Immunobiology and immunopathology of human gut mucosa: Humoral immunity and intraepithelial lymphocytes

GASTROENTEROLOGY

SPECIAL

REPORTS

AND

1989;97:1582-84

REVIEWS

Immunobiology and Immunopathology of Human Gut Mucosa: Humoral Immunity and Intraepithelial Lymphocytes P. BRANDTZAEG, T. S. HALSTENSEN, D. KVALE, T. 0. ROGNUM, H. SCOTT,

K. KETT, P. KRAJCI, and L. M. SOLLID

Laboratory for lmmunohistochemistry and lmmunopathology (LIIPAT), Institute of Pathology, University of Oslo, The National Hospital, Rikshospitalet, Oslo, Norway

A

n intestinal secretory immune system was first observed by Besredka (1) in rabbits after oral immunization with killed Shig a bacillus; the experimental animals were protected against fatal dysentery by “coproantibodies” that seemed to appear irrespective of the serum titers. Davies (2), who in 1918 studied soldiers stationed around Jerusalem, subsequently obtained evidence for specific gut immunity in humans; he noted that bacterial agglutinins could appear in dysentery stools earlier than in peripheral blood. The molecular basis for such coproantibodies was obscure until 1965 when Tomasi et al. (3) substantiated that external secretions contain a unique immunoglobulin (Ig) now called secretory IgA or SIgA. Considering the extensive surfaces and the particular properties of the vulnerable epithelia covering most mucous membranes, it seems advantageous that a specialized mucosal immune system has developed throughout the phylogeny. The secretory Ig system is the best defined effector mechanism of mucosal immunity. It depends on a fascinating cooperation between the local B-cell system and the secretory epithelium, which contribute, respectively, J chain-containing polymeric Ig (pIgA and pIgM) and the epithelial pig receptor called “secretory component” or SC. Recent observations have indicated that interactions between activated leukocytes (e.g., T cells and macrophages) and the secretory epithelium may upregulate the X-dependent pig transport as part of a local immune response. Indirect evidence has suggested, moreover, that lymphoepithelial interactions (perhaps involving the numerous intraepithelial lymphocytes in the gut) may also be involved in modulation of mucosal immunity by normal down-regulation and pathological up-regulation of systemic types of immune responses to harmless luminal antigens. This review

will focus on recent progress in these aspects of mucosal immunity and speculate about the putative biological significance. Citations will mainly be based on human studies when possible, and emphasis will be placed (perhaps unduly) on current work in our own laboratory.

Mucosal Immunobiology Basis of Specific Humoral Gut

Immunity

in the

Exocrine tissues constitute quantitatively the most important mediator organ of specific humoral immunity. The generation of secretory antibodies depends on a striking local preponderance (70% 90%) of IgA immunocytes (plasma cells and plasma blasts) in the absence of overt inflammation (Figure 1); the gut is the major contributor to this unique defense system (Figure 2). There are normally about lOlo Ig-producing immunocytes per meter of human small bowel (4). Taking the total length of the intestine into consideration, this is a great number compared with 2.5 X lOlo that has been determined for

Abbreviations used in this paper: AX, antigen-presenting cell; BALT, bronchus-associated lymphoid tissue; C, complement; FAE, follicle-associated epithelium; GALT, gut-associated lymphoid tissue; IBD, inflammatory bowel disease; IEL, intraepithelial lymphocyte: IL-Z,interleukin 2; MHC, major histocompatibility complex; pig, polymeric immunoglobulin; pIgA, polymeric (or dimeric) immunoglobulin A; pIgh4, polymeric immunoglobulin M; PP, Peyer’s patch: SC, secretory component; SIgA, secretory immunoglobulin A; SIgM, secretory immunoglobulin M; TCC, terminal complement complex; TCR, T-cell antigen receptor; Tcs, contrasuppressor T cells; Th, helper T cell; Ts, suppressor T cell; UC, ulcerative colits. 6 1989 by the American Gastroenterological Association 0016-5085/89/$3.50

December

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a $ E ‘; g

400

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IgA-producing

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IgM-producing

cella: a

IgG-producing

cells:

IgD-producing

cells: m

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$ . d

300-

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t

P

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200

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I Figure

Figure

1. Paired immunofluorescence staining for IgA and IgG in a section of normal human colonic mucosa. All of the numerous immunocytes in the lamina propria produce IgA except those indicated by arrows, which produce IgG. Immunoglobulin A is also seen in the crypt epithelium (C] as sign of external transport. Gut lumen is at the top. (~400,)

bone marrow, spleen, and lymph nodes collectively (5). A rough estimate indicates that 70%-80% of all Ig-producing cells in humans are located in the intestinal mucosa (Figure 3). A comparable estimate (P-80%) has recently been reported for mice (6). Immunocytes adjacent to exocrine glands produce mainly pIgA (dimers and larger polymers) containing a disulfide-linked polypeptide called the “joining” or J chain (7). Such pIgA can be transported through the glandular epithelium along with J chaincontaining polymeric IgM (pIgM) via the pig receptor constituted by transmembrane SC (8,9). J chain and SC may in fact be regarded as “lock and key” factors in secretory immunity; glandular pig transport thus depends on an intimate interaction between the B-cell system and exocrine epithelia (Figure 4). More pIgA is in this way translocated to the gut lumen every day (40 mg/kg body wt) than the total daily production of IgG, which is about 30 mgikg body wt (10). The transglandular secretion of pIgA into 1 m of

mammarv

(II.

I

Bland

1

oland

oland

2. Densities of Ig-producing immunocytes of different classes in various normal human secretory tissues. Schematic representation based on immunohistochemical data from the authors’ laboratory.

human small intestine has been estimated to be almost 0.8 g/day (ll), which is similar to the daily output from a lactating mammary gland (12). Human bile contributes only about 1 mg/kg- day to the intestinal IgA pool (10) because human hepatocytes lack SC as a pig receptor (see below) in contrast to the rat counterparts (8). This is an important species difference that regrettably is not always apparent from the literature. Regulation of local humoral immunity has to be geared for generation of early memory B-cell clones with prominent J-chain expression and preference for IgA production (7,8). The result is not only a “first line” defense mediated mainly by SIgA antibodies; in addition, the abundance of locally prozo-30% lone

7040%

marrow

0

D

Figure

Spleen

3. Schematic illustration of the distribution (%] of Igproducing immunocytes, all classes included but the respiratory and upper alimentary tract was not taken into account.

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Transmembrane

SC =-

Free

SC

and bound

: Glandular

epithelium

J chain

Plasma

cell

Figure 4. Model for SC-mediated epithelial transport of dimeric IgA. [l] Synthesis and core glycosylation of transmembrane SC in rough endoplasmic reticulum (AER) of epithelial cell. [Z] Terminal glycosylation b) in Golgi complex. [3] Expression of SC at basolateral cell surface. [4] Complexing of SC with J-chain-containing dimeric IgA. [5] Endocytosis of ligand-receptor complexes and excess SC. [6] Transcytosis of vesicles. [7] Cleavage and release of SIgA and free SC. The cleavage mechanism and the fate of the cytoplasmic tail of transmembrane SC are unknown (?). During the external translocation, covalent stabilization of the IgA-SC complexes regularly occurs (two disulfide bridges indicated in SIgA between SC and one of the IgA subunits).

