Intestinal immunity and inflammation: Recent progress

GASTROENTEROLOGY

PROGRESS

1988:91:746-68

ARTICLE

Intestinal Immunity and Inflammation: Recent Progress CHARLES 0. ELSON, MARTIN F. KAGNOFF, CLAUDIO FIOCCHI, A. DEAN BEFUS, and STEPHAN TARGAN Departments of Medicine and Pathology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia; University of California at San Diego, La Jolla, California; Cleveland Clinic Foundation, Cleveland, Ohio; McMaster University, Hamilton, Ontario, Canada; and University of California at Los Angeles, Los Angeles, California

The immune system is absolutely essential to our health. It allows us to live in an ocean of potential and real pathogens-witness the recent experience with the acquired immune deficiency syndrome. At the same time the immune system has enormous potential for harm if turned against self or if it simply gets out of control. This may explain why a large fraction of lymphocytes appear to be in the business of regulating other lymphocytes. These points apply to the immune system in general but are especially germane to the intestinal immune system, which must deal continuously with enormous numbers of bacteria and other antigens and which may play a role in a number of poorly understood inflammatory diseases of the intestine. The past decade has been one of extraordinary progress in immunology. We now recognize that the immune system is composed of sets of lymphocytes, with each set having a different function. The exact number of sets that exist in the immune system is still being defined. Lymphocyte sets recognize and interact with one another and with nonlymphoid cells via a number of cell surface molecules, with the most important of these being molecules coded for in Received July 26, 1985. Accepted December 17,1985. Address requests for reprints to: Charles 0. Elson, M.D., Box 711,MCV Station, Richmond, Virginia 23298-0001. This report derives from the AGA Workshop on Intestinal Immunity and Inflammation held at Ft. Lauderdale, Florida, on October 8-10,1984.The financial support for the Workshop by the American Gastroenterological Association, the National Institutes of Health (AM R13 33006), and the National Foundation for Ileitis and Colitis is gratefully acknowledged. Dr. Elson is the recipient of Research Career Development Award AM00992 from the National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases. Dr. Befus’ present address is: Gastrointestinal Research Group, Department of Microbiology and Infectious Diseases, Health Sciences Center, The University of Calgary, Calgary, Alberta, Canada. 0 1986 by the American Gastroenterological Association 0016-5085/86/$3.50

the major histocompatibility complex. The sets communicate with one another also by releasing a variety of soluble molecules or mediators. These factors, known as lymphokines, not only function to signal other cells, but also to amplify those signals, and thus increase the numbers of cells involved in any given response. The interactions among sets of lymphoid cells are complex. Many interactions appear to be arranged in circuits similar to those among neurons in the nervous system. In fact, the immune and nervous systems share a number of common features (1). Much of the recent progress in immunology is based on work done on systemic lymphoid tissues such as the spleen or peripheral lymph nodes. Although these results seem to apply generally to intestinal lymphoid tissues as well, it is clear that the mucosal and systemic immune systems are distinct in many ways. Although most of the same sets of lymphocytes that are present in the peripheral lymphoid system are present also in the intestine, certain sets seem to preferentially localize to the intestine and others are poorly represented. In the sections that follow we will focus on selected areas of recent progress in intestinal immunology, in each instance considering how newer, basic information about mucosal immunology might apply to a better understanding of inflammatory diseases of the intestine in which the immune system appears to be playing a prominent, if not predominant, role.

Abbreviations used in this paper: ADCC, antibody-dependent cellular cytotoxicity; BF, binding factor; CH, gene coding for the constant region of the immunoglobulin heavy chain; IBD, inflammatory bowel disease; IEL, interepithelial lymphocyte; IFN, interferon; IL, interleukin; LPL, lamina propria lymphocyte; MHC, major histocompatibility complex; NK, natural killer; RMCP, rat mast cell protease; sIg, surface immunoglobulin; VH, gene coding for the variable region of the immunoglobulin heavy chain.

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a

P

“2N

CLASS

MHC antigen: Human : Mouse: Figure

HLA H-2

CLASS II

I

A,B,C K,D, L,Q

?J’-‘2

HLA H-2

Op, DO, DR I-A, I-E

1. Schematic representation of class I and class II MHC molecule structure of mouse and humans. Class I molecules consist of a single chain, with three domains (I, II, III). The first and second domain are variable (y], whereas the third domain is fairly constant (C). The class I molecule is closely associated with PZ microglobulin (pzm). Class II molecules have an (Y- and p-chain structure, with each chain having a variable (Vcu, VP) and a constant (Ccu,Cp) domain. The domain structure is maintained by intrachain disulfide bonds F-S).

Genetic Control of Immune Responses at Mucosal Surfaces Genes that map to the major histocompatibility complex (MHC) in humans (HLA on chromosome 6) and in mouse (H-2 on chromosome 17) as well as genes associated with the immunoglobulin heavy chain locus (chromosome 14 in humans; chromosome 12 in mice) regulate T cell- and antibodymediated immune responses that may be highly relevant in the pathogenesis of specific intestinal diseases-particularly celiac disease and inflammatory bowel disease (IBD) (2). Major Histocompatibility Molecules

Complex

The term “histocompatibility” refers to how these genes were discovered rather than to their actual purpose. Genes in the MHC code for cell surface molecules that are involved in cell-cell recognition and signalling among lymphoid cells as well as between lymphoid and nonlymphoid cells. The precise structure of these histocompatibility antigens is now known, allowing them to be classified into several broad groups, two of which are shown in Figure 1. This classification into class I and class II molecules also clusters these molecules into functional groups: class I molecules are present on all nucleated cells and are important in the presen-

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tation of cell surface-associated antigens (e.g., viral antigens) to T cells. T cells that recognize cell surface antigens in conjunction with class I MHC molecules have the CD8 marker (formerly termed T8 or Leu-2) on their cell surface (3,4). From a functional viewpoint, most of these T cells mediate cytotoxic or suppressor functions. Class II MHC molecules are present on B cells, macrophages, dendritic reticular cells, and activated T cells. Under some conditions class II molecules are found also on other cell types including intestinal epithelial cells. T cells that recognize soluble antigens in conjunction with class II molecules have the CD4 marker (formerly termed T4 or Leu-3) on their cell surface (3,4). Most of these T cells manifest helper or inducer functions (5). The wide variety of pathogens and antigens that the immune system, and thus these MHC molecules, must interact with presumably accounts for the extensive polymorphism of the genes coding for these molecules: populations expressing many different MHC molecules have a greater chance of having MHC molecules that can interact effectively with a specific pathogen or antigen, and thus a greater chance of being protected. Class II Major Histocompatibility Molecules

Complex

Because of the central role of class II MHC molecules in immune induction, these molecules and how they work have attracted much attention. Two avenues of investigation have led to the definition of their role in the immune system. The first avenue has been the study of immune response gene effects. Simply put, inbred strains of animals can be shown to segregate into high or low responders when immunized with certain antigens. The immune response genes determining such responsiveness have been localized frequently in the subregion of the MHC that encodes class II molecules. The second avenue has been the study of genetic restrictions to interactions among lymphoid cells; i.e., cells from different mouse strains cannot interact in certain immune responses unless they share the same class II genes. Both of these phenomena are thought presently to reflect the functions of MHC-encoded class II molecules. There is substantial evidence that immune response genes associated with the MHC are the structural genes for class II molecules (I-A and I-E molecules in mouse; DP, DQ, and DR molecules in humans]. An example of specific evidence to support this derives from studies in the bm12 mutant mouse (6,7), especially from an elegant study in which it has been shown that a nonresponder status to the antigen sheep insulin can be converted to a responder status by transfer of a 14 base pair

