activated macrophages from normal mice can limit their own cytokine synthesis by the production of IL-10 (Fiorentino et al., 1991 b), macrophages (e.g., stimulated by endotoxins) could directly contribute to an overproduction of cytokines in IL10T mice. Accordingly, we found that spleen cells from IL10T mice produced up to 20-fold more IL-6 and TNFa than splenic cells from normal mice upon LPS activation in vitro (data not shown). Any of these factors could contribute directly or indirectly to the inflammatory process that, once started, could lead to the complex pathological alterations found in the intestine of IL10T mice. Intestinal microbial flora is thought to play an important role for the initiation and perpetuation of inflammatory bowel disease in humans (Sartor, 1990) and is of prime importance for the onset of ulcerative colitis-like disease in IL-2-deficient mice, which are free of symptoms when kept in a germ-free environment (Sadlack et al., 1993 [this issue of Cell]). Similarly, we observe that IL-10-deficient mice develop an attenuated disease restricted to the proximal colon when kept in a facility with a defined microbial environment (SPF). This striking difference to the conventionally housed mutants suggests that the inflammatory response in IL10T mutants is triggered by enteric microbial flora, the composition of which determines the severity of symptoms. However, as far as we have tested, mutant mice raised under conventional conditions were free of common intestinal pathogens (data not shown), so we have no clue as to whether a particular pathogen might be responsible for the more severe inflammation seen in conventionally kept as compared with SPF animals. It will be interesting to determine which organism(s), environmental factor(s), or both cause the difference in the severity of symptoms between the two groups. Since the difference in body weight between mutants and littermate controls starts to develop at the age of 3-4 weeks, i.e., the time of weaning, the onset of the disease in IL10T

Cell 272

mice probably begins right after the colonization of the gut with adult microflora. Anemia and growth retardation of IL10T mice from conventional conditions could be readily explained by disturbed nutrient resorption as a result of the severe alterations in the small and large intestine of these animals. In accord with this notion, we found reduced iron levels in serum and depleted iron stores in spleen and bone marrow of IL10T mice, suggesting that the anemia is caused by iron deficiency. Since the intestinal pathology is less severe in anemic mutants from SPF conditions, factors other than malabsorption, possibly an overproduction of certain cytokines, might contribute to the anemia. Anemia can be induced in mice by the administration of TNF(~ or IL-I~ (for review see Means and Krantz, 1992). Apart from IL-10, the cytokines IL-4 and TGFI31 inhibit macrophage functions in vitro, acting synergistically in combination with I L-10 (for review see Sher et al., 1992). While IL-4-deficient mutants appear healthy (KLihn et al., 1991), TGFI31-deficient mice suffer from inflammation in multiple organs, mainly heart, liver, lungs, and muscle (Shull et al., 1992). The present results establish that IL-10 is only of regional importance to prevent inflammation, in contrast with the broad anti-inflammatory action of TGFI31. So far, it is an open question whether this difference in anti-inflammatory activity reflects a true compartmentalization of IL-10 function or whether TGFI31 compensates for IL-10 in IL10T mice in all organs except the intestine. Interestingly, similar to IL-10-deficient mice, mutants deficient for IL-2 also develop a chronic bowel inflammation that is initiated and perpetuated by enteric microflora, probably by a different pathogenetic mechanism (Sadlack et al., 1993). The intestinal immune system, which is continuously involved in immune responses against varieties of antigens, may be particularly vulnerable to any perturbance of the various regulatory mechanisms that prevent immunopathologic reactions. In conclusion, the present data indicate that IL-10 has an essential rote in controlling intestinal immune responses directed to enteric antigens. Breeding of IL10T mice with mutants specifically deficient for T cells, B cells, or both or cytokine-deficient mice should lead to the identification of cell types and cytokines crucial for the development of the chronic enterocolitis seen in the IL10T mutant strain. In addition, the introduction of IL10T mice into a germ-free environment will allow the screening of microorganisms for their potential to induce the disease. As an animal model, IL10T mice could contribute to the understanding of the pathogenesis of human idiopathic inflammatory bowel disease. Experimental Procedures Generation of IL10T Mice The IL-10 gene-targeting vector was constructed by blunt-end ligatlon of a 24 bp DNA linker into a filled Espl site in the first exon of the IL-10 gena (Kim et al., 1992) contained within a subcloned 1.4 kb Bglll fragment. The linker (5'-GTGAACATCGATAGCTAGCTCGAG-3') interrupts the coding sequence behind codon 4, and translation should be terminated at a linker-derived stop codon. The n e o expression cassette from pMClneopA (Thomas and Capecchi, 1987) was inserted as a XhoI-Sall fragment into aXhol site of the linker 3'of the termination