duced IgA antibodies is probably crucial for immunologic homeostasis within the lamina propria. Immunoglobulin A may block the triggering of nonspecific biological amplification mechanisms because it lacks potent effector functions such as classical complement (C) activation (13). At the same time it would be conducive to preservation of health to dampen potentially phlogistic IgG and IgE reand T-cell mediated hypersensitivity sponses against harmless luminal antigens to avoid undue local tissue damage caused by these systemic types of immunity. Although there is little direct evidence that such “oral tolerance” exists in humans, it most likely does so in view of the fact that the vulnerable gut mucosa normally shows no inflammation and virtually no IgG response despite the influx of small amounts of intact dietary antigens after meals (13). It is still obscure how the particular inductive and suppressive immunoregulatory mechanisms are achieved in the gut. The local mucosal site with its lamina propria and epithelium, the organized lymphoepithelial nodules, and the larger lymphoid aggregates like the Peyer’s patches (PPs), are probably all involved in various ways (13). The latter structures (initially believed to be glands), which were described as early as 1677 by the Swiss medical

scientist Johann Conrad Peyer (1653-1712), are continuously being subjected to various types of studies to reveal their supposedly major role in mucosal immunity (9,13). The concept of a “common mucosal immune system” (14) has gained wide acceptance (Figure 5). Antigenic priming of organized gut-association lymphoid tissue (GALT), mainly including the PPs, may give rise to specific secretory immunity not only in the gut but also in the respiratory tract and in the lacrimal, salivary, and lactating mammary glands (13,15). Experimental evidence in animals has suggested that the dissemination of IgA precursor cells to distant sites is much more limited in bronchusassociated lymphoid tissue (BALT) than in GALT (16), and the regulatory mechanisms operating in GALT, BALT, and tonsils may in fact differ in several ways (16,17). Altogether, it seems that GALT plays a fairly unique and dominating role in mucosal immunity. Migration

of Mucosal

Lymphocytes

An important basis for intestinal immunity is the migration of specifically primed T and B cells from GALT through lymph and peripheral blood and

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Peyer’s

Figure

patch

Distant

intestine

Other

5. Schematic illustration of B-cell traffic and differentiation in the secretory immune system. Bold arrows indicate major, thin arrows intermediate, and broken arrows minor pathways. Most stimulated B cells from Peyer’s patches end up as terminally differentiated Q-producing immunocytes in the distant gut lamina propria, some in other secretory tissues and mesenteric lymph nodes, but only few in peripheral lymph nodes and in the dome areas of the patches themselves. Stimulated B cells from lymph nodes may to some extent join those from Peyer’s patches on their migration to secretory sites.

then primarily to the lamina propria surrounding the crypts of Lieberkiihn (Figure 5), which are the glands responsible for pig transport into the gut lumen (Figure 4). Extravasation of lymphoid cells seems to depend primarily on their receptors for endothelial determinants (“vascular addressins”), which to some extent are tissue specific (18-20); but additional adhesion molecules (21) and other local factors are probably also involved (22). Different addressins are expressed by high endothelial venules in peripheral lymph nodes and PPs. Small amounts of the PP-type of vascular addressins are also present on ordinary venules in the gut lamina propria (20); this may contribute to the low-rate continuous mucosal extravasation of GALT-derived lymphoid cells (21). It is noteworthy that intestinal intraepithelial lymphocytes (IELs), which are generated at least in part in GALT (23,24), show selective affinity for the addressin characteristic of PPs (25). High endothelial venules of mesenteric lymph nodes apparently share vascular addressins of peripheral lymph nodes and GALT (18). This fact may explain why migratory spillover from the secretory to the systemic immune system seems to take place mainly in the mesenteric nodes (Figure 5) as indicated from the relatively high expression of J chain and IgA2 subclass shown by IgA immunocytes terminating there (26). However, some migration of T lymphocytes and B cells with activated J-chain gene, probably occurs from stimulated peripheral lymph nodes to secretory sites (Figure 5) because parenteral immunization can lead to low-level priming of an intestinal immune response (13).

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Whereas B cells localize in the lamina propria and proliferate and differentiate there (13), many T cells find their way into the intestinal epithelium. The latter phenomenon may be partly antigen-independent because IELs are observed before birth (27); but luminal antigens clearly determine the magnitude of the intraepithelial migration (28). The follicle-associated epithelium (FAE) covering the human PPs contains many T cells in particular, especially near the antigen-transporting M (“membrane”) cells (29). Particular Properties of Intraepithelial Lymphocytes It has been claimed that there are more lymphocytes in the gut epithelium than in any lymphoid organ (30). Two as yet unproven functions have been ascribed to the IELs, namely major histocompatibility complex (MHC)-restricted or unrestricted cytotoxicity (30-32) and suppression of mucosal hypersensitivity reactions (33). Human IELs are mainly positive for the pan-T-cell marker “cluster of differentiation” No. 3 or CD3 (Table l), and they show a striking predominance (80%-90%) of T8 (CD8+) cells (“cytotoxic/suppressor” phenotype), whereas the lamina propria contains mainly the T4 (CD4+) (“helper/inducer”) subset (Figure 6). Most human IELs are negative for the H366 antigen (38), which is a putative marker of MHC-restricted cytotoxic T8 cells (42), and they show no spontaneous cytotoxicity (43). These results may reflect that human IELs are mainly functional suppressor (Ts) cells, although the concept of immunologic suppression remains quite controversial (44,45). Both suppressor and

Table

1. Characteristics Lymphocytes Intestine

of lntraepithelial T in the Normal Human

Small

CD3+CD8+

80%-90%

CD3+CD4+ CD3+CD8-CD4HML-I+ (105050 kDa] HML-l+CD3+ HML-l+CD8+ HML-l+CD4+ CD8+CD7+ CD4+CD7+ CD8+H366+ CD45/UCHLl+ (antigen primed?)

6%-12%” -6%b -94% -88% -77% -9% 70%-80% 8%-12%a,c Few

CD45R/ZH4+ (naive?) TCRa@+ TCRy/S+

- 44%d Virtually none -90% - 2%”

Data are from Selby et al. (34), Hirata et al. (35), Malizia et al. (36), Cerf-Bensussan et al. (37), Trejdosiewicz et al. (38), Spencer et al. (39), Brandtzaeg et al. (401, and Halstensen et al. (41). 0 There may be a higher proportion in colonic epithelium. b Increased to 28% in celiac disease. ’ Increased to 30% in celiac disease. d Increased to 69% in celiac disease. e Increased to ZO-30% in celiac disease.

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Figure 6. Immunofluorescence staining of CD4 and CD8 in two adjacent cryostat sections of normal jejunal mucosa (a) and mucosa from a patient with untreated celiac disease (b). Note that T4 cells (“helper/inducer” phenotype) are distributed mainly in the lamina propria, whereas T8 cells (“suppressor/cytotoxic” phenotype) show a striking selectivity for the epithelium. The density of intraepithelial T8 cells in celiac disease is remarkably increased, especially in the surface epithelium (at the top). Epithelial basement membrane indicated by broken lines. (x200.)

helper activites have been indicated from in vitro studies of human IELs in cocultures with peripheral blood mononuclear cells, apparently depending on the T- to B-cell ratios (46,47). The FAE of human PPs contains a much higher proportion of T4 cells than the ileal villous epithelium, i.e., 40% vs. 10% (29), perhaps reflecting a larger population of functional helper (Th) cells. Human T4 cells that express larger amounts of the CD45IU2H4 marker apparently belong to a naive (unsensitized) population, whereas those high in the smallest (180 kilodalton) CD45 marker (UCHLl) probably represents antigen-primed memory cells (48,49). The same distinction seems to hold true for T8 cells (50). Normal human intestinal mucosa has been reported to contain relatively fewer 2H4+ T cells than peripheral blood, i.e., 38% vs. 68% (51,52),