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DNA sequence within the MHC class II subregion corresponding to three amino acid substitutions from one gene (I-Eab) to another (I-A,b”12) (i.e., gene conversion) (8). Major controversy remains, however, with respect to the specific mechanisms by which class II molecules mediate immune response gene function. One hypothesis of how class II MHC molecules present antigen to T cells is that the antigen or its fragments bind directly to the class II molecules, and thus both class II molecules and antigen are presented together to the T-cell receptor. Studies on the immune response to myoglobin, a globular protein whose sequence and conformation are well known, have indicated that T-cell antigen recognition is more limited than B-cell antigen recognition. In murine strains with different H-2 haplotypes, a limited number of different antigenic sites appear to be able to trigger immune response gene-controlled T-cell responses (9). In contrast, antibody can be induced to many different sites on the myoglobin molecule. Berzofsky and coworkers (9,lO) have presented a striking demonstration of class II molecule influence on T-cell specificity. When they analyzed a group of 14 myoglobin-specific murine T-cell clones for epitope specificity and genetic restriction, all those clones that were specific for glutamic acid residue 109 on myoglobin were noted to recognize antigen in conjunction with I-Ad, whereas all those specific for lysine at position 140 recognized antigen in conjunction with I-Ed. Other studies using synthetic peptides showed that the information necessary to stimulate T cells specific for lysine at position 140 on myoglobin was contained in an 11 amino acid peptide that must contain the site needed to interact with class II molecules as well as the site recognized by the T-cell receptor for antigen. Inhibitors of antigen processing (e.g., chloroquine or leupeptin) can inhibit the presentation of native myoglobin but not of a peptide fragment encompassing residues 132-153 and not of an unfolded form of intact myoglobin to the same T-cell clone (10). One conclusion of this work is that antigen processing is needed to unfold, not to reduce the size of the antigen. This suggests that such unfolding may expose critical binding sites (e.g., hydrophobic ones) that are normally buried within water-soluble globular proteins such as myoglobin. Such sites may well be necessary to interact with class II MHC molecules or with the lipid membrane of the antigen-presenting cells. Class II MHC molecules appear to play a similar role in the function of the immune system in humans as in the mouse, although less is known in this regard. These molecules may well be important in the pathogenesis of some human diseases, as wit-

GASTROENTEROLOGYVol. 91.No.3

nessed by the association between certain HLA class II molecules and a number of diseases with an apparent immunologic pathogenesis (11). Some recent findings have major implications with regard to the noted associations between HLA class II molecules and specific disease susceptibility. Studies on HLA class II structural variants indicate the presence of striking polymorphisms within HLA haplotypes. Thus, the HLA-DR 1-14 specificities determined by serology are now recognized as broad public serologic markers expressed on different class II molecules from distinctly different haplotypes. Nepom et al. (12) have defined at least six different DR4associated haplotypes and five different DRw8associated haplotypes by two-dimensional gel analysis of DR and DQ P-chain electrophoretic variants. Southern blot analysis of restriction fragment length polymorphisms among 17 DR4 homozygous cells indicated that phenotypically identical cells (i.e., serologic and mixed lymphocyte reactions suggest HLA identity) are genotypically very diverse. Other studies showed marked DQ Q- and p-chain polymorphisms among DR4 homozygous typing cells. It appears that specific haplotypes associated with class II restricted T-cell recognition or with HLAassociated disease may, in fact, be but a small subset of any particular serologic HLA specificity: all 12 juvenile rheumatoid arthritis patients who were homozygous for DR4 in Nepom’s series expressed specific haplotype markers characteristic of only two of six known DR4-associated haplotypes. Moreover, 7 of the 12 patients were heterozygous for a very rare combination of two specific DR4-associated haplotypes, suggesting a possible role for combinatorial class II specificities. In other studies, insulindependent diabetes mellitus in individuals with the DR4 serologic specificity was associated with a specific DR4 haplotype marked by a polymorphism in the DQ p-chain (Nepom G, personal communication). Among the human intestinal diseases of presumed immunologic pathogenesis, celiac disease has the strongest association with certain HLA haplotypes. Now recent studies suggest that the association is strongest with class II HLA antigens. Studies from Italy report a 100% association between celiac disease and the HLA specificity DQw2 (formerly known as DC3) (13).However, no segregation studies in families have been reported as yet, and 59 of the 60 patients in the Italian study also had either DR3 or DR7. Pena and coworkers (14) have now confirmed the previously recognized association between celiac disease and HLA-DR3/DR7 heterozygosity in Holland as well as an increase in DR7/DR5 and DR3/DR7 heterozygosity in celiac disease in Spain: in eight of nine multiple-case families, all children

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with celiac disease shared both HLA DR antigens. Factors other than gluten and DR antigens may be important in disease expression. JmmunogJobuJin Complex

Heavy Chain Gene

A second genetic locus that is associated with the regulation of immune responses is the immunoglobulin heavy chain complex. This complex in mice consists of eight tightly linked, heavy-chain, constant-region genes and -100 heavy-chain, variable-region genes that can be divided into seven or eight major V gene families (15). Studies in mice have shown that the immune response to A-gliadin (i.e., a wheat gliadin component known to activate celiac disease and whose primary amino acid sequence is known from amino acid sequencing studies and sequencing of a cDNA clone) is regulated both by genes that map to H-Z (I-A subregion) and genes at or linked to the immunoglobulin heavy chain allotype locus (16,17). H-2 genes appear to predominate over immunoglobulin-linked genes in determining the response to A-gliadin; other murine background genes did not appear to be important. Genes associated with or in the immunoglobulin heavy chain complex may be associated with human diseases. In humans there is a striking association between the presence of antigliadin antibody in individuals with celiac disease on a gluten-free diet and the IgG2 immunoglobulin heavy chain allotype marker G2m(n) on chromosome 14 (18), although at the population level, no association was found between celiac disease and a particular Gm phenotype or haplotype (19). These studies suggest that genes at or linked to the G2m(n) allotype locus on chromosome 14, likely immunoglobulin variable-region genes, determine the specificity of antigliadin immune responses in individuals with celiac disease. Other studies have probed for other environmental factors that may be important in celiac disease. The primary amino acid sequences of A-gliadin and two -y-gliadins have been screened against a data bank of proteins. This demonstrated that A-gliadin has a significant region of sequence homology with an adenovirus 12encoded protein (20). Adenovirus 12 is a human adenovirus usually isolated from the intestinal tract (20). The region of homology between A-gliadin and the adenovirus 12 protein has been synthesized. Kagnoff et al. have speculated that encounter of the immune system with a protein produced during viral infection may be important in the pathogenesis of celiac disease because of a chance immune cross-reactivity between a viral protein and dietary gliadin. After the ingestion of gliadin, gliadin peptides associated with intestinal

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mucosal structures were envisioned as a target of immune injury. They further postulated the presence in celiac disease of a specific HLA haplotype that governs the host immune response to determinants perhaps shared by gliadin and a viral protein. A second intestinal inflammatory disease may also be associated with certain immunoglobulin allotypes. Recent studies have demonstrated a significant difference in the overall distribution of Gm phenotypes and Gm haplotypes in a well-defined Northern European white population with Crohn’s disease living in the United States compared with matched controls (21). Individuals having the phenotype Gm(a,x,f;b,g), the haplotype Gmasx;g, and the Glm marker Glm(x) were demonstrated to have a significantly increased risk of Crohn’s disease. The association between Gm markers and Crohn’s disease might reflect a genetically determined difference in how Crohn’s disease patients respond to antigens that are important in the pathogenesis of that disease. However, studies relating Gm allotypes to Crohn’s disease in a less-well-defined disease population in Holland have not revealed a similar association (Pena AS, personal communication). This discrepancy raises the possibility that more than one agent or different agents may be involved in the pathogenesis of Crohn’s disease in different populations, or it simply may reflect the difference in diagnostic criteria and disease distribution required for inclusion in those studies. In summary, the information presented in this section indicates the powerful role that studies in immunogenetics and molecular biology will play in determining how the immune system may be involved in the pathogenesis of specific intestinal diseases, particularly celiac disease and IBD. Within the next few years, it is possible that the specific peptide sequences within gliadins responsible for activating celiac disease and the mechanisms through which they interact with specific cells in the immune system will be determined. Similar powerful approaches using monoclonal antibodies and molecular biology likely will be applied in the future to define important mechanisms in other intestinal diseases.