codon The following 500 bp, including codons 5-55 of exon 1, were deleted by digestion with BamHI-Bglll and religation. A homology region of 4.2 kb (BgllI-Hindlll fragment) and an HSV TK expression cassette (Mansour et al., 1988) were added. An additional termination codon was created in exon 3 by cutting, filling, and religating an EcoRI site that was thereby destroyed. The linearized vector was introduced into E14-1 ES cells by electroporation. ES cells were transfected, cultured, and selected as previously described (K~ihn et al., 1991). Of 298 G418 and gancyclovir-resistant colonies, 20 were identified as recombinants by polymarase chain reaction as described previously (Kitamura et al., 1991) using an IL-10 primer located upstream of the short arm of homology (5'-TAGGCGAATGTTCTTCC-3')and a primer complementary to a sequence of the neo gene (5'-CCTGCGTGCAATCCATCTTG-3~, using an annealing temperature of 59°C was used. The recombination events in exon 1 and exon 3 were verified by Southern blot analysis of EcoRI-digested genomic DNA from polymerase chain reaction-positive colonies using a 0.6 kb XbaI-Bglll fragment (probe A) and a 1.1 kb EcoRI fragment (probe B) as probes (Figure 1A). All polymerase chain reaction-positive colonies showed the correct integration of one copy of the n e o gene into one allele of the IL-10 gene, and 70% of them cointegrated the mutation in exon 3. Hybridization with a neo probe confirmed the presence of a single copy of the neo gene. The presence of duplications in the targeted locus were excluded using additional probes (data not shown). ES cells carrying the IL10T mutation were injected into blastocysts of C57BIJ6 mice as described (Bradley, 1987), the resulting male chimeras were mated to C57BL/6 females, and heterozygous IL10T mice were intercrossed to generate homozygous mutants. The genetic background of these animals is a mixture of the strains 129/Ola and C57BL/6, since the ES cell line used is derived from strain 129/Ola and heterozygous littermates were crossed randomly to generate larger numbers of IL10T and control mice. All animals were typed for the IL10T mutation by Southern blotting of EcoRI-digested DNA isolated from tail biopsies, using probe A. The mice were kept in a conventional animal facility at the Institute for Genebcs (Cologne, Federal Republic of Germany) and at the DNAX Research Institute (Palo Alto, California) or under SPF conditions at the Max Planck Insbtute for Immunobiology (Freiburg, Federal Republic of Germany). Flow Cytometric Analysis Cells from thymus, spleen, bone marrow, and peritoneum were stained with monoclonal antibodies (MAbs) and analyzed with a FACScan cytometer using the Lysis 2 program (Becton-Dickmson). The following MAbs were used in the flow cytometric analysis: allophycocyanin and fluorescein-conjugated RA3-6B2 (anti-CD45R/B220; Coffman, 1982), fluorescein-conjugated sheep antimouse IgD (Nordic Immunological Laboratories), phycoerythrin-conjugated anti-lgM (R33-24-12; Gr(Jtzmann, 1981), phycoerythrin-conjugated GK1.5 (anti-CD4; Dlalynas et al., 1983), fluorescein-conjugated 53-6.72 (anti-CD8; Ledbetter and Herzenberg, 1979), and biotinylated-conjugated 53-7.3 (anti-CD5, Ledbetter and Herzenberg, 1979). Biotinylated MAbs were detected in a second staining step with streptavidin-phycoerythnn. CD4÷ T cells were isolated by staining with fluorescein isothiocyanate-conjugated GK1.5 and sorted using a FACStar Plus cell sorter. Dead cells were excluded by propidium iodide staining. Cells present in the lymphocyte gate defined by light scatter (FSrster et al, 1989) were analyzed. Mitogen Stimulations and Measurement of Cytokine Production Spleen cell suspensions from IL10T mice and littermate controls were depleted from erythrocytes and stimulated for 48 hr with 3.5 i~g/ml Con A (Pharmacia) in CG medium (Camon Labor-Service)at a density of 3.5 x 106 cells per milliliter for the measurement of IL-4, IL-5, IL-10, and IFN7 production. The cultures contained, in addition, the anti-II_-I 0 antibody SXC1 (10 I~g/ml)(Mosmann et al., 1990) as indicated Groups of animals infected subcutaneously with 500 stage 3 larvae of N. brasdiensis (Kassai, 1982) were compared with nomnfected mice 8 days after infection. F l u o r e s c e n c e - a c t i v a t e d cell-sorted CD4+ T cells were stimulated for 48 hr in CG medium at a density of 106 cells par milliliter in rnicrotiter plates coated with the anti-CD3 antibody 145-2C11 (Leo et al., 1987). Stimulation of splenic and peritoneal cells (2 x 106 cells per milliliter) with LPS (Sigma) were performed with 2 ilg of LPS per milliliter for 36 hr in CG medium. Concentrations of IL-4, IL-5, IL-6,

