Accumulation of immunoglobulin-containing cells in the gut mucosa and presence of faecal immunoglobulin in severe combined immunodeficient (scid) mice with T cell-induced inflammatory bowel disease (IBD)

Abstract

Scid mice transplanted either with a gut wall graft or with low numbers of purified CD4⁺ T cells from immunocompetent syngeneic donor mice show clinical signs of IBD 3–4 months post-transplantation. The disease is mediated by mucosa-infiltrating CD4⁺ TCRαβ⁺ T cells. The pathology of 52 individual colon segments obtained from 20 gut wall- or CD4⁺ T cell-transplanted diseased scid mice was evaluated by histology and the numbers of infiltrating immunoglobulin-containing cells were determined. In particular, cells positive for IgM, IgA and non-inflammatory immunoglobulin isotypes such as IgG1 and IgG2b were found to accumulate in colon segments displaying the most severe histopathology, including inflammatory cellular infiltration, epithelial hyperplasia and ulcerative lesions. Compared with colon segments of normal C.B-17 mice, the lesional scid colon shows increased levels of cells positive for the IgG classes. Faecal extracts of the CD4⁺ T cell-transplanted scid mice revealed the presence of all six murine immunoglobulin isotypes. Disease progression was accompanied by an increased level of excreted IgM and IgG3 and decreased levels of IgA. It is concluded that locally secreted immunoglobulins may play an immunomodulating role in the pathological changes observed in the present model of T cell-induced inflammatory bowel disease.

INTRODUCTION

Scid mice develop an IBD 3–4 months post-engraftment of a full thickness gut wall graft (GWG) or after injection of purified CD4⁺ T cells from immunocompetent congenic donors [1,2]. T lymphocytes in the gut mucosa of normal mice include both CD4⁺ and CD8⁺ TCRαβ⁺ and TCRγδ⁺ subsets [3,4]. In contrast, only CD3⁺CD4⁺ TCRαβ⁺ T cells are observed in the colonic mucosa of GWG or CD4⁺ T cell recipient scid mice [1,2], suggesting a central role for CD3⁺CD4⁺ TCRαβ⁺ T cells in the pathogenesis of IBD. Phenotypically, these T cells express an activated, memory and mucosa seeking Th1 phenotype, i.e. they are CD44⁺, CD45RB^low, LPAM-1⁺, l-selectin^low and produce enhanced levels of Th1-like cytokines [1,2,5–8]. The T cells migrate selectively between gut-associated lymphoid tissues, mesenteric lymph nodes and the spleen of the recipient. In previous work we observed the occurrence of immunoglobulin-containing cells in the colonic lamina propria of diseased scid mice [2]. However, it remains unclear how locally secreted immunoglobulins in the present model of IBD relate to the pathological lesions of the colonic mucosa.

In the present study we have typed and quantified local immunoglobulin-containing cell numbers in the lamina propria of individual colon segments of scid mice with clinical symptoms of IBD and compared immunoglobulin-containing cell numbers and immunoglobulin isotypes in colon segments displaying different lesional stages. Also, we have analysed the presence of immunoglobulin in the faeces of clinical non-diseased and diseased CD4⁺ T cell transplanted scid mice.

MATERIALS AND METHODS

Mice

C.B-17 (H-2^d) and C.B-17^scid/scid (scid) mice were purchased from Bomholtgaard (Ry, Denmark). The mice were kept in a controlled microbial environment at the animal facilities of the University of Ulm and the Panum Institute. Donor and recipient mice were 5–6 weeks old at the time of grafting.

Induction and assessment of disease

The procedures for atraumatic grafting of GWG from immunocompetent congenic C.B-17 donors onto the back of 6–8-week-old scid mice and the transplantation of low numbers of purified CD4⁺ T cells have been described in detail previously [1,9].

Transplanted mice were monitored for weight loss, rectal prolaps and diarrhoea biweekly. Mice were killed for histological examination when they displayed two out of four of the following signs of disease development: a 20% loss in body weight, profound rectal prolapse, loose stools and bloody diarrhoea. Faecal samples were recovered from groups of control mice, non-transplanted, 6-month-old scid mice, and CD4⁺ T cell-transplanted scid mice with or without clinical signs of IBD.