Regulation of Antibody Synthesis in the Intestine The interaction of an antigen with gutassociated lymphoid tissues can either immunize or tolerize. Such immunization frequently takes the form of a secretory IgA antibody response, but intestinal immunogens can also stimulate serum IgM and IgG antibodies as well. Alternatively, an intestinal antigen can render the animal less responsive to that

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same antigen when the animal is subsequently immunized parenterally with it. This has become known as oral tolerance; it occurs with a variety of different antigen types and in a variety of animal species. The most common, but not the only, mechanism involved is the generation of suppressor T cells by antigen feeding (22,23). A possible third outcome is the induction of secretory IgA antibodies simultaneous with the induction of systemic tolerance. Evidence in favor of this third possibility has been demonstrated experimentally (24,25); however, it is probably not a very frequent occurrence (26,27). The reader is referred to recent general reviews for a more detailed description of the experimental work summarized by the above statements (28,29). The focus here will be on the induction and regulation of IgA responses. Immunoglobulin A is the major immunoglobulin present in secretions. It adds specificity and memory to a variety of nonspecific host defense factors, such as gastric acid, bile, digestive enzymes, mucus, and intestinal motility. Immunoglobulin A interacts with, and in some instances, synergizes with these nonspecific host defense factors, such as mucin or the bacteriocidins (30). Immunoglobulin A protects the host by excluding and/or neutralizing antigens, toxins, and organisms (81-88). The capability of inducing IgA responses at will to any given intestinal pathogen or antigen has been a desirable, but as yet unattained goal, because of a lack of understanding of exactly how the IgA response is initiated and regulated. It seems unlikely that IgA antibody is induced to each antigen resident in or traversing the intestine, because the number and quantity of such antigens is simply too enormous. What factors determine which antigens in the intestine will elicit an IgA response, and why, is far from clear, but some recent developments are yielding a general outline. T-Cell Regulation

of IgA Responses

Much of the recent progress in this area is based on earlier observations that IgA responses are dependent on the presence of functional T cells (34-36).These observations, coupled with the known role of T cells as central regulatory cells in the immune system, have focused attention on the role of T cells in IgA responses. Moreover, because Peyer’s patches are known to be an enriched source of IgA B-cell precursors (37), this component of gut-associated lymphoid tissues has seemed a likely place to examine the T-cell regulation of IgA responses. In fact, early studies on Peyer’s patch T cells did indicate that Peyer’s patches contain T cells that preferentially help in producing IgA antibody responses (38). Subsequent studies using T-cell clon-

IN

Figure 2. T-cell regulation of IgA responses operates at several different stages of B-cell differentiation. The switch T cell acts upon an early B cell bearing IgM on its surface, causing a rearrangement within the immunoglobulin heavy chain gene so that the B cell “switches” from IgM to IgA. Classic carrier-specific T helper cells (Tu) can stimulate B cells committed to IgA as well as B cells committed to other isotypes such as IgM or IgG. In contrast, IgA-specific Tu cells stimulate mature IgA+ B cells but not B cells of other isotypes. Any given IgA response presumably involves the sum of the effects of all of these various T-cell effects.

ing techniques (39-41) have confirmed this and have shown that T cells specific for IgA can exert their effects at several different levels of IgA B-cell maturation, as shown in Figure 2. Switch T Cells T cells cloned from the Peyer’s patches of mice have been shown to cause a switch in B cells expressing surface IgM (sIgM+) to B cells expressing surface IgA (s&A+) (40,41). These “switch” T-cell clones have the surface markers characteristic of helper/inducer T cells. Interestingly, they also have receptors for the Fc portion of IgA, even though their target cells bear sIgM and not sIgA. In fact, switch T cells have no effect on sIgA+ B cells: switch T-cell clones do not stimulate either proliferation of or IgA secretion by sIgA+ B cells, making unlikely the possibility that these T cells merely act on I3 cells that have already undergone DNA rearrangement of their immunoglobulin genes but have not yet expressed the a-chain gene product. A switch T cell

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has been identified also in humans (42). The latter was discovered in a patient with mycosis fungoides and Sezary syndrome who had serum IgG and IgA but no serum IgM. Coculture of patient T cells with normal B cells caused a switch from IgM synthesis to IgG and IgA synthesis. In addition, T cells from this patient cocultured with B cells from a patient with the hyper IgM syndrome caused a switch in the isotype synthesized in vitro to IgA and IgG, even though B cells from this patient could only synthesize IgM when cocultured with normal T cells. Because of recent advances in DNA technology, the genes coding for the immunoglobulin molecules have now been identified and their structure analyzed (43). In the case of the heavy chain immunoglobulin genes, the genes encoding the constant regions (Cu) lie downstream (3’) from the genes encoding the variable regions (Vu). In the mouse, the sequence of genes encoding the heavy chains of different isotypes is mu (IgM), delta (IgD), gamma (IgG), epsilon (IgE), and alpha (IgA) (43). In humans there is a somewhat different gene order. The initial Cn gene expressed early in B-cell development is the first in the sequence, i.e., mu. For the other isotypes to be expressed, DNA rearrangements must occur such that the DNA for mu and any other intervening Cu genes are deleted. In the mouse, a switch from IgM to IgA thus requires the deletion of all the Cn genes except alpha, the last gene of the sequence. The exact mechanism by which T cells are able to signal B cells to undergo these rearrangements of CH genes is unknown. B cells are thought to be able to rearrange CH genes in the absence of T cells as well (44,45), and the relative importance of the T-cell pathway vs. the non-T-cell pathway is also unknown. It is clear, however, that the Peyer’s patch is a site where Cn switching is being greatly facilitated, by switch T cells and perhaps by other, as yet undefined, mechanisms. IgA-Specific

Helper T Cells

B cells undergo these switching events early in their development. T-cell regulation of B cells does not stop at this point but continues throughout B-cell maturation. Isotype-specific T-cell regulation has been demonstrated for IgE, IgA, and IgG responses (39,46-48). In regard to IgA responses, T cells cloned from Peyer’s patches have been shown to provide preferential help for IgA responses to sheep red blood cells fed to mice (39). These Peyer’s patch T-cell clones fell into one of two groups: those that had helper activity for IgM and IgA, but not IgG, and those that provided a small amount of help for IgM and IgG but considerably more help for IgA. Again, these Peyer’s patch T-cell clones had

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helper/inducer surface markers and IgA-Fc receptors, similar to the murine switch T-cell clone described above. However, these T-cell clones acted only on mature B cells that already expressed IgA on their cell surface membrane (49). Helper T cells specific for IgA also appear to exist in humans (50,51). Mayer et al. (51) fused human peripheral blood T cells with a human T-cell lymphoma line. One of the resulting T-cell hybridoma clones secreted a factor that specifically enhanced IgA secretion when cocultured with either normal B cells or with monoclonal lymphocytic leukemia B cells that expressed only membrane IgA (51). Other T-cell hybridoma products were not IgA specific, increasing the production of all isotypes. It should be noted that stimulation of all isotypes by cloned helper T cells has been found repeatedly in murine systems, and is the rule rather than the exception (52). This fact helps to make the important point that classic helper T cells that provide help for all isotypes are likely involved to some extent (in addition to isotype-specific helper T cells) in responses that are relatively isotype restricted; the role of each T-cell type and the interactions between them are unknown at this point. In addition, suppressor T cells specific for IgA responses have been convincingly demonstrated in some systems and undoubtedly also play a role in down-regulating IgA-restricted antibody responses (47,531, but exactly what role they play in normal mucosal responses remains to be defined. Lastly, different types of antigens may be regulated by different mechanisms. In some recent studies, Murray et al. (54,55) have demonstrated that T-cell-derived B-cell growth and differentiation factors, including gamma interferon (IFN r), interleukin 2 (IL2), and B-cell growth factor II exhibited differential effects on the induction of IgA as opposed to IgM responses to the type 2 antigen, dextran Bl355 (54,55). Mechanism Regulation

of Isotype-Specific

T-Cell

How are T cells able to specifically regulate B cells bearing a certain isotype such as IgA? A number of possible mechanisms are displayed in Figure 3. First, T cells may have a receptor on their surface distinct from their antigen-specific receptor that recognizes IgA heavy chains present on the B-cell membrane. Second, some variable region idiotypes may nonrandomly associate with certain Cn region isotypes, and thus T cells regulating idiotype via idiotype-specific receptors would necessarily regulate isotype as well. Third, T cells bearing isotypespecific Fc receptors may preferentially regulate B cells secreting that isotype. All three of these mech-

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ELSON ET AL.