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and our findings have indicated a still lower mucosal proportion of this putative naive subset (40). Also studies of interleukin 2 (IL-2) receptor (Tat antigen or CD25) on intestinal T cells from normal monkeys have suggested a remarkable level of stimulation (52,53). This observation harmonizes with the fact that many T cells in normal (and especially in diseased) human intestinal mucosa express UCHLl, both in the epithelium (Table 1) and in the lamina propria (40). The IELs bear, in addition, a lymphocyte marker (HML-1) that was thought to be fairly unique for mucosal sites (37). This notion has recently been challenged (54); HML-1 positivity may rather reflect a certain state of stimulation (55), which is in keeping with the UCHLl expression (Table 1) and the morphologic features of the IELs (56). However, they generally seem to be negative for markers of recent activation such as MHC class II and IL-2 receptor (34,36,38). Nevertheless, CD7 (RFT2) is normally expressed on most intraepithelial T8 cells (Table l), supporting the notion that this IEL subset is indeed stimulated (36,38). Intestinal IELs are normally found mainly along the basement membrane, apparently crossing it in either direction (57). This may reflect that they leave the epithelium rapidly by reentering the lamina propria; and it is possible that their immunoregulatory function(s) at least to some extent take place in the latter compartment. There has been some recent controversy about the nature of the T-cell receptor (TCR) for antigen employed by the IELs. Two studies in mice indicated that intraepithelial T8 cells expressed exclusively the TCRyIG rather than the conventional TCRLUIP(58,59); this finding was taken to support the notion that the IELs are mainly cytotoxic (31). However, we have recently shown (4Q,41) that most human IELs employ the TCRa/P (Figure 7); this feature, along with the prominent UCHLl expression, suggest that they are mainly traditional memory T cells (40). What remains unexplained is their remarkable enrichment of the T8 subset (Figure 6) and its biological signficance. Another intriguing feature is the small fraction (median, 2%; range, OS-39%) of TCRy/G+ IELs, that are mainly (-75%) CD3+CD4_CD8-, employ the variable 61 gene much more frequently than their counterparts in peripheral blood (41), and increase intraepithelially in celiac disease (41). Cellular Interactions Promoting ImmunogJobuJin A and J-Chain Expression The appropriate regulatory events of homeostatic intestinal immunity most likely take place both in the organized lymphoepithelial structures of

December 1989

Figure

7. Immunofluorescence staining for T cells expressing TCRLUIBin normal jejunal mucosa. Note that positive lymphocytes are found both in the lamina propria and in the villous epithelium. Epithelial basement membrane indicated by broken line. (x500.)

GALT (such as PPs) and additionally at the secretory sites (Figure 8). It is fascinating that GALT is enriched for precursors of IgA- and J-chain-producing immunocytes (13). Some mechanism(s) apparently exist in PPs that facilitates expansion of early memory clones while inducing little terminal B-cell differentiation locally (Figure 5); the final maturation to plasma cells will therefore mainly take place under the influence of topical antigen and T cells after &A-precursor dissemination to the lamina propria adjacent to the glands (13,601. Most information about mucosal regulatory T cells has been obtained in experimental animals (9,13). Helper T cells that can mediate B-cell isotype switching directly from IgM to IgA expression have been cloned from murine PPs (61). Observations on human B cells have indicated that such a direct differentiation pathway may mainly lead to IgA2 production (62), which is the predominant isotype expressed in the distal gut (63). The switch Th cells seem to be autoreactive and are apparently triggered directly by MHC class II determinants (64). In vitro experiments with human appendiceal lymphoid cells have supported an important role of class II (HLA-DR) molecules in the induction of IgA and IgM production (65). HLA-DR determinants are expressed both on the FAE (66) and on villous epithe-

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lium (Figure 9) in the normal human gut (33), and also on various histiocytic cells and B lymphocytes (13,66,67); it is therefore not possible as yet to know which accessory elements are most important for mucosal T-cell regulation in vivo. Nevertheless, the spatial requirements for immunoregulatory cell-tocell interactions seem to be fulfilled both in the organized parts of GALT and in the mucosal secretory tissue (Figure 8). The murine switch Th cells are apparently unable to induce terminal B-cell differentiation; but Fccureceptor positive postswitch Th cells favoring terminal differentiation with IgA production have also been cloned from murine PPs (9). Moreover, Th cells that promote IgA production by both switch and postswitch effects have recently been cloned from the human appendix (68). The involvement of various T cell-derived lymphokines in mucosal IgA induction is currently in the focus of interest, although it is still unclear at which regulatory level these peptides act (69). Synergistic interactions between various lymphokines seem to be important, for example, the combined effect of IL-4 and IL-5 (70) and the induction of IL-2 receptors on B cells by IL-5 (71). It is of interest that IL-2 and interferon-y have been shown to synergize in selective induction of an IgA response to a bacterial polysaccharide (72). Activated T cells may also induce B-cell transcription of the J-chain gene via IL-2 (73). Antigen Uptake, Processing, Presentation

and

T cells can only respond to antigens that are recognized by their receptors (TCRa/P or TCRyIG) in the context of “self” MHC molecules, although the putative restriction elements for TCRyIG are still obscure. The polymorphic MHC proteins bind processed (degraded and unfolded) antigens in a genetically restricted way and therefore have a crucial guidance function in the immune system. Only cells expressing MHC molecules can serve as antigenpresenting cells (APCs), but they are not necessarily responsible for the uptake and processing of the antigen (74). T4 cells are promoted by MHC class II positive APCs, whereas T8 cells generally seem to depend on class I molecules; the two modes of antigen presentation apparently require different processing pathways (75). The importance of particular APCs within GALT has been emphasized in several studies (76,77). Certain dendritic cells in combination with T lymphocytes from murine PPs have been found to augment remarkably IgA production in contrast to similar cells from spleen (78). It is interesting in this context that we have found a much greater propor-

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Luminal antigens = 0 MHC class II molecules =A Regulatory signals = Secretory component: Dimeric IgA = Figure 8. Schematic illustration of possible sites of interaction between lymphoid cells (T and B), gut epithelium (dotted), and various accessory histiocytic cells. [l] Luminal antigen is transported into Peyer’s patch through M cell of follicle-associated epithelium and is presented to T cell by subepithelial MHC class II positive dendritic cell after being processed in macrophage. [2] Antigen is transferred directly to dendritic cell, [a] Antigen that has been processed in the gut lumen is transported and presented to T cell by class II positive epithelial cell. [4] Autoreactive T cell is stimulated by class II positive epithelium to induce B-cell differentiation (e.g., switching to IgA expression). [5] Activated T cell provides various regulatory signals to B cell. [6] Antigen presentation performed by follicular dendritic cell. T-cell activation and B-cell differentiation takes place in follicular germinal centers, but it is unknown (?) whether the intrafollicular or extrafollicular compartment is more important as a source of stimulated B cells migrating to secretory tissues. [7] Antigen uptake and presentation by class II positive villous epithelium affords further T-cell stimulation at secretory site. [6] Such stimulation can likewise follow absorption or penetration of antigen and presentation by dendritic cell. [9] Interferon-y (IFN-y) and other cytokines released by activated leukocytes enhance local epithelial class II expression. [lo] Such cytokines may also induce terminal B-cell differentiation and [ll] increased expression of secretory component, thereby [12] promoting external transport of dimeric IgA.