Histology and immunohistochemistry

The large intestine was removed and fixed in Bouins' fixative (picrylic acid, formalin, acetic acid). Proximal, medial and distal segments of colon were isolated and studied separately. The colon specimens were embedded in paraffin, sectioned, and stained with haematoxylin–eosin. For detection of immunoglobulin-containing cells, 5-μm sections were incubated with biotin-conjugated rabbit anti-mouse immunoglobulin antisera, anti-IgA (Southern Biotechnology, Birmingham, AL), rabbit anti-IgM (Serotec, Oxford, UK), anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 (Zymed Labs, San Francisco, CA). Horseradish peroxidase (HRP)-conjugated streptavidin (Amersham, Aylesbury, UK) was used as a secondary layer. The sections were reacted for 15 min with 0.5% freshly made H₂O₂ to neutralize endogenous peroxidase activity and overnight with antisera or rabbit serum (negative controls) at 4°C. HRP-conjugated streptavidin (RPN 1231, batch 56; Amersham) diluted 1:100 was used as secondary layer for 30 min at room temperature. Reactivity was visualized by incubation in DAB (KemEnTec, Copenhagen, Denmark) with freshly prepared 0.2% H₂O₂ for 10 min.

Bouin-fixed, paraffin-embedded pellet sections of IgM⁺, IgA⁺ and IgG⁺ mouse hybridomas were used as specificity controls for the anti-mouse immunoglobulin reagents mentioned above. The antisera reacted strongly with the relevant immunoglobulin hybridoma class. The anti-IgA, -IgG1 and -IgG2a antisera did not show any cross-reactivity. The anti-IgG2b and -IgG3 antisera showed weak cross-reactivities with the IgA and the IgM hybridomas, respectively. The anti-IgM antiserum showed weak to moderate cross-reactivity with the IgA and the IgG hybridomas. Consequently, only cells in the colonic lamina propria which reacted strongly with the anti-IgM reagent (see Fig. 1B) were considered positive.

Fig. 1.

Inflamed colonic lamina propria stained for cells containing different immunoglobulin classes and subclasses. Colon specimens from scid mice transplanted with gut wall from C.B-17 mice were stained for (A) IgA; (B) IgM; (C) IgG1; (D) IgG2a; (E) IgG2b; and (F) IgG3. Immunoglobulin-containing cells are present as clusters of darkly stained cells. Notice the accumulation of IgG2a-containing cells in the marginal zone of an epithelial ulceration (D). (Mag. × 250.)

Histopathological grading and counting of immunoglobulin-containing cells

For histopathological classification 10 consecutive microscopical fields of six individual sections per colon segment were studied at ×125 magnification equalling approx. 72 mm² per individual colon segment. Pathological changes were verified at × 250 magnification. The gut histology was compared with that of normal scid mice. Three types of lesions were scored: mild, infiltration, i.e. increased mononuclear cell infiltration in the lamina propria; moderate, hyperplasia, i.e. enlargement of crypts with increased mitotic activity, changes which were always combined with infiltration; severe, epithelial lesions including small and larger ulcerations, changes always combined with infiltration and hyperplasia. For counting of immunoglobulin-containing cells, 10 consecutive microscopical fields of the tunica mucosa were examined per immunoglobulin type and colon segment at ×250 magnification equalling 3 mm² per colon segment.

Preparation and dot blot of faecal extracts from diseased and control mice

Faeces was collected from C.B-17 control mice, non-transplanted control scid mice, and CD4⁺ T cell-transplanted scid mice either with or without clinical signs of IBD, and prepared as described by others [10]. In short, the collected faeces was frozen at −20°C, followed by vacuum drying. PBS (20 μl) containing 5% (w/v) dry milk (Difco Labs, Detroit, MI) and 1 mm PMSF (Sigma Chemical Co., St Louis, MO) were added per mg dry weight faeces. After vigorous vortexing and centrifugation at 2 × 10⁵ g for 30 min, the supernatant was collected and stored at −20°C. Murine IgA, IgM, IgG1, IgG2a, IgG2b and IgG3 antibodies were used as positive controls (PharMingen, San Diego, CA) at concentrations of 2.5, 0.5, 0.1 and 0.02 μg/ml. The faecal extracts were diluted in PBS prior to application onto a nitrocellulose membrane (Amersham). Three microlitres were dotted of all samples. The membrane was blocked in PBS containing 3% (w/v) dry milk for 2 h at room temperature, washed in PBS and incubated for 2 h at room temperature with the biotin-labelled antibodies also used for immunohistochemistry, diluted 1:1000 in incubation buffer (PBS containing 0.3% (w/v) dry milk). The membrane was then washed in PBS and incubated with peroxidase-conjugated streptavidin (Dako, Glostrup, Denmark) diluted 1:2000 in incubation buffer. After washing, the blot was developed using ECL Western blotting detection reagents and ECL Hyperfilm (Amersham). In separate experiments, the presence of α₁-antitrypsin (α₁-AT) was evaluated in serum and faecal samples by dot blotting with rabbit anti-human α₁-AT MoAb (Zymed) and peroxidase-conjugated swine anti-rabbit immunoglobulin (Dako). The blots were developed as described above.