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Tcell T-ccl I

recognizes isotype via Figure

Bcell

a receptor specific for Ig heavy chain determinants

Tcell

B cell

a receptor specific for

Ig idiotypes characteristic

Tcell

B cell

an Fc receptor specific for isotype

of an isotype

3. Possible mechanisms by which T cells might regulate a specific isotype such as IgA: (1) T cells might bear a second receptor, one that recognizes immunoglobulin isotypes on the B-cell surface. (21 If certain variable region genes (idiotypes) and constant region genes (isotypes) were linked at a genetic level, T cells might regulate isotypes by regulating idiotypes. (3) T cells with isotype-specific Fc receptors on their surface could recognize and regulate B cells secreting that isotype.

anisms remain possible and they are not mutually exclusive. The weight of evidence available today, however, has established that the last mechanism, involving T cells with isotype-specific Fc receptors, does definitely play a role in isotype-specific regulation. Fc Receptor-Positive Regulation

T Cells in Isotype

The evidence in favor of isotype regulation by T cells bearing Fc receptors is strongest in relation to IgE responses. Ishizaka and coworkers (46) have identified a subset of T cells that bear Fc receptors for IgE. These T cells are associated with and appear to release glycoprotein IgE binding factors (BFs) that can either help or suppress IgE B cells. It appears that the protein component of the IgE binding factor is identical in both the helper and suppressor factor; whether the IgE binding factor helps or suppresses is determined by its glycosylation. The presence of IgE-BF appears closely related to the presence of T cells bearing IgE-FcR; indeed the BF may well represent IgE-FcR in whole or in part. Not as much is known about the regulation of IgA by IgA-FcRf T cells. The IgA-specific T-cell clones of Kiyono et al., described above, all bear IgA-FcR (39). The function of these helper T-cell clones can be inhibited by blocking their IgA-FcR with large amounts of IgA myeloma proteins (56). Moreover, T-cell hybridomas derived from these clones secrete an IgA binding factor that stimulates IgA responses at low doses and suppresses IgA responses at high doses (57). Somewhat similar results have been obtained by Yodoi et al., who have developed a murine T-cell hybridoma, TZD4, that bears both IgA-FcR and IgG-FcR. When the IgA-FcR of this T-cell hybridoma are triggered with IgA myeloma proteins, the hybridoma produces an IgA-BF that suppresses IgA responses (53). Conversely, if the

hybridoma is triggered with IgG myelomas, a suppressive IgG-BF is produced instead (58). These properties of the T2D4 hybridoma appear to reflect events occurring in normal murine T cells, because when normal T cells are stimulated with the mitogen concanavalin A in the presence of excess exogenous of the cells express IgA-FcR and a IgA* -15%20% suppressive IgA-BF appears in the culture supernatants (59). In contrast, ‘T cells stimulated with concanavalin A alone neither express IgA-FcR nor secrete an IgA-BF. The stimulation of IgA-FcR bearing suppressor T cells by exposure to exogenous IgA had previously been shown by Hoover and Lynch (60), who showed that mice bearing IgA plasmacytomas or mice injected with large quantities of IgA myeloma protein develop IgA-FcR+ suppressor T cells. Moreover, such suppressor T cells are able to specifically suppress the IgA, but not the IgM or IgG, response to an antigen that is fed to mice (61). A similar development of isotype-specific suppressor T cells has been found as well in mice bearing IgG or IgE plasmacytomas (62). Thus, the numbers of FcR+ T cells can be increased by the local presence of the relevant isotype, and the evidence is rather strong that such cells play some role in isotype regulation. At the same time it is clear that there is a good deal of functional heterogeneity among cells bearing IgAFcR. For example, the murine switch T cell bears an IgA-FcR, but this cell is functionally distinct from the IgA-FcR+ T-cell clones that act on later stages of B-cell development. Moreover, there is no simple relationship between sites of IgA synthesis and numbers of IgA-FcR+ cells (63). Regulation of Antibody Synthesis Intestinal Lamina Propria

in the

The intestinal lamina propria is the site where the terminal differentiation of IgA B cells into IgAsecreting plasma cells occurs. The regulatory T cells

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discussed above presumably exert their influence over B cells in the lamina propria, but information about the regulation of antibody responses by lamina propria T cells is scanty. A substantial proportion of lamina propria lymphocytes are T cells, and roughly two-thirds of these have the helper/inducer phenotype in both mice (64) and humans (65).Consistent with this, helper activity for immunoglobulin synthesis among lamina propria T cells is the predominant regulatory activity (66).To date, IgA-specific T-cell help has not been evident in lamina propria isolates in either the mouse or humans (66,67), but it is possible that isotype-specific effects may be difficult to identify in the pokeweed mitogen system used in these studies. Even less is known about regulation of antibody responses in the inflamed intestinal lamina propria, such as in IBD, and whether altered immunoregulation in the intestinal lamina propria might play a role in the pathogenesis of such diseases (68). Interestingly, the ratio of the CD4+ helper/inducer (OKT4, Leu-3) and CD8+ suppressor/cytotoxic (OKT8, Leu-2) T-cell subsets resident in the human lamina propria does not change even in a setting of intense inflammation, a finding that has prompted some to conclude that immunoregulation in the intestine is normal in IBD (69). Although such data are important, the interpretation needs to be tempered by the realization that the usefulness of the CD4+ helper/inducer and CD8+ suppressor/cytotoxic T-cell subset markers has major limitations in humans. From the work of Thomas et al. (76-72), it is now clear that a good deal of heterogeneity exists within these T-cell subsets. Although the CD4’ T-cell subset does contain helper/inducer cells, it also contains T cells that can suppress immunoglobulin synthesis. Likewise, although the CDs+’ subset does contain suppressor T cells, it also contains T cells that can contrasuppress, i.e., augment immunoglobulin synthesis. This work has now been confirmed and extended in studies done on human lamina propria T-cell subsets: CD4+ cell helper, CD4+ cell suppressor, CD8+ suppressor, and CD8+ cell contrasuppressor activities have all been identified in T cells isolated from the noninflamed human lamina propria (73). The last of these functional activities, contrasuppression by CD8+ cells, is particularly prominent in lamina propria CD8+ cell isolates. A contrasuppressor circuit has been demonstrated in the mouse (74,75), and has been postulated to be important in certain microenvironments, such as the intestine, where it would allow a continuing immune response to occur that would normally be shut off by suppressor mechanisms. This contrasuppressor activity is presumably responsible in part for the predominance of helper activity in human lamina propria T-cell iso-

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lates, even in those from inflamed intestine. The possibility that contrasuppressor activity might prevent inhibition of immunoglobulin synthesis and thus contribute to further inflammation in the intestine in IBD is an intriguing notion.

Lymphokine Regulation of Cellular Immunity in the Intestine Some of the most crucial defense functions of the body, such as generation of antigen-specific cytotoxic cells and antibodies, are under the control of immunoregulatory cells with enhancing or suppressive activity. These regulatory cells, in turn, as well as the immune effector cells, are under the influence of soluble mediators, which regulate their activation, maturation, proliferation, and functional specificity. Such soluble mediators or factors are often produced and released by the same cells upon which these factors will act. These soluble mediators, termed lymphokines or monokines, are extremely important in the regulation of the immune system, because each specific cellular function of this system appears to be controlled by different assortments of factors (76). Thus there are lymphokines that regulate the growth and differentiation of B cells (77,78), and others that exert similar effects on T cells. Although a large number of lymphokines have been reported (79), it is now clear that many different functions, each attributed to unique lympokines, are actually mediated by relatively fewer molecules having multiple activities. This point is well exemplified by the monokine interleukin 1 (ILl).