tion of APCs of the interdigitating cell phenotype relative to
those in BALT (83). Follicular dendritic cells are known to play an important role in the induction of memory B cells (84). Finally, it is possible that the way antigen normally enters the mucosa has a profound effect on local immunity. The M cells of FAE lack SC and generally also HLA-DR expression (66); they therefore are probably without an IgA-transporting and antigen-presenting function. The M cells rather seem to perform nonselective and to some extent receptormediated inwards transport of luminal antigens and immune complexes (85). The remaining FAE, like the villous epithelium (Figure 9), shows a striking apical expression of HLA-DR and also a granular appearance of such protein in the cytoplasm (66). In view of recent information about the peptidebinding properties of MHC molecules, it is tempting to speculate that antigen that has been adequately processed in the gut lumen is taken up by HLADR-expressing epithelial cells on a genetically restricted basis and perhaps subsequently presented directly to primed T lymphocytes (33). The biologi-

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Figure

9. Immunofluorescence staining for MHC class II (HLADR) determinants in villus of normal jejunal mucosa. The villous epithelium shows striking DR expression related to the brush border and staining of less intensity along the basolateral membranes and in the cytoplasm. Note numerous positive cytoplasmic granules, especially apically (arrows). There are a few DR-positive histiocytic cells in the lamina propria. (X 650.)

cal implications in terms of positive or negative immune regulation will be discussed later. Transport of Polymeric the Gut Lumen

Immunoglobulins

to

Positive intestinal immune regulation has to favor the development of B-cell clones with a prominent potential for J-chain expression; this is a prerequisite for SC-mediated external transport of locally produced pIgA and pIgM (8) by the crypt epithelium (Figure 4). Cell biology studies have shown that rabbit SC is produced as a transmembrane protein family 25-30 kilodaltons greater than secreted free SC (86). The relatively large cytoplasmic extension (- 15 kilodaltons) seems to function as an effector domain guiding SC on its migration through the epithelial cell, as recently revealed in experiments with mutant transmembrane SC (87). The production of core glycosylated transmem-

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brane SC takes place on the rough endoplasmic reticulum and needs 30-60 min for maturation in the Golgi apparatus (Figure 4); the subsequent migration to the basolateral plasma membrane and the transport of pIgA to the luminal cell face apparently occur very quickly, and this route seems to be followed by at least 90% of the SC (88,89). Experiments with the HT-29 colonic carcinoma cell line have demonstrated that human SC is likewise produced as a transmembrane precursor (-95 kilodaltons) that, by addition of sugars, becomes about 20 kilodaltons larger than the secreted form (90). Also, transmembrane SC depends on the presence of J chain in pIgA and pIgM to perform specific epithelial uptake of these polymers (91). Cloned complementary deoxyribonucleic acid of rabbit SC has demonstrated that it contains five extracellular domains with remarkable homology to each other and to variable or constant regions of Ig chains (92). The amino acid sequence of human free SC has suggested the existence of similar homology regions (93), and we have recently confirmed this by cloning of its complementary deoxyribonucleic acid (94). Secretory component thus belongs to the “Ig superfamily,” which includes a variety of cell-surface recognition structures. Like other members of the Ig superfamily, SC expression can be regulated by lymphokines. Both recombinant interferon-y and tumor necrosis factora up-regulate the intracellular pool and epithelial membrane expression of functional SC in the HT-29 cell line (95,96). The effects of these two cytokines on SC expression are additive in a dose-dependent manner (97). Interferon-y is secreted by T cells during immune responses, whereas activated macrophages are the best recognized source of tumor necrosis factor-a. Our observations therefore suggest that both these cell types may promote the external transport of pIgA and pIgM and thereby enhance the efferent limb of the secretory immune system. Our immunohistochemical studies on human gastric mucosa harmonize with an immune responserelated up-regulation of SC-mediated epithelial transport; signs of increased SC expression and enhanced uptake of IgA are seen in fundic and antral glands in gastritis specimens with dense mononuclear cell infiltration (98). We have noted similar epithelial features in celiac disease lesions (99); enhanced external transport may thus explain that the serum level of IgA is considerably less raised in these patients than the increase of their jejunal IgA-cell population (100). Nevertheless, the SC-mediated epithelial transport capacity may be insufficient in certain patients with unusual local IgA-cell proliferation, resulting in excessive amounts of pIgA in serum (101,102).

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JlsJurwm

SphWl Epldldymla, Prostate Kidney Lung Myomstrlum Endometrium Llvst COIWI

Figure

10.

Northern blot analysis of SC messenger ribonucleic acid extracted from various human tissues as indicated. Total ribonucleic acid (20 pg) was separated by electrophoresis, and hybridized with a nick-translated human transmembrane SC complementary deoxyribonucleic acid probe. Adapted from KrajCi et al. (94).

The availability of complementary deoxyribonucleic acid probes specific for human SC messenger ribonucleic acid (94), has made it possible to confirm by Northern blot analysis that our previous immunohistochemical studies (8) have given a true idea about SC expression in various exocrine tissues. Impressive amounts of messenger ribonucleic acid can be detected in various segments of the intestinal tract (Figure lo), in harmony with the prominent IgA-transporting requirment. Interestingly, no messenger ribonucleic acid is detected by this method in the liver (Figure lo), confirming that there is no SC-mediated IgA transport through human hepatocytes (8). Suppression Reactions

of Mucosal Hypersensitivity

When secretory antibodies are unable to perform adequate antigen exclusion, the internal body environment should preferably be protected against potentially harmful systemic types of immune reactions elicited by IgG, IgE, or T cell-mediated delayed-

type hypersensitivity. There is experimental evidence in animals suggesting that such protection may be afforded by suppressive mechanisms collectively called “oral tolerance” (103). This phenomenon of hyporesponsiveness to harmless nonreplicating luminal agents that do not bind strongly to the epithelium (104) probably involves multiple immunoregulatory events and may to some extent depend on the liver. The down-regulation is apparently different for humoral immunity and delayedtype hypersensitivity (105). Antigen handling by an intact gut epithelium seems to be critical (1061, and direct injection of antigen into the PPs is incompatible with the induction of oral tolerance (24). A role of intestinal antigen “processing” has thus been suggested-at least for suppression of delayed-type hypersensitivity (107)-but the cellular elements involved (possibly epithelium or special mucosal macrophages) are still obscure. In the normal respiratory mucosa of rats, macrophages have been found to mediate immunologic down-regulation (80). There is evidence in humans that systemic IgG hyporesponsiveness to common dietary antigens develops over time (108), and it is tempting to propose that oral tolerance induced via the intestinal mucosa in some way involves the numerous intraepithelial T8 cells (Figure 11). Intraepithelial lymphocytes predominate in the small intestine and are located at a site that may render them easily exposed to luminal antigens, particularly those from food. Bland and Warren (109,110) found that MHC class II positive columnar cells from rat villous epithelium could present ovalbumin to primed lymph node T cells which thereby were induced to proliferate; this apparently class II-restricted immune response led to antigen-specific suppression involving a T8 subset. Mayer and Shlien (111) applied human colonic epithelial cells in autologous or allogeneic mixed lymphocyte responses and found also preferential stimulation of T8 lymphocytes. The responding cells did not express a putative cytotoxicity marker (9.3 antigen) and showed no cytotoxic effect, but they caused nonspecific suppression. It was possible to block this response with a rabbit antibody to human MHC class II determinants, suggesting that such molecules in some way were involved in the induction phase. Despite these fascinating observations, there is much to be learned about immunoregulatory interactions between the gut epithelium and IELs, both with regard to the possible requirement for thirdparty cells and the actual MHC restriction elements involved (33). T8 cells are generally thought to be class I-restricted, but some human CDs+ TCRcUIP+Ts clones derived from lepromatous leprosy lesions were suggested to be class II-restricted (112). Excep-