Statistical analysis

Statistical significance was evaluated using the Mann–Whitney U-test.

RESULTS

GWG-induced IBD in scid mice

As previously reported, scid mouse recipients of a GWG or low numbers of purified CD4⁺ T cells from congenic donor mice develop, within 3–4 months post-transplantation, an inflammatory, hyperplastic and ulcerative bowel disease [1,2]. The scid recipients in the present study were killed and their colons were examined histologically and by immunohistochemistry when the animals showed at least two of the four clinical signs of disease development described in Materials and Methods.

Localization of immunoglobulin-containing cells in the colonic lamina propria

The colonic histopathology of GWG CD4⁺ T cell-transplanted scid mice has been described elsewhere [1,2] and reviewed recently [11,12]. Therefore, the primary aim of the present study was to quantify and type the immunoglobulin-containing cells which infiltrate the colonic lamina propria of clinically diseased mice in colon segments without pathology or with varying degrees of inflammatory pathology.

Figure 1 shows examples of immunoglobulin-positive cells in the lamina propria of the colon from GWG recipient scid mice. IgA⁺ cells were mostly localized as cell clusters deep in the lamina propria basal to the colonic crypts and glands (Fig. 1A). The overlaying epithelial cells often contained substantial amounts of intracellular immunoglobulin, indicating the active transport of this immunoglobulin class through the epithelium [13]. IgM⁺, IgG1⁺ and IgG2⁺ cells were localized as single cells or in small cell clusters both in the basal and in the mid-propria adjacent to the basement membrane of the crypts (Fig. 1B–E). Figure 1D shows IgG2a⁺ cells accumulating in the marginal zone of an epithelial ulceration. This particular ulceration was examined on serially cut sections for all six immunoglobulin classes and subclasses, but only IgG2a⁺ cells were observed to accumulate at the site of ulceration (data not shown). IgG3⁺ cells were scarcely represented in the inflamed mucosa and were always present as single cells scattered throughout the lamina propria (Fig. 1F).

Quantification of immunoglobulin-containing cells in the colonic lamina propria

Table 1 shows the actual number of immunoglobulin-positive cells in areas of 3 mm² colonic lamina propria of seven colon segments obtained from healthy congenic C.B-17 mice and 52 segments from GWG or CD4⁺ T cell-transplanted scid mice showing clinical signs of IBD. Due to the very low numbers of IgG3⁺ cells these are not included in Table 1. In the normal C.B-17 colon, IgA⁺ and IgM⁺ cells dominated the lamina propria and very few IgG⁺ cells were observed. In scid mice with IBD, the numbers of IgA⁺ cells were significantly increased in segments showing moderate (P < 0.02) and severe (P < 0.03) lesions compared with segments without any lesional changes. Table 1 shows that segments showing moderate histopathology contained increased numbers of IgM⁺ cells compared with segments showing either mild (P < 0.02) or no lesions (P < 0.02). Likewise, segments showing severe histopathological changes contained increased numbers of IgM⁺ cells compared with segments showing mild (P < 0.01) or no lesions (P < 0.002). Increased numbers of IgG1⁺ cells were found in severely affected segments compared with lesions with mild (P < 0.01) or no changes (P < 0.002) (Table 1). Despite the apparent increase in IgG2a⁺ cells in segments with severe changes, this increase was not significant according to the Mann-Whitney U-test. Table 1 shows that the numbers of IgG2b⁺ cells were increased in segments displaying severe histopathological changes compared with segments with moderate (P < 0.02), mild (P < 0.0002) or no changes (P < 0.002).

Table 1.