lnterleukin

1

Macrophages are monocytes that have been activated by some in vivo or in vitro process. Activated macrophages possess extraordinarily diverse and extensive secretory capacities, and among the multiple products synthesized and released by these cells is ILl. Interleukin 1, previously called LAF for lymphocyte-activating factor, is a monokine of crucial importance in mediating the interactions between macrophages and T lymphocytes. Interleukin 1 was originally described by Gery et al. (80) as a murine macrophage-derived factor capable of amplifying the proliferative response of mouse thymocytes to suboptimal doses of mitogens. This is also a property of human ILl, and the capacity of enhancing proliferation of thymocytes to submitogenic doses of lectins constitutes the basis of the assay routinely used to measure IL1 activity (81). Interleukin 1 itself is not mitogenic, as shown by

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ELSON ET AL.

its inability to independently support the long-term growth of T-cell lines, but it enhances proliferation through its ability to induce the lymphokine IL2. Indeed, the conversion of the IL1 maturational signal into the IL2 proliferative signal (82), and the induction of the expression of receptors for IL2 (83) by IL1 are essential to the process of T-cell activation. In turn, T lymphocytes, stimulated by either specific or nonspecific signals, release a macrophage-activating factor (84). This interrelationship between macrophages and T cells leading to mutual activation is a typical example of the role played by soluble immunoregulators in the complex interplay among lymphoid cells. In addition to its immunoregulatory functions, IL1 is also a mediator of inflammation. Endogenous pyrogen (also called LEM for leukocyte endogenous mediator), the substance responsible for the appearance of many manifestations of inflammation such as fever, release of neutrophils from the bone marrow, elevation of acute phase reactants, and lowering of plasma iron and zinc concentrations, is functionally and biochemically indistinguishable from IL1 (85). Thus IL1 is capable of modulating a vast array of inflammatory events, and it is likely to be involved in the pathogenesis of many inflammatory phenomena at diverse anatomic sites, including the intestinal mucosa. Because macrophages are prominent in inflamed intestine, such as in Crohn’s disease and ulcerative colitis, they may be actively secreting ILl. Whether or not IL1 plays a role in the pathogenesis of IBD can only be speculated upon at present. Interleukin 2 With the expansion of our understanding of the role of lymphokines in immune regulation, it has become apparent that some of these soluble factors act in the afferent phase of the immune response, others in the effector phase, while still others seem to be active in both phases of the immune response. As an example of the latter, IL2 (or TCGF, T-cell growth factor), has been shown to be a molecule essential for proliferation, differentiation, and clonal expansion of T cells (86,87). After appropriate antigen presentation by accessory cells and exposure to the maturational signals of ILl, T cells will produce and release IL2, which, in turn, will activate other T cells to become effective cytotoxic, helper, and suppressor cells (Figure 4) (86). Resting T cells must first express IL2 receptors in order to respond to this signal; a monoclonal antibody (anti-Tat) (881, which binds to and blocks the human IL2 receptor on human T cells, also inhibits T-cell activation (89). Interleukin 2 receptor appearance and disappearance mirror the rate of T-cell proliferation, and three conditions are

GASTROENTEROLOGY Vol. 91, No. 3

Macrophage

::.:.:. + @

J

??Antigen

ILI

Activated la+ @77&T macrophage

cell

\

T cvtotoxic

/

T heber

0 dC Clonal expansion

T sucwessor . .

@ J S

of T cells

Figure 4. Schematic diagram of cell-lymphokine interaction. Activated macrophages present antigen and IL1 signal to T cells, which become activated and produce macrophage-activating factor, probably represented by IFN y, and ILZ. Interleukin 2, in turn, acts on activated subsets of T cells that express IL2 receptors, inducing clonal expansion of T-cell populations of various functional characteristics.

essential for the progression of the T-cell cycle: IL2 concentration, IL2 receptor density, and duration of IL2 receptor interaction (90). The influence of IL2 may extend beyond the T-cell system, because activated B cells also express receptors for, and proliferate in response to, this powerful immunomodulator (91). Advances in the understanding of the immunoregulatory role of IL2 have been followed by the realization that disorders jn the release or response to IL2 may occur in vivo, and perhaps contribute to the pathogenesis of a variety of conditions clinically manifested as immune deficiency, autoimmunity, and neoplasia. Evidence for defective production or response to IL2, or both, have been shown in animal models of autoimmunity (92) as well as in human primary immunodeficiency syndromes, aged subjects, metastatic cancer patients (93-96), and patients with systemic lupus erythematosus and rheumatoid arthritis (96).

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1986

Interleukin

INTESTINAL IMMUNITY

3

Interleukin 3 (IL3) is a lymphokine that is biochemically and functionally distinct from IL1 and ILZ, and it appears to function as a differentiation factor for immature T lymphocytes as well as a variety of hematopoietic cells (97). Interleukin 3 is produced predominantly by helper T cells, and has the unique property of inducing the appearance of 20 a-hydroxysteroid dehydrogenase, an enzyme associated with T-cell lineage differentiation. An additional peculiar characteristic of IL3 is that of generating and maintaining in vitro, granulated toluidine blue-positive cells resembling basophils and mast cells. All of the studies on IL3 have been performed in animals. Therefore, the importance of this lymphokine to humans and its relationship to other soluble mediators of the immune response remain to be defined. Interferon Interferon, originally described as a substance with antiviral activity (98), is actually not a single substance, but rather a class of inducible molecules with a broad spectrum of activities. Thus, besides its well-recognized local and systemic antiviral effects, IFN also affects cellular differentiation, growth, surface antigen expression, morphology, and immunoregulation (99). Interferons are usually classified in three distinct types: IFN a or IFN /?, and immune or IFN y. All three types are antigenically distinct and are induced by different stimuli. Interferon y, which is usually acid labile and produced by mononuclear cells activated by immune mechanisms (loo), has much greater antiviral and antigrowth potential than the classical IFNs, and possesses a multitude of immunoregulatory functions of great importance (99). Therefore, the present considerations on IFN will be restricted to IFN 7. Although IFN may affect humoral immunity, its major effects are on cell-mediated immunity, as demonstrated by inhibition of lymphocyte proliferation (lOl), augmentation of natural killer (NK) cell activity (102), activation of macrophages for tumoricidal and bacteriocidal activities (103), enhancement of surface antigen expression, such as class II MHC molecules (log), and induction of IL2 receptors on T cells (105). Within the previously mentioned framework of macrophage-T lymphocyte interaction, it has been proposed that IFN y has macrophage-activating activity (106), although probably not all lymphocytederived MAF is attributable to IFN y. Recent evidence supporting this concept has been provided by Nathan et al. (107), who identified IFN y as the lymphokine responsible for stimulating human mac-

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rophage oxidative metabolism and antimicrobial activity. Such macrophage activation may be crucial to the development of defense against pathogenic organisms, and deficient levels of IFN y may result in defective activation of tissue macrophages, leading to persistent bacterial infection, as proposed by Nogueira et al. (108). Inadequate macrophage function has been postulated as a cause of IBD, and now this hypothesis is testable directly by measuring the effect of lymphokines on cells isolated from human intestinal mucosa. Lymphokine

Interactions

As the number of known lymphokines has increased, it has become apparent that each had the capacity of influencing the function of other lymphokines under in vitro experimental conditions, suggesting that different lymphokines could be mutually interacting among themselves (109). The functional interrelationships of lymphokines and monokines is now beyond doubt, and, in fact, appear to be integrated in “cascades” that are necessary for the functional expression of an immune response (Figure 4) (110). Role of Lymphokines

in Intestinal

Immunity

The information summarized above comes from studies utilizing systemic lymphoid tissues or peripheral blood. Considering the key role that lymphokines play in the immune system, it is reasonable to assume that they are involved in all immune reactions, whatever the site. However, each anatomic environment is unique, possibly adding still another level of complexity to the function and interaction of the various soluble mediators discussed above. This applies particularly to the intestinal mucosa, with its peculiar distribution and compartmentalization of immunocompetent cells (11 l), and its continuous handling of large quantities of antigens and mitogens under both normal and pathological conditions. Thus, a thorough understanding of how the various lymphokines function in the intestinal mucosa will require the use of intestinal lymphoid cells, as these can differ from those in the peripheral blood and are the cells actually exposed to the modulatory influences of locally produced factors (112). It seems likely that lymphokines are intimately involved in the expression of the normal mucosal immune response in health and may be involved in the abnormal response in diseases such as IBD. Observations in other clinical conditions, where significant immunologic abnormalities may be related to defects of lymphokine function, such as primary immunodefi-