IL-10-Deficient Mice Develop Chronic Enterocolihs 273

iL-10, TNF(~, and IFN'y m the culture supernatants were determined by ELISAs (Kendall et al., 1983) by comparison of serial ddutions of the supernatants and standard cytokme preparations. The ELISAs for IL-4, IL-5, and IFN? were performed as described (Schmitz et el., 1993). For determination of I L-6, IL-10, and TNF(x, plastic plates were coated with MAb (2-5 p_g/ml) MP5-20F3 (anti-lL-6; Mosmann et al., 1990), SXC2 (anti-lL-10; Mosmann et al., 1990), or MP6-XT3 (anti-TNF~; Pharmingen). Diluted samples were added, and the bound cytokine was detected with the biotinylated MAb (1 p_g/ml)MP5-32C11 (anti-lL-6; Mosmann et al., 1990), SXC1 (anti-lL-10; Mosmann et al., 1990), or MP6-XT22 (anti-TNF(~; Pharmingen). The assays for IL-4, IL-5, IL-10, and IFN'y were performed in the presence of 0.05% Tween 20. Standards of IFN'y and IL-5 were purchased from Genzyme Corporation; standards of IL-6 and IL-10 were a gift from A. O'Garra (DNAX), and a TNF(~ standard was obtained from R. Devos (Roche Research). As the IL-4 standard, we used a preparation of recombinant IL-4, 1 U being defined as the concentration reducing half-maximal proliferation of the T cell line CTLL2 (Gillis and Smith, 1977); 1 U of IL-10 is defined as the amount inhibiting the IFN? production of the T cell line HDK1 by 50o/0 (Fiorentlno et al., 1989).

Acknowledgments

T Cell-Dependent Immune Response and Determination of Immunoglobulin Isotypes

Bhattacharya, A., Dorf, M E., and Springer, T, A. (1981). A shared alloantigenlc determinant on la antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication. J. Immunol. 127, 2488-2495.

IL10T and control mice were injected intraperitoneally wzth alumprecipitated NP14-CG (100 ~g per mouse), and sera were collected after 6 and 14 days. For measurement of the secondary antI-NP response, the animals were boosted 7 weeks after the primary immunization with 100 p.g of NP~4-CG per mouse, and sere were taken after 6 days. For the determination of NP-binding antibodies by ELISA, plastic plates were coated with NP14-bovine serum albumin (10 ~g/ml). Diluted serum samples were added, and the bound ~mmunoglobulin isotypes were detected as described (KDhn et al., 1991), except that goat antibody to mouse IgG2a (Southern Biotechnology Associates) was used for the detection of IgG2a. The measurement of the total concentrations of ~mmunoglobulin isotypes in sera was performed as described previously (K~hn et al., 1991), except that plates were coated with sheep antibody to mouse IgG2a (Dunn Labortechnik) and developed with goat antibody to mouse IgG2a (Southern Biotechnology Associates) for the detection of IgG2a.

Blood Parameters Blood was collected from the tad vein, and hemoglobin concentrations were determined photometrically at 546 nm after dilution into lysls buffer (3 mM K3(Fe(CN)6, 1.5 mM KCN, 5 mM Na2BO4, 0.1% Nonidet P-40; Boll and Heller, 1991). A reference curve was established with mouse hemoglobin (Sigma) solutions of known concentration. Cell numbers were determined in a hemocytometer.

Histological and Immunohistological Analysis Tissue specimens were fixed in 40/oformaldehyde containing 1% acetic acid and embedded in Histosec (Merck) The en block-fixed gastrointestinal tract was dissected into stomach, duodenum, proximal and distal jejunum, ileum and colon, and rectum. Sections were stained with hematoxylin-eosin, periodic acld-Schiff reaction, the Prussian blue reaction for stored iron, and the Gomori silver impregnation method according to standard protocols. Immunostaining of MHC class II antigen and mouse immunoglobuhns was performed on sections from paraffin-embedded tissue by use of the avidin-biotin-peroxidase method (Hsu et al., 1981) after overnight incubation at 4°C with the primary antibodies. The MAb M5/114.15.2 (Bhettacharya et al., 1981) reacting with mouse i-Ab,~,e I-Ed,k and rabbit antisera directed against the I~, 7, or (z heavy chains of mouse immunoglobuhns (Nordic Immunological Laboratories) were used as primary reagents. As bridging reagents, blotlnylated F(ab)2 fragments of immunoafflnity-purified mouse antibodies against rat IgG (Jackson Laboratory) or sheep antibodies against rabbit IgG (Sigma) were used. Biotinylated antibodies were detected by immersing the sections for 40 min in a solution of streptavidin-biotin-peroxidase complexes (DAKO Diagnostica) followed by the diaminobenzldine reaction. Finally, the sections were counterstained with hemalum.

We thank H. Mosmann for transfer of the IL10T mice into SPF cond=tions; A. O'Garra, R. Devos, and R. Coffman for cytokine ELISA reagents; K. Moore for the IL-1O gene; R. Harder for nematode larvae; H. te Rlele and M. Hooper for ES cells; the Genetics Institute for a leukemia inhibitory factor-producing cell line; B. Hampel, C. Gottlinger, and C. Hachenberg for technical assistance; and U. Ringeisen for preparing the figures. We are grateful to I. Horak for sharing results about IL-2-deflcient mice prior to publication. This work was supported by the Bundesministerium fLir Forschung und Technologie through the Genzentrum KSIn by Land Nordrhein-Westfalen, and by the Fazit Foundation. The Heinnch Pette Institute is supported by the Frele und Hansestadt Hamburg and the Bundesministerium f~ir Gesundheit. The DNAX Research Institute is supported by the Schering-Plough Corporation. Received June 25, 1993; August 9, 1993.

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