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Induction Y

Figure

Abrogation

of tolerance

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of tolerance

IgA-IgA

11. Hypothetical scheme for induction and abrogation of locally induced tolerance which normally affords down-regulation of both delayed type hypersensitivity (DTH) and IgG (By] and IgE (Be) responses with concomitant maintenance of local IgA (Ba) response. Left: presentation to T8 suppressor cells of antigen that has been processed by epithelial cell and/or by specialized mucosal macrophage, or direct epithelial presentation of luminally processed antigen, results in dominating regulatory signals conducive to suppression (heavy wavy split arrow]. Little or no help is provided by T4 cells to By and BE cells (broken wavy arrow), whereas Ba cells receive substantial help from appropriate T4 cells (wavy thin arrow]; the latter cells are either preferentially stimulated by antigen-presenting dendritic or epithelial cells, or both, or released from T8-induced suppression by signals from a contrasuppressor T8 subset. Right: when there is a break in the epithelial lining, undue stimulation of antigen-presenting cells, and/or enhanced epithelial expression of MHC class II determinants with ensuing increased presentation of luminal antigens, the result is general overstimulation of T4 cells (changed quality of arrows]. This may give rise not only to increased production of dimeric IgA, but also to excessive IgG (and IgE) responses and delayed-type hypersensitivity.

tions to the general rule have also been reported in murine test systems (113). Interestingly, it has been possible by means of soluble antigen to induce cytotoxic class II-restricted T8 cells that apparently exerted clonal killing of B cells presenting the same antigen (114). This could be one way IELs might perform suppression after returning to the lamina propria. Alternatively, as a great proportion of the intraepithelial T8 cells apparently are primed (4O), and therefore express various adhesion molecules (49,50), the cells may have sufficient affinity to be retained in the epithelium and be stimulated by antigen in the context of MHC class II molecules, even without being CD4+. This might cause clonal anergy rather than activation and therefore lack of some essential lymphokine(s) (115). Class II positive keratinocytes have in fact been shown to render antigen-specific T cells unresponsive (116). Finally, it cannot be excluded that class II-dependent autoreactive T4 cells are also involved in an immunoregulatory suppressive circuit (117). One interesting and testable possibility for interaction between intraepithelial T8 cells and lamina propria T4 cells is the following hypothetical sequence of events: processed luminal antigen taken up via apical MHC class II molecules on the epithelium (Figure 9) is presented to specifically primed T8 cells that are. retained unresponsive (anergic) within the epi-

thelium; idiotypic peptides derived from the (Y-and Preceptor chains of the IELs are subsequently presented to antiidiotypic T4 cells in the context of class II molecules; expansion of such autoreactive clones may in various ways result in antigen-specific clonal suppression without the involvement of a unique Ts-cell lineage (118). It is puzzling how local IgA responses can be elicited and maintained if suppression is a general phenomenon induced by soluble nonreplicating antigens. One possibility is that GALT generates antisuppressor (119) or contrasuppressor T cells (Tcs) specifically interfering with the inhibition of IgAantibody production (120). Murine PPs have been reported to contain a population of Vicia villosa lectin-adherent contrasuppressor T cell that preferentially support IgA responses (121). Also human Tcs have been isolated by binding to Vicia villosa, and they have been shown to be a subset of T8 cells (122). The possibility thus exists that the prominent intraepithelial T8 population may include Tcs (Figure 11). Recent observations have indeed indicated the presence of Tcs of the CD8 phenotype in normal human gut mucosa; these cells augmented IgA and IgM production in an in vitro assay, apparently by acting on a CD4+ subset when this was overrepresented (123).

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BRANDTZAEG ET AL.

GASTROENTEROLOGY Vol. 97, No. 6

12. Changes of gastrointestinal immunocyte populations as indicated by relative increases in cell numbers per mucosal tissue unit in various gut diseases compared with normal counterpart. Immunocyte class distributions in percentages are indicated above the columns. Based on data from the authors’ laboratory. Modified from Brandtzaeg et al. (124).

Aspects of Mucosal Immunopathology Putative Importance of Break in Oral Tolerance Abrogation of hyporesponsiveness to nonreplicating luminal antigens may be involved in the pathogenesis of celiac disease, chronic gastritis, and inflammatory bowel disease (IBD), as suggested by the disproportionate although highly varying local overproduction of IgG (Figure 12) seen in all these In experimental animals, antigen disorders (13,124). feeding combined with some sort of damage to the gut epithelium (106)seems to be incompatible with induction of oral tolerance. The same is true when APCs are excessively activated by stimuli such as muramyl dipeptide, estrogen, or a graft-versus-host reaction (103). All these situations apparently favor general overstimulation of Th cells (Figure 11). Also aberrant epithelial MHC class II expression may be involved in abrogation of oral tolerance (Figure 11). Preferential activation of T4 rather than T8 lymphocytes was observed after in vitro stimulation with antigen presented by colonic epithelial cells obtained from IBD (1251,in which epithelial HLA-DR is markedly increased (126-l29).Enhanced

and differential (DR>DP>DQ) epithelial HLA class II expression (99,13&132) may likewise be involved in exaggerated immune responses to gluten and other dietary antigens in celiac disease (13,124) and to autoantigens in IBD (133,134). Interactions between gut epithelium and T cells may in this way contribute to a class II-associated predisposition, which is particularly well documented in celiac disease. The primary association seems to be related to a particular DQ polymorphism where peptidebinding residues of both the (Y-and the Pchain may have functional importance for the pathogenesis (135). Immunopathology of Celiac Disease The first immunopathologic feature observed in celiac lesions after challenge with gluten, is a dose-dependent migration of lymphocytes into the jejunal epithelium (136,137). The proportion of CD3+UCHLl+ IELs is significantly raised (40),and the density of such putative memory T cells is strikingly increased both in the surface and crypt epithelium of untreated patients compared with normal controls (Table 2). There is also an elevated

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INTESTINAL

1989

Table 2. Mucosal Distribution of Putative AntigenPrimed CD&/LJCHLl+ T Cells in Celiac Disease (Median Number per Millimeter of Muscularis Mucosae) Controls Surface epithelium Crypt epithelium Lamina propria

84 (ZO]b 18 556

Treated patients

Untreated patients

106 (321b 22 466

a Modified from Scott et al. (131). b Number epithelium (density) in parentheses.

80 (80)b 40 802

per millimeter

of

mitotic activity and blastoid transformation of many IELs (138). About three times more intraepithelial T4 cells show a sign of stimulation by expressing the CD7 marker (36), although the T4- to T8-cell ratio is apparently unchanged compared with normal, both in the epithelium and in the lamina propria (34,36). A recent study, however, has reported raised numbers of T4 cells isolated from the lesion (139). Noteworthy is an increased fraction of CD3+CD4CD8- intraepithelial cells (Table l), the biological significance of which is as yet unknown (39). These IELs obviously represent TCRyl “double-negative” 6+ cells, which also are increased to 20%30% in celiac disease (41; MacDonald TT, personal communication). This subset shows only -10% CD8 expression compared to -25% in normal jejunal epithelium, whereas a remarkably high expression (-67%) of the variable Sl gene product is observed (41). The possibility that the TCRy/G+ IELs are cytotoxic and contribute to villous atrophy is a fascinating challenge for further research, but a putative contrasuppressive function should also be explored. Increased epithelial expression of both SC and MHC class II (HLA-DR) determinants was first observed in 1981 (99) in jejunal lesions of celiac disease and dermatitis herpetiformis; bright immunohistochemical staining for SC extended from the crypt to the surface epithelium, whereas DR positivity extended from the surface epithelium deep into the HLA-DR hyperplastic crypts. Increased epithelial expression in celiac patients has subsequently been reported to take place quite rapidly after gluten challenge (140); it is most likely mediated by cytokines released from primed T cells and perhaps macrophages. This possibility has been supported by our observations on the intestinal HT-29 epithelial cell line, which initially shows differential (DR>DP>DQ) HLA class II expression in response to recombinant interferon-y (141). Tumor necrosis factor-a can further intensify this expression (96,97). The report that rat intraepithelial T cells can mediate epithelial class II expression is also of interest in this context (142). Differential HLA class II expression in the jejunal