756

ELSONETAL.

ciency syndromes, systemic lupus erythematosus, rheumatoid arthritis, and aging (93-96), support the possibility that similar defects in lymphokine function may be involved in IBD. Recently, the production of and response to soluble mediators by intestinal mucosal mononuclear cells in the epithelium (IEL) or the lamina propria (LPL) have been examined. The interactions between IEL and intestinal epithelial cells via solution mediators has been studied by Cerf-Bensussan et al. (113) in a rat model. These authors showed that the number of class II MHC molecule (Ia) positive intestinal epithelial cells increases with the age of the animals, and that the Ia-antigen expression by enterocytes correlates with an increase in the number of IEL. Furthermore, they showed that IEL stimulated in vitro with mitogens release soluble factor(s) capable of inducing expression of Ia-antigen in a long-term, Ia-negative intestinal epithelial cell line of rat origin. The characteristics of the factor(s), e.g., kinetics of production, lability at pH 2.0, and growth inhibitory activity, suggest IFN y as the molecule responsible for the Ia-inducing activity. They concluded that because of the close physical association between IEL and epithelial cells, the former may have important modulatory activity on the function of the latter. Such modulatory activity may be particularly important if viewed as part of the relationship between IEL and antigen handling at intestinal mucosal sites. Circumstantial evidence supporting such a relationship is provided by the observation that antigenic stimulation leading to an increase in the number of IEL also leads to enhanced class II molecule expression by epithelial cells (114), which raises the exciting question of a possible role for epithelial cells in antigen processing and presentation to the intestinal mucosal immune system. In addition, an increased number of IEL is observed in a variety of intestinal diseases, such as celiac disease, cow’s milk intolerance, parasitoses, and graft-vs.-host reaction. Common to these conditions may be an increased intestinal mucosal permeability or an augmented antigenic exposure by the gut mucosa, both requiring processing of a massive antigenic overload. Such may also be the case in IBD, in which an increased number of antigen-presenting “veiled” cells has recently been documented (115), as well as an augmented presence of HLA-DR class II molecules on epithelial cells overlying segments of inflamed, but not of normal, colonic mucosa (116). No reports exist on the production of IL1 by intestinal mucosal cells. As outlined previously, this monokine is intimately involved in the pathogenesis of a variety of inflammatory processes (ll7), and some indirect evidence exists to suggest that IL1

GASTROENTEROLOGYVol. 91,No.3

might also be involved in chronic intestinal inflammatory conditions, particularly IBD. Such evidence derives from reports of an activated monocytemacrophage system in IBD, as reflected by enhanced turnover of these cells, and elevated serum, fecal, tissue, and intracellular levels of lysozomal enzymes (118-120). Activated human tissue macrophages, such as alveolar macrophages, are known to spontaneously produce and secrete IL1 in vitro (121), and an analogous situation could be present in regard to human intestinal mucosal macrophages. Using a thymocyte proliferation assay (81), preliminary evidence of strong IL1 activity has been detected in supernatants of unstimulated human intestinal mucosal macrophage cultures (Fiocchi C, unpublished observations). Another crucial regulatory molecule, IL2, has been recently studied by Fiocchi et al. (122) using human intestinal LPL. The high rate of spontaneous proliferative activity of these cells suggests response to locally produced IL2, but these authors detected no evidence for IL2 activity in culture supernatants of unstimulated LPL. Significant levels of IL2 activity, however, were observed in the supernatants if the LPL were cultured with phorbol myristic acetate, a substance known to significantly augment the level of IL2 by cells already producing it at low levels. More remarkably, even though LPL from Crohn’s disease- and ulcerative colitis-involved mucosa produced comparable levels of IL2, these levels were significantly lower than those found in culture supernatants of LPL derived from histologically normal control tissues. Inflammatory bowel disease LPL that totally failed to produce IL2 when stimulated with phorbol myristic acetate were, nevertheless, able to produce it after stimulation with phytohemagglutinin, proving that IBD LPL had not lost their intrinsic capacity to produce IL2. Autologous IBD and control peripheral blood lymphocytes, under identical phorbol myristic acetate-stimulated conditions, did not produce any significant amounts of IL2, suggesting that LPL are in a different state of activation, possibly due to previous antigen exposure in situ. In addition, as production and response to IL2 is intimately involved with the stage of T-cell activation, these data also suggest that T lymphocytes of IBD and control intestinal mucosa may be in a different state of activation. In addition to the production of IL2 by human intestinal LPL, studies have begun on the response of these cells to IL2. Lamina propria lymphocytes from noninflamed intestine proliferate when cultured with IL2 in vitro in the absence of any other exogenous stimuli, whereas under the same conditions peripheral blood lymphocytes do not proliferwith the LPL being ate (123).This is consistent

September

1986

activated in situ, as mentioned above. Interestingly, LPL isolated from the active lesions of Crohn’s disease had a reduced proliferative response to IL2, although LPL from the uninvolved margin of the same patients had a normal response (123).Thus, both the production of and response to IL2 by LPL from inflamed intestine in IBD patients is impaired. One of the major functions of IL2 is the development of the effector phase of the immune response, and a variety of specific and nonspecific functions, such as helper, suppressor, and cytotoxic activity are dependent on IL2 for full expression. It is of some interest, therefore, that incubation of LPL from noninflamed intestine with IL2 stimulates high levels of nonspecific cytotoxicity against a variety of tumor target cells, with the degree of cytotoxicity being strictly dependent upon and proportional to the amount of IL2 present in the cultures (124). The function of IFN in immunologic events involving intestinal mucosal mononuclear cells probably involves other aspects of cell-mediated immunity, such as modulation of cytotoxic phenomena. Reports by Flexman et al. (125) and Targan et al. (126) describe enhancement of NK activity after IFN treatment by, respectively, rat small bowel IEL and human colonic mucosal mononuclear cells, although the observed levels of augmentation were less than those usually detected using spleen or peripheral blood cells. These findings are in contrast with recent data of Fiocchi et al. (124), who reported that IFN was unable to induce cytotoxicity by human LPL. Differences in the type and origin of the effector cells, anatomic source, cell fractionation techniques, methods of incubation with IFN, and the type of IFN used to stimulate the intestinal mononuclear cells probably have significant influences in these apparently discordant results. These preliminary reports are obviously initial approaches to the potential role of IFN in the induction of cytotoxicity at the gut mucosal level, and more work is needed both in animal and human systems. Besides IFN, other factors are apparently involved in the function of mucosal mononuclear cells. Tagliabue et al. (127), for example, have shown that Peyer’s patch lymphocytes from mice not only express natural cytotoxicity against WEHItarget cells, but also that such natural cytotoxicity is augmented by IL3 containing supernatants. This report is quite provocative in view of the capacity of IL3 to serve as a growth factor for murine basophils and mast cells (97) and the shared morphologic and functional characteristics of these cells with intestinal mucosal T cells and NK cells (128). These observations, therefore, are compatible with the hypothesis that soluble mediators may act selectively on

INTESTINAL IMMUNITY

AND INFLAMMATION

757

different subpopulations of intestinal mucosal mononuclear cells. As compared to the massive amount of information available on the role of soluble mediators of the systemic immune response in animal and human systems, the preliminary information regarding the intestinal mucosal immune system constitutes no more than initial steps toward an area that promises a wealth of new data on the mechanisms of immunoregulation in the gastrointestinal tract. Efforts should be devoted to the continued investigation of the functional characteristics of local immunocompetent cells, the locally and systemically produced factors that may modulate their action, and the possible specificity of soluble mediators for lymphocyte subsets in the intestine.