Accumulation of immunoglobulin-containing cells in the colonic lamina propria of CD4⁺ T cell-transplanted clinically diseased scid mice and normal C.B-17 mice in relation to local histopathology

Faecal extracts from diseased scid mice contain elevated levels of immunoglobulin

To test whether immunoglobulin accumulated in the faeces of clinically diseased mice, the immunoglobulin contents of faecal extracts from CD4⁺ transplanted healthy scid mice, diseased scid mice and C.B-17 control mice were evaluated by isotype-specific dot blotting technique. The results are shown in Table 2, and demonstrate the presence of IgA, as well as the other five murine immunoglobulin classes and subclasses. The faecal levels of IgA in clinically healthy and diseased CD4⁺ T cell-transplanted scid mice were comparable to the IgA levels of C.B-17 control mice. No IgA was found in the faeces of non-transplanted scid mice. Clinical diseased scid mice displayed elevated faecal levels of IgM compared with healthy CD4⁺ T cell-transplanted scid mice. Low levels of IgM were present in the faeces of control scid mice, probably due to spontaneous immunoglobulin leakiness [14,15]. IgG2a was found in the faeces of both clinically healthy and diseased CD4⁺ T cell-transplanted scid mice at higher levels than in C.B-17 control mice. Clinically diseased mice displayed increased levels of faecal IgG2b compared with transplanted, still healthy scid mice (Table 2). Despite the small numbers of IgG3⁺ cells found in the intestinal mucosa (see above), clinically diseased scid mice displayed highly increased levels of faecal IgG3 compared with transplanted, still healthy scid mice. Together, these data show that disease progression was accompanied by decreased faecal amounts of IgA and an increased amount of IgM, IgG2b and, in particular, IgG3.

Table 2.

Immunoglobulin in faecal extracts

To investigate whether the diseased colon is leaky for a non-immune serum protein, the presence of α₁-AT in the faecal extracts was investigated. Sera from C.B-17 mice, non-transplanted scid mice and clinically diseased scid mice were all strongly positive for α₁-AT, whereas faecal extracts from C.B.-17 mice and non-transplanted scid mice showed only marginal levels. In contrast, faecal extracts from clinically diseased mice showed highly increased levels of α₁-AT (data not shown). This observation suggests that at least some of the faecal immunoglobulin was leaking out in the gut lumen through the diseased gut wall.

DISCUSSION

The present results confirm and extent previous data showing accumulation of immunoglobulin-containing cells in the colonic lamina propria of scid mice which develop IBD after transplantation of gut wall or CD4⁺ T cells from normal congenic donor mice [1,2]. Furthermore, CD4⁺ T cell transplantation resulted in intestinal secretion and excretion of immunoglobulins. Clinical disease was found to be accompanied by a change in the pattern of immunoglobulin isotypes in the faeces of the scid recipients. Previous data have clearly demonstrated that the IgM secretion in scid mice engrafted with CD4⁺ T cells is derived from leaky recipient B cells [15,16]. However, the origin of the immunoglobulin-containing cells present in the colonic lamina propria of GWG-transplanted scid mice is not clear. Cytokines secreted by the locally expanding CD4⁺ T cells [1] and the increased antigen load in the lamina propria, caused by epithelial lesions, might bypass the effects of the autosomal recessive scid mutation in the GWG recipients. The specificity of locally secreted immunoglobulin is not known. The antibodies might react with microbial constituents present in the gut lumen or with epithelial-derived constituents liberated into lamina propria due to epithelial hyperproliferation and destruction. Cross-reactivity between the luminal microflora and epithelial components might also occur (see below). Thus, it was interesting to note that cells positive for the complement-fixating immunoglobulin, IgG2a [17,18], were the only immunoglobulin-positive cell subset observed in the marginal zone of an ulcerative lesion (see Fig. 1D). Since we did not stain for B cell-specific surface markers, it is probable that some of the immunoglobulin-positive cells encountered may not represent plasma cells but rather phagocytic cells (swamp cells) which have picked up immunoglobulin not necessarily produced locally in the colonic lamina propria (P. Brandtzeag, personal communication). However, our estimation of the relative distribution of immunoglobulin isotypes in the colonic lamina propria remains unaffected by the occurrence of such swamp cells.