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epithelium of celiac patients does in fact seem to be related to the number of intraepithelial putative memory (UCHLl+) T cells, positivity for DP and DQ being seen only when this number is high (131). Local stimulation of T cells may therefore be an early event in the celiac lesion (Figure 13). One consequence is probably cytokine-induced crypt hyperplasia (138,143) and another may be increased epithelial permeability (144) with ensuing overstimulation of the B-cell system. In untreated adult celiac disease the average numbers of jejunal IgA, IgM, and IgG immunocytes per mucosal “tissue unit” are raised 2.4, 4.6, and 6.5 times, respectively (Figure 12). These results have recently been supported by tissue culture studies (145). The local immune response is, nevertheless, dominated by a typical secretory IgA and IgM response with preserved Jchain expression and a relatively high proportion of IgA2-producing immunocytes (146). Secretory component expression is also enhanced, and immunohistochemical staining indicates increased epithelial IgA and IgM transport (147). As expected, only trace amounts of IgG antibodies to gluten and other food

??

Initiating

Luminal

event(s)?

antigens

y

u and

PiI

IgGl

and

lgG2

__--

presentation

of luminal

antigens

6____.“‘-

rl?sponse ElII

Polymeric

(secretory)

IgA ond

IgM response

1

m Altored lmmunologlcal homeortash Figure

13. Putative immunopathological mechanisms in celiac disease. [l] Initiating event(s) attracts T cells to the epithelium. [2] There is stimulation of T cells and macrophages and perhaps an increased proportion of CD4+ helper cells. [3) Cytokines released from stimulated mononuclear cells induce epithelial HLA class II expression. [4] Class II positive epithelium mediates enhanced uptake and presentation of luminal antigens and induces help rather than suppression. [5] Overstimulation initially gives rise to a secretory immune response. [6] Additional minor (broken arrow) IgGl and IgG2 responses give rise to overproduction of IgG antibodies to luminal antigens. [7] Immune reactions involving IgG antibodies contribute to altered immunologic homeostasis manifested clinically as increased mucosa permeability and morphologic changes. For symbol key, see Figure 14.

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antigens appear in intestinal juice in contrast to the IgA and IgM counterparts (148). There is evidence to suggest, however, that the disproportionately raised number of IgG-producing cells present in the celiac lesion (Figure 12)reflects an imbalanced immune response to gluten and probably also to other dietary antigens. We found in an adult celiac patient that a much larger percentage of the jejunal IgG (5.7%)than IgA (1.6%)cells were producing antibodies to gluten (4). Another preliminary report based on children with celiac disease indicated gluten specificity for 11% 10.5% and 6.5% of the jejunal IgG, IgM, and IgA cells, respectively (149). These results have recently been supported by measurements of gluten-specific antibody Furthermore, release in jejunai tissue cultures (150). in the celiac children we treated who were challenged with gluten, a significant inverse correlation appeared between the time to clinical relapse and the number of jejunal IgG immunocytes seen after challenge (151). Some antigen specificity of the local IgG response or genetic influence is also suggested by the disproportionate increase of IgG2 compared with IgGl cells seen in adult celiac disease (152). Overstimulation of T4 helper cells inducing undue local and systemic IgG responses to gluten and other dietary antigens may cause altered immunowhich precipitates important logic homeostasis, pathogenic mechanisms in the mucosa (Figure 13). Immunoglobulin G antibodies have been shown both in vitro (153) and in vivo (151) to increase mucosal permeability for luminal antigens. The biological properties of IgG imply that it may exert both Cmediated inflammatory and perhaps killer cell-mediated cytotoxic activities in the course of abrogation of oral tolerance as discussed in the previous section The initiating event of the deleterious development proposed above, resulting in celiac disease in genetically predisposed individuals, may be a virus infection that perhaps initially leads to increased epithelial HLA class II expression. Adenovirus serotype 12 is of particular interest because one of its proteins (Elb) shows significant amino acid sequence homology with an antigenically active gluten peptide (155). Such a molecular mimicry may accelerate the postulated immunopathologic mechanisms of celiac disease, possibly by activating both class Iand class II-dependent antigen-presenting epithelial pathways (75). As there is a remarkably strong class II association in this disorder (135), disturbed interactions between the jejunal epithelium and regulatory T cells may play a decisive role in the pathogenesis. The epithelial barrier is nevertheless fairly well preserved in celiac disease; this fact combined with enhancement of secretory immunity probably

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Table 3. Distribution

(%I of Total and CD4+ Putative Naive CD45R/2H4+ T Cells in Peripheral Blood and Intestinal Mucosa of Patients With Crohn’s Disease and Controls” Total

PBL Mucosa

CD4 positive

P

C

P

C

70 16

68 38

48 8

38 10

C, controls; P, patients; PBL, peripheral a Modified from James et al. (51).

blood

lymphocytes.

constitutes the basis for the reversibility of the mucosal lesion usually seen after gluten elimination. Immunopathology Disease

of Inflammatory

Bowel

The etiology of IBD is unknown. Various microbial agents have been proposed, but there is no definite proof for the infective theory. Regardless of initiating factor(s), however, much circumstantial evidence suggests that immunologic mechanisms are involved in the pathogenesis of both ulcerative colitis (UC) and Crohn’s disease. Although the initial histologic lesion has not been clearly defined, patchy necrosis of the surface epithelium (156), focal accumulations of leukocytes adjacent to glandular crypts (157),and also an increased number of IELs (158) have been described as putative early alterations, especially in Crohn’s disease (159). The established Crohn’s disease lesion has been found to contain a significantly reduced proportion of putative naive T cells (Table 3), thus suggesting a preferential accumulation of antigen-primed memory cells (Figure 14). This has been supported by a raised proportion of cells with early activation markers (160). Only small IBD-related mucosal alterations of the T4- to T8-cell ratios were noted in some studies (35,161), but a significant increase of T4 lymphocytes in the lamina propria of severely inflamed UC lesions was recently reported (162). Interestingly, the same authors described a striking increase of T4 and a decrease of T8 cells in the epithelium compared with normal colon (162). There is also discrepant information in the literature with regard to the functional properties of T cells isolated from IBD lesions (52,159,163,164). Nevertheless, a remarkably intensified epithelial expression of HLA-DR (126128,163,164)suggests class II induction by cytokines released from activated T cells (129). In addition, numerous HLADR-positive (presumably activated) macrophages are present in the lamina propria (67). On the basis of the above information it may be speculated that an early immunopathologic event in