Mast Cells in the Intestine Mast cells (because of their virtually ubiquitous distribution throughout the body, responsiveness to diverse stimuli, and collection of potent mediators) are involved in most, if not all, inflammatory responses and pathologic conditions. The central theme of current mast cell research is that all mast cells are not identical. For example, in the rat, mucosal mast cells in the intestine have a number of characteristics different from systemic or peritoneal mast cells (129-131). The same type of differences may also exist in humans (132-134). Differences in mast cell characteristics are likely to have importance in the pathogenesis and, ultimately, the therapy, of intestinal and other diseases. Until recently, and to some extent even yet, mast cell function has been viewed with the conceptual blinkers of relating solely to IgE-mediated immediate hypersensitivity reactions. It is clear, however, that mast cells are involved not only in immediate hypersensitivity but also in the late-phase component of allergic reactions (133,135), in delayed-onset hypersensitivity reactions (136), and in regulation of immune responses (137). Moreover, mast cells can express direct cytotoxic activity (138) and can potentiate eosinophil (139) or macrophage cytotoxicity (140). The spectrum of cells that respond to mast cell mediators is vast. As an example with but one mediator (histamine), at least two receptor types have been recognized (Hl, H2), and these are expressed on a variety of cells, including lymphocytes, macrophages, neutrophils, basophils, eosinophils, smooth muscle cells, parietal cells, and others (141, 142). If one considers other mast cell mediators, including prostaglandins and leukotrienes, there must be few cell types that would not directly or indirectly be affected by local mast cell secretion. Presently, the major clinical interest in mast cells

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ELSON ET AL.

GASTROENTEROLOGY Vol. 91, No. 3

has been restricted to allergic reactions (143), parasitic infection (142,144), mastocytosis, and inflammatory states such as fBD (143) and pulmonary fibrosis (145).

Table

2.

Mast Cell Heterogeneity in the Rat: Factors That Induce or Modulate Secretion0 Mast cell source Factor

Mast Cell Structure

and Content

Mature mast cells contain many electrondense granules that exhibit metachromatic staining properties. This staining property is due to the proteoglycans present in the granules. The initial unequivocal evidence that rat mucosal and peritoneal mast cells ate distinct was based upon the dye-binding characteristics of the granule proteoglycans of these cells after the use of different fixation and staining techniques (129) (Table 1). Using routine formalin fixation, mucosal mast cells are difficult to stain, whereas peritoneal mast cells stain quite well. Rat peritoneal mast cells contain heparin proteoglycan, whereas rat intestinal mucosal mast cells possess no heparin (129-131), but do contain a chondroitin sulfate (probably diB; Stevens, Lee, Seldin, Austen KF, Befus AD, Bienenstock J, unpublished observations) that is distinct from the chondroitin sulfate E of cultuted mouse bone marrow mast cells (146). Two mast cell types have been recognized in the human intestinal and respiratory tracts using the same histologic techniques (132, Table

1. Heterogeneity of Mast Cells Among Different Species and Sites Mast cell type Rat

-

Human

Bone marrow

urn

propria

Lung/gut

culture

_

+

+ and -

+ -

_

+ ?

Peritone-

Characteristic Histochemistry Formalin sensitivity Mediators Proteoglycans Heparin Chondroitin sulfate Neutral proteases Chymase Ctiboxypeptidase Tryptase Histamine (pg/cell) Serotonin Arachidonic acid metabolites Proliferation T-cell dependency LTB,, leukotriene Dz. + , - indicate

A

Mouse

Intestinal lamina

Type 1 + -

8-24 + PGD,

-

(Ty& ?I Type II ? ? 1-2 + ?

+

_ + 1-2 _ LTC, PGD,

?

? ? ? 0.1-1.0 + LTB, LTC, PGDz

+

Bq; LTC*, leukotriene C,; PGDZ, prostaglandin present or absent, respectively.

Secretagogues Antigen, anti-&E Neutrophil cationic proteins, C3a, C5a, dextran, polylysine 48/80, bee venom 401 Ionophores Substance P Vasoactive intestinal peptide, somatostatin, bradykinin, neurotensin Dynorphin, p endorphin, a neoendorphin Modulation of secretion Phosphatidyl serine Adenosine Cromoglycate, AH9679,

Intestinal lamina propria

Peritoneum

++ ?

++ ++

0

0

++ ++ ++ ++

0

++

+I+ + +

0 t 0

theophylline Doxantrazole, quercetin

’ 0, +, + + indicate degree of responsiveness. enhanced or inhibited, respectively.

t , & indicate

147,148). However, whether these histochemically distinguishable mast cell subtypes in humans are functionally distinct, as has been found in the rat (see below and References 149,150), remains to be established (151). The proteoglycans are not only the major constituents of granules, but their polyanionic character is important also in interactions with other moieties stored preformed in the granules. These other preformed moieties include histamine, serotonin, neutrophil and eosinophil chemotactic factors, neutral proteases, acid hydrolases, peroxidase, superoxide dismutase, and late-phase inflammatory factor of anaphylaxis (152). In the rat, peritoneal mast cells possess a neutral protease type I, RMCP-I, whereas intestinal mucosal mast cells have a distinct protease, RMCP-II. RMCP-II secreted by mucosal mast cells can be detected in the circulation (153). The precise roles of these proteases are unknown, although it is interesting that they degrade type IV collagen of the basal lamina underlying epithelia (152,153). The pathologic implications are obvious. An additional group of mediators is not stored preformed but is synthesized de novo by mast cells after activation. These include prostaglandins, leukottiienes, and platelet-activating factor (152,154,155). Whether or not all the mediators described above are present in all mast cell subtypes is unknown. Intestinal mucosal mast cells contain histamine, serotonin, a form of chondroitin sulfate, and Rh4CPII, but convincing evidence does not exist about

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INTESTINAL IMMUNITY

1986

other preformed mediators (Table 2). Moreover, no studies of arachidonic acid metabolism or plateletactivating factor have been conducted on purified mucosal mast cells. Evidence from long-term cultures of murine mast cells (Table 1) that seem to be analogous to mucosal mast cells, shows that leukotriene Cq is a major arachidonic acid metabolite and prostaglandin Dz is also produced (146). Before the role of mucosal mast cells in normal intestinal physiology or disease can be clarified, it is essential that their assortment of mediators be defined. Mast Cell Activation

and Inhibition

Many stimuli induce mast cell activation and secretion (156)(Table 2).Antigen induces mediator secretion by cross-linking specific IgE, and to a lesser extent, IgG antibodies on the cell surface. New, exciting information shows that in the mouse, antigen-specific T-cell factors also induce mast cell secretion (157) but that the mechanisms of exocytosis differ from IgE-antigen-induced secretion and involve preferential release of serotonin (158). Other mast cell secretagogues include neutrophil cationic protein, C3a and C5a anaphylatoxins, compound 48/80, concanavalin A, dextran, polylysine, and ionophores (156).In the rat the responsiveness to compound 48/80and bee venom peptide 401 defines functionally distinct mast cell subtypes; peritoneal mast cells respond, intestinal mucosal mast cells do not (129,149). The responsiveness of mast cell subtypes to other secretagogues has been incompletely defined. Various neuropeptides and hormones, such as vasoactive intestinal peptide, neurotensin, substance P, and somatostatin, induce mediator release by rat peritoneal mast cells (159) (Table 2).This fits well with recent evidence of mast cell innervation (160). Interestingly, only substance P induces mediator release from intestinal mucosal mast cells, despite the large array of peptide hormones that stimulate peritoneal mast cells (161).Similarly, certain opioid-peptides, e.g., dynorphin, induce histamine secretion from peritoneal mast cells, but none has activity on isolated rat intestinal mucosal mast cells (162).The underlying mechanisms of these differences and their clinical and biologic significance remain to be determined. Therapeutically it is essential to know if antiallergic drugs inhibit secretion by all mast cell subtypes or are restricted in action to only certain subtypes. In the rat, many drugs inhibit peritoneal mast cell secretion (e.g., disodium cromoglycate, AH9679, theophylline) but are without effect on intestinal mucosal mast cells (150) (Table 2). Doxantrazole (150) and the flavonoid, quercetin

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(163), inhibit antigen-induced histamine release by both mast cell subtypes. Isolated (but not purified) human intestinal mast cells, which contain two histochemically distinct subtypes (Table l), are largely unresponsive to disodium cromoglycate [-20% inhibition of mediator release, (134)]. Whether this represents potent inhibition of only a minor mast cell subtype in the mixture, or merely experimental variation and not mast cell inhibition, will only be determined when distinct histochemical subtypes can be purified and studied separately. The development of antiallergic drugs specific for different mast cell subtypes not only may have great therapeutic importance but also will help elucidate the roles of mast cell subtypes in disease. Ontogeny,