Increased numbers of IgM-containing cells were found in both the moderate and severe colonic lesions in diseased scid mice. This was paralleled by an increase in the faecal IgM levels in clinical diseased mice. Despite the low numbers of IgG3-containing cells in the colonic lamina propria (data not included in Table 1), increased levels of IgG3 were found in the faeces of clinical diseased mice (Table 2). Since murine IgM and IgG3 are strongly complement-activating, it could be speculated that these immunoglobulin classes play a direct role in the observed epithelial lesions by the activation of complement on the surface of the epithelial cells, thereby leading to their destruction (see below). However, significant numbers of cells containing typical non-inflammatory potentially protective immunoglobulins (IgA, IgG1 and IgG2b) were also present in some of the colon segments which showed the most severe histopathological changes (see Table 1), although their levels appeared to decrease in the faecal extracts of clinical diseased animals. The local accumulation of non-inflammatory immunoglobulin classes and isotypes is also surprising in view of recent data showing a dominant Th1 cytokine profile in the colonic lamina propria of scid mice with IBD [8]. The presence of non-inflammatory immunoglobulin-positive cells might reflect immunohomeostatic, protective mechanisms in the ongoing inflammatory processes, whereas the luminal decrease of IgA in clinical diseased animals might reflect a decrease in immunoglobulin-mediated mucosa protection. The luminal immunoglobulin isotypes are probably produced by immunoglobulin-containing cells in the lamina propria, as immunoglobulin-positive cells are not found in the mesenteric lymph nodes or in the spleen (Claesson, unpublished). The presence of α₁-AT in the faecal extracts only in clinically diseased mice suggests that many immunoglobulins passively leak out in the gut lumen through lesions in the epithelial membrane.

Epithelial proliferation and ulcerations in CD4⁺ T cell- and GWG-induced IBD always include massive cellular infiltration in lamina propria adjacent to the ulcerative lesions [1,2]. These changes, together with accumulations of IgG-containing plasma cells, are consistent with previous observations in humans showing that IgG-containing cells might increase up to 30 times in lesional areas in Crohn's disease and ulcerative colitis (UC) when compared with control specimens [19,20]. In human IBD, locally secreted immunoglobulin may thereby contribute to the colonic tissue damage through activation of complement and via antibody-dependent cellular cytotoxicity. In human UC, locally secreted IgG has been demonstrated to react with intracellular proteins in the colon mucosa [21]. In addition, plasma cells in UC have been demonstrated to accumulate in the lamina propria and secrete complement-fixing IgG1 antibodies which react with components of the epithelial cell brush border [22–24].

The importance of local immunoglobulin secretion in murine IBD development has been questioned by the resent demonstration that mice deficient for both IL-10 and B cells acquire a severe colitis indistinguishable from that occurring in mice only deficient for IL-10 [25]. The pathogenic T cell subset in this model of IBD displays the phenotype of a lamina propria-infiltrating activated memory CD4⁺ T cell, i.e. a phenotype resembling that described in our model of IBD [1,2]. However, IBD in IL-10-deficient mice differs fundamentally from the CD4⁺ T cell-induced development in IBD scid mice. First, in IL-10-deficient mice IBD develops spontaneously from 3 weeks old and proceeds for more than 6 months. In GWG and CD4⁺ T cell scid recipients, the first signs of IBD occur several months after engraftment [1,2,11]. Second, the IBD of IL-10-deficient mice kept under specific pathogen-free (SPF) conditions is less severe and only present in the proximal colon segment [26], whereas IBD in scid mice is present throughout the colon irrespective of SPF status. Third, apart from IL-10 deficiency, the knock-out mice possess an intact T cell immune system in contrast to the complete lack of mature T (and B) cells in young non-transplanted scid mice [27]. A role for protective immunoglobulins in IBD was recently suggested from experiments with TCR-α/B cell double-deficient mice, which developed a faster and more severe IBD than TCR-α single-deficient mice [28]. The authors suggested that this lack of immunoglobulin may lead to an impaired clearance of apoptotic cells from the lamina propria and an increased level of circulating autoantigens which accelerated the disease development. Since increased levels of apoptosis in the lamina propria of scid mice with IBD is a typical feature [29], it could be speculated that the increased levels of immunoglobulin observed in the severely affected areas of the colon participate in the clearance of apoptotic cells, thereby reducing the concentration of potentially harmful autoantigens.

In conclusion, we found accumulations of immunoglobulin-containing cells of the IgA, IgM, IgG1 and IgG2b classes and subclasses in the lamina propria of colonic mucosa displaying the most severe histopathological changes. Immunoglobulins were also found in the faeces of diseased mice, and diseased progression was associated with increased levels of IgM, IgG2b and IgG3 and decreased levels of IgA. The present data support the hypothesis that locally secreted antibodies modulate the development of CD4⁺ T cell-induced IBD.