December

INTESTINAL IMMUNITY

1989

Luminal Initiating

antigens

131

ond of

El

C = complement Figure

antigens

event(s)? &

Q = increased

Luminal

1575

0

luminal

ontigens

Altered mmndogical homeostasls

= decreased

activation

presentation

T = T cells

LT = leukotriene

B = B cells

1 Id+ = macrophages

PG = prostaglandin

TX = thromboxane

14. Putative immunopathological network of inflammatory bowel disease. [l] Initiating event(s) attracts T cells to the epithelium. [2] There is stimulation of T cells and macrophages and perhaps an increased proportion CD4+ helper cells. [8] Cytokines released from stimulated leukocytes induce epithelial HLA class II expression. [4] Class II positive epithelium mediates enhanced uptake and presentation of luminal antigens and induces help rather than suppression; such a development will be accelerated if luminal antigens gain access through a break in the epithelium (bold arrows). [5] Overstimulation initially gives rise to a secretory immune response. [6] Cytokines from stimulated leukocytes induce altered endothelial receptor mechanisms which attract B cells belonging to the systemic immune system. [7] Such extravasation along with reduced suppression gives rise to overproduction of IgGl and IgG2 (different proportions in ulcerative colitis and Crohn’s disease]. [8] The IgG response is accompanied by preferential production of monomeric IgAl (see Figure 16). [9] The changed relationship between secretory and systemic types of local immunity leads to altered immunologic homeostasis favoring further inflammation and tissue damage. [lo] Immunoglobulin G (auto)antibodies and complement factors reach the epithelial surface after transudation from vessels or local production, and immune complex formation leads to complement activation and subsequent epithelial damage. [ll] Immune complexes and activated complement stimulate macrophages to produce complement factors and to release leukotrines and other biologically active substances. [12] Immune complexes and activated complement mobilize numerous neutrophils (crypt abcesses) whose lysosomal enzymes and toxic oxygen radicals may damage the epithelium. [18] Mediators released from activated macrophages influence smooth muscles [spastic contractions) and give rise to vasodilatation and increased permeability, which may also be directly induced by complement activation in vessel walls. [14] Transudation of serum IgG antibodies and complement factors contributes substantially to the altered immunologic homeostasis in the mucosa.

IBD is abrogation of oral tolerance to luminal antigens (Figure 11). Activation of T cells and macrophages probably leads to aberrant epithelial HLA-DR expression; and it has been suggested that antigen presentation by epithelial cells from IBD lesions

results in stimulation of T4 helper rather than T8 suppressor cells (125). A raised number of T4 cells in and beneath the epithelium may be further conducive to intensified B-cell activation and increased Ig production (Figure 14).

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GASTROENTEROLOGY Vol. 97, No. 6

15. Hypothetical explanations for changes of the mucosal @A-cell population in IBD, from preferential polymer (J’) to monomer (J-) production. [l] Endothelial recognition mechanism is altered from mucosal type to inflammatory type, which allows extravasation of B cells from the systemic (IgAl-J-) rather than from the mucosal (IgAZ-J+ and &Al-J+ immune system. [Z] Switching of heavy-chain genes may generate IgAl-JJ cells from IgG-JJ precursors. [3] Overstimulation of lymphoid follicles may enhance local generation of mature memory cells (IgG-J- and IgAl-J-1; in contrast there is the normally dominating generation of early memory cells which express J chain and migrate to glandular sites via lymph and peripheral blood circulation (cf. Figures 6 and 6).

The established IBD lesion is in fact a typical B-cell lesion (163,164). We and others have found that the mucosal IgA- and IgM-cell populations are increased several times and that there is a disproportionate rise of IgG immunocytes depending on the severity of the lesion, both in Crohn’s disease (Figure 12) and in UC (162). It therefore seems that the local humoral immune response initially engages the secretory IgA and IgM system to enhance the “first line” of defence (4,13,124). Later on, however, more and more features of systemic immunity evolve locally associated with an increasing degree of inflammation This development may be considered the establishment of a second line of defence aiming at elimination of antigens massively penetrating into the mucosa (4,13,124). In addition to the dramatically raised proportion of IgG immunocytes, the IgAl-producing cells are increased to the extent that they become much more frequent than the IgA2 counterparts (165,166). It is also noteworthy that J-chain expression is reduced, both in IgG (7) and IgAl cells and to a lesser extent in IgA2 cells (167). This change apparently reflects local accumulation of mature B cells because cytoplasmic J chain seems to be a marker of relatively immature memory clones (78). Significance of Locally Altered Humoral Immunologic Homeostasis The mature B-cell clones accumulating in IBD lesions may originate directly from the circulation because of altered endothelial recognition mechanisms (Figure 15). At a certain level of activation, lymphoid cells show reduced expression of receptors for mucosal endothelium (168), and endothelial recognition determinants (“vascular addressins”)

are subjected to modulation by cytokines like interferon-y (169). Such addressins identified at inflammatory sites have in fact been shown to be different from those found in normal mucosal tissue (19,21). An additional mechanism may be that mature B-cell clones expand locally; many of the IgAl-producing immunocytes are perhaps derived from J-chain negative IgG precursors by sequential switching of heavy-chain genes (Figure 15). A third possibility is generation of mature memory clones in overstimulated lymphoid follicles present in the gut wall (Figure 15). We have found, even in the normal state, that there is an IgG-predominant population of immunocytes with down-regulated J-chain expression associated with solitary lymphoid nodules and PPs (60). It is possible that this population is expanded in IBD-perhaps as a result of ulceration of the FAE, which may be an early lesion (159). The consequences of the change to a systemic type of humoral immunity in IBD lesions are probably conflicting in terms of preservation of health. A second line of defence established within the gut wall may promote immune elimination and thereby limit dissemination of antigenic and possibly replicating agents, but it will at the same time disturb the normal immunologic homeostasis in the mucosa (Figure 14). Local down-regulation of J chain in IgA immunocytes (167) shifts the production from polymers to monomers (7,165), and this change jeopardizes secretory immunity (8). However, the overall production of pIgA is quantitatively maintained in IBD because of the great total increase of mucosal IgA immunocytes (Figure 12); an increased SIgA response has in fact been reported after oral immunization with cholera toxin B subunit in patients with Crohn’s disease (170). Nevertheless, epithelial IBD lesions often show decreased SC expression

INTESTINAL IMMUNITY

December 1989

1577

Terminal pathway

C5 C3b inactivation

nC9 \

C5b-9(m) TERMINAL Figure

SC5b-9(m) COMPLEMENT TCC

SC5b-9 COMPLEX

16. Schematic depiction of various steps in complement activation. Left: Early activation (the inflammatory phase). Classical pathway is antibody-dependent; alternative pathway is induced by various components of the target surface. Both pathways produce C3 convertase that cleaves C3 to yield C3a and C3b. The latter split-product participates in C5 cleavage, which initiates terminal pathway activation by generation of C5a and C5b. C3b is partly degraded to the inactivated iC3b. The larger fragment C3c is thereafter released to the fluid phase, leaving C3dg attached to the target. Antibody to the C3c fragment can be used on extensively washed tissue to demonstrate bound C3b/iC3b. Right: Terminal activation [the transmembrane pore). C8b reacts with C6, C7, C8, and C9 to form TCC. On biological membranes TCC is generated as the cytolytic (pore-forming] Cgb-9(m). Conversely, in the fluid phase (i.e., in plasma] S-protein binds to TCC at the C8b-7 level, and cytolytically inactive soluble SCgb-9 results. S-protein may also be present in membrane-incorporated TCC SGb-9(m) (179).

(128,147), which probably results in patchy defects of SIgA (and SIgM) secretion and thereby topically reduced immune exclusion. The local overproduction of IgG may be of paramount pathogenic significance. Although IgG antibodies have a potent capacity for immune elimination by promoting phagocytosis and cytotoxicity, they may maintain inflammatory and tissue-damaging processes through C activation (see below) and by arming of killer cells. Production of IgG antibodies against various fecal bacteria and especially Escherichia coli strains has been reported in several studies of IBD lesions (159,163,164). Immunoglobulin G autoantibodies may also be produced locally, particularly in UC (134). Hybridoma generation of antibody-producing cells from mesenteric lymph nodes draining IBD lesions has suggested frequent specificities for E. coli and mycobacteria but not for food and autoantigens (171). It seems, therefore, that persistent immunopathologic reactions are maintained in IBD lesions by ubiquitous micorbial components present in the gut lumen, but some antigens may be more important in Crohn’s disease (possibly mycobacterial) and others

in UC (possibly autoantigens). Recent observations showing relatively more colonic IgGl production in the latter than in the former disorder, and the opposite finding for IgG2 (172,173), further attest to some disease specificity of the local IgG responses. This disparity between the two disorders is also seen in the serum IgG-subclass levels (174); it may reflect dissimilar antigenic or mitogenic exposure in the gut, different genetic “makeup” of the patients, or a combination of the two. Crohn’s disease has been suggested to be associated with certain IgG allotypes or Gm-linked genes, but this has not been confirmed in other studies (175). Putative Role of Local Complement Activation Both IgGl and IgG2 (in addition to IgG3) can induce C activation and also arm cytotoxic cells, although the former subclass, which is preferentially produced in UC (172,173), is more potent. Early immunohistochemical localization studies of C factors in IBD were based on antisera that did not distinguish between native and activated compo-

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GASTROENTEROLOGY

ET AL.

nents, and tissue specimens containing unpredictable amounts of extracellular proteins were used. As we have previously pointed out (176), it is quite uncertain whether the observed Clq (177) and C3 (177,178) deposits beneath the epithelium represented immune complexes rather than retained native C components. Activation of C on biological membranes (Figure 16) generates the terminal C complex (TCC) in the form of the cytolytic pore-forming C5b-9(m) (180). Conversely, when C activation occurs in the extracellular fluid phase, S-protein (vitronectin) binds to C5b-7, C5b-8, and C5b-9 and the cytolytically inactive soluble form (SC5b-9) of TCC is generated (181). Identical neoepitopes that can be detected by monoclonal antibodies are expressed by C5b+(m) and SC5b-9. We have used such an anti-TCC to trace sites of C activation in IBD lesions (182). Moreover, localization of C3c in extensively washed tissue specimens has been taken to indicate recent (within hours) or ongoing C activation (183). C3c is part of C3b but is rapidly released to the fluid phase during C3b inactivation (Figure 16). Terminal complement complex without associated C3c, therefore, reflects relatively old deposits. We found that walls of submucosal blood vessels contained significantly more C3c and TCC positivity in IBD than in histologically normal colonic control tissue (182,183). We have also observed C3c and TCC deposits, sometimes along with bound IgGl, on the luminal phase of epithelial cells in specimens from active UC (184). Contrary to the situation in the submucosal vessels, epithelial C3c was more widely distributed than TCC, suggesting that C3 activation had preceded TCC deposition, perhaps induced by IgGl autoantibodies. Intense apical TCC positivity was often observed on partly disrupted epithelial cells in the absence of S-protein, apparently signifying the presence of cytolytically active C5b-9(m). Beneath disrupted epithelium in severely inflamed UC specimens there were, in addition, variable amounts and irregularly distributed TCC positivity, and TCC was always present in crypt abscesses. We have not as yet performed similar studies of Crohn’s disease specimens. On the basis of these observations it may be visualized that C activation is involved both in the epithelial destruction and in perpetuation of the inflammatory reaction (Figure 14). Regardless of the mode of C activation and the nature of the target attacked, generation of biologically active products (C3a, C5a, and TCC) will cause inflammation. In addition to these well-known effects, other inflammatory mechanisms may also be triggered. C3b, iC3b, C3c, and TCC (Figure 16) have been shown to stimulate release of prostaglandin E, and thrombox-

Vol. 97, No. 6

ane A, from human macrophages, and C3a and C3b induce production of thromboxane B, (185-187). C3a, C5a, and C5adesArp have, moreover, been found to enhance the release of leukotriene B, in tissue cultures (188). The elevated levels of leukotriene B,, prostaglandin E,, and thromboxane B, found in UC lesions (189,190) may therefore be explained by C activation. Both leukotriene B, and anaphylatoxin C5a are highly chemotactic for granulocytes and are probably involved in the massive mucosal mobilization of such cells seen in UC; and lysosomal enzymes and toxic oxygen radicals released from these cells may also attack the epithelium (Figure 14). A break in the surface barrier caused by C activation obviously leads to bombardment of the underlying mucosal tissue with a battery of luminal antigens and subsequently intesified immunopathologic alterations (Figure 14). If epithelial breaching in IBD is the result of an initiating event-explained, for example, by a mucin defect (191) or virus infectionabrogation of oral tolerance and immunologic overstimulation with altered homeostasis may be an early consequence (Figure 14).

Epilogue This review has discussed certain local immunologic components in a rather isolated manner. Possible epithelial destruction by various types of cell-mediated cytotoxic mechanisms, including delayed-type hypersensitivity, has virtually not been considered. This is not to exclude the possibility that such immunopathology is involved in celiac disease and IBD; but our present knowledge to this end is at best meager (159,192). It should also be pointed out that there is an incresing awareness that local immunologic activity may be influenced by neuropeptides, nerves, and mast cells (193). Such interactions are most likely at play in the gut mucosa, both normally and in various diseases. Complete definition of the local immunopathologic network-including secondary phenomena-obviously requires further painstaking work along various lines of exploration. This effort certainly seems to be justified; mapping of important pathogenic steps is needed to improve rational treatment in IBD (192) and may finally lead to identification of the etiologic factor(s).

Conclusions The best defined part of mucosal immunity is the secretory antibody system; it depends on local production and selective epithelial transport of pIgA and pIgM. To this end there is a fascinating cooperation between the local immune system and the glandular epithelium by their production of two key

INTESTINAL IMMUNITY

December 1989

factors, J chain and SC, respectively. Mucosal immune regulation is normally geared for generation of early memory B-cell clones with prominent J-chain expression and preference for IgA production. Moreover, cytokines released from activated T cells and macrophages probably stimulate the secretory epithelium to perform enhanced SC-dependent transport of J-chain-containing pIgA and pIgM when their local production is increased. There is also circumstantial evidence to suggest that interactions between the gut epithelium and T cells are involved in mucosal down-regulation of systemic types of immunity to nonreplicating luminal antigens. Immunoglobulin G responses and delayed-type hypersensitivity may thereby be minimized to avoid undue complement activation, increased mucosal penetrability, inflammation, and tissue damage. It is possible that abrogation of such hyporesponsiveness to usually harmless luminal antigens contributes to the pathogenesis of various gut diseases. Increased epithelial expression of MHC class II determinants, enhanced stimulation of putative helper T4 lymphocytes, and disproportionate local overproduction of IgG are features that may reflect such a deleterious development. In celiac disease, expansion of a particular TCRy/G+ IEL subset may also be involved in the pathogenesis. The putative superimposed effects of the activities of neuropeptides, nerves, mast cells, and various cytotoxic cells on mucosal immunobiology and persistent local immunopathology are at present difficult to visualize.

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Received November 3, 1988. Accepted May 29, 1989. Address requests for reprints to: Prof. Per Brandtzaeg, LIIPAT, Rikshospitalet N-0027 Oslo 1,Norway. Studies in the authors’ laboratory are supported by the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities, and the Anders Jahre Foundation. The authors thank Dr. 0. Fausa, Medical Department A, The National Hospital, for providing much of the patient material.