Differentiation,

and Proliferation

Mast cells can be detected in the rat embryo. Within the first weeks after birth the distribution and abundance of mast cells changes in various organs (164),but the significance of this is unknown. Rat intestinal mast cells and RMCP-II levels are low at birth, peak by 6 wk of age, and then decline gradually during adulthood (165). Infection with many helminth parasites induces a dramatic T cell-dependent proliferation of intestinal mucosal mast cells (129,142,144). Precursors of mucosal mast cells probably originate in the bone marrow but can be found in the intestinal mucosa, mesenteric lymph nodes, thoracic duct lymph, and even the spleen (166).Their frequency in Peyer’s patches is low. Parasitic infection stimulates the production of the T-cell lymphokine IL3, which stimulates mast cell differentiation and proliferation. The T-cell dependency of intestinal mastocytosis lies in the production of IL3 and perhaps other factors, but T cells do not themselves become mast cells. The precursor does not appear to be a “granulated lymphocyte,” a cell type so common in the intestinal epithelium (167,168). There is no convincing evidence that distinct precursors exist for different mast cell subtypes, and perhaps the most attractive hypothesis to explain the ontogeny of mast cell heterogeneity is that microenvironmental factors selectively induce the expression of distinct portions of the genome in a common precursor, leading to phenotypically distinct subtypes. Mast Cell Function The role of the mast cell in any inflammatory disease or infection will be difficult to define because the possibilities are so diverse. Both histamine and serotonin may act as neurotransmitters, thereby modifying local nervous control. Lymphocytes, mac-

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ELSONET AL.

rophages, neutrophils, eosinophils, platelets, bone marrow stem cells, smooth muscle cells, and goblet cells are targets of certain mast cell mediators. Moreover, Perdue and Gall (169) have shown that changes in epithelial cell function during intestinal anaphylaxis can be inhibited by doxantrazole, the antiallergic compound that blocks rat intestinal mucosal mast cell function (150). Therefore, direct or indirect communication occurs between mast cells and intestinal epithelial cells. It will be interesting to determine if mast cell-mediated killing (138) is relevant to changes in epithelial cell turnover in intestinal diseases. Whether mast cell-epithelial interactions underly the pathophysiology of some diarrhea1 diseases or IBD is as yet unexplored. Other communication networks include those among mast cells, fibroblasts, macrophages, and lymphocytes. Mast cells and histamine levels are elevated in the lesions of Crohn’s disease-lesions marked by granulomas and fibrosis. Histamine can activate suppressor T cells that reduce the size of granulomas (170). Thus, mast cell secretion may be relevant to the chronic granulomatous responses of Crohn’s disease. Given the potential activities of mast cells on a wide variety of targets, as well as the diversity of cells and products that modulate mast cell function, it is obvious that in any working model of inflammatory diseases, mast cells must be prominent. The elucidation of such a model is complicated by our present ignorance of the nature and extent of mast cell heterogeneity in humans. Research must continue to identify the diversity of cellular interactions possible among these different cell types, and to dissect not only the role of lymphocytes and mast cells but also that of eosinophils, macrophages, epithelial cells, goblet cells, smooth muscle cells, and local neurons in the pathogenesis of intestinal inflammatory diseases.

Natural Killer and Other Cytotoxic Cells in the Intestine An important efferent limb of the immune response involves cells that are capable of killing a variety of target cells such as allogeneic normal cells (MHC nonidentical), virally infected autologous cells (MHC identical), autologous blast cells, and tumor cells. There are different types of cytotoxic cells that are distinguished by their cell surface phenotypes, their mode of induction or activation, the target cell they kill in in vitro assays, and the mechanism by which they recognize and lyse target cells (171-173). These different cytotoxic cells can be grouped into three broad classes. Cytotoxic T-cell precursors are activated by cell surface antigens (e.g.,

GASTROENTEROLOGY Vol. 91,

No. 3

viral antigens) in conjunction with the host’s self class I MHC molecules or by allogeneic class I MHC molecules. Interleukin 2 also plays an important role in the activation of cytotoxic T lymphocytes. Mature cytotoxic T cells recognize target cells bearing the same allogeneic class I MHC molecules or foreign antigen in conjunction with self class I MHC molecules used for stimulation. Natural killer cells are cells circulating in the blood that require no induction to express cytotoxic function. They recognize target cells through an “NK receptor” interacting with an “NK structure” on the target cell, which is not MHC-restricted. Natural killer cell activity in human peripheral blood cells is closely associated with a subpopulation of lymphocytes that are large in size and possess azurophilic cytoplasmic granules. This subpopulation of large granular lymphocytes can be isolated by density gradient centrifugation. Natural killer cells appear to be involved in host resistance or surveillance against a wide variety of tumors and viral infections. Antibody-dependent cellular cytotoxicity (ADCC) can be performed by a variety of cells such as T cells, NK cells, granulocytes, and macrophages-all of which have receptors for the Fc portion of antibody. The specificity of the antibody determines which target is recognized and eventually lysed, i.e., there is no “specific” effector cell receptor. Lysis ensues when the antibody “bridges” target and effector cells. The evaluation of cytotoxic activity in the intestinal mucosa has become more of a challenge since the discovery that a variety of cell types can be cytotoxic. For example, there is a multitude of effector cells capable of killing the “NK target” cell known as K562 (Table 3). As mentioned, these cells can be distinguished by cell surface markers (e.g., T cell vs. NK cell), the conditions inducing cytotoxic potential (e.g., IL2 vs. IFN), and the mechanism of recognition [e.g., NK receptor (NKCC) vs. antibody (ADCC) vs. T-cell receptor (T-cell)]. There may also be heterogeneity among cytotoxic cells of a single class such as NK cells (174). For example, cells bearing NK surface markers vary in their NK lytic activity; the use of such surface markers to study NK cells in the intestine vs. the blood is complicated by the possible modulation of surface molecules that occurs during culture (I 75). The heterogeneity among cytotoxic cells is exemplified also by the responses to IL2 by two phenotypically different cytotoxic cells. Exposure of NK cells to high doses of IL2 (100 U/ml) for 2 h induces activated NK cell lysis, whereas exposure of T cells to IL2 requires at least 48 h to induce de novo T-cell killing (activated NK vs. lymphokineactivated killer] (Table 3). Recent delineation of the NK lytic mechanism and discovery of soluble lytic factors (Figure 5) will allow the molecular definition

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1986

INTESTINAL IMMUNITY AND INFLAMMATION

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Table 3. Cells That Can Kill K562 Target Cells Cytotoxicity type NK Activated

Cytotoxic activity generated by

NK

IFN. IL2

Time

Cell type

Mediated through

1-z h

LGL LGL

NK receptor NK receptor

-

LGL T cell LGL-11+ DR+ T cell ? T cell

Antibody ? Non-NK receptor

ADCC LAK NK-like

IL2 MLR

48-nh

NC CTL

IL3 MLR

3-5 days

6 days

Other targets Virally infected cells [a) B cells (b) Hematopoietic stem cells (c) Fresh tumors Any Fresh tumors

? T-cell receptor

WEHI; 18-h assay All MHC targets

ADCC, antibody-dependent cellular cytotoxic cells; CTL, cytotoxic T lymphocytes; ILZ, interleukin 2; IL3, interleukin 3; IFN, interferon: LAK, lymphokine-activated killer cells; LGL, large granular lymphocyte; MHC, major histocompatibility complex: MLR, mixed lymphocyte reaction; NC, natural cytotoxic cells; NK, natural killer cells.

of this lytic process,

which can be compared to other lytic mechanisms in intestinal lymphoid cells. The use of single-cell assays with highly enriched effector populations has demonstrated the existence of cells that can recognize and bind targets but that are With this not capable of lysing them (176,177). approach, the lytic efficiency (the amount of killing per potential effector cell) can be measured. Recent studies have shown that NK cells have many biologic activities other than killing. Table 4 illustrates the multiplicity of biologically related

functions that NK cells perform. In addition to the killing of tumor cells, they may have roles in stem cell development, immunoglobulin regulation, direct defense against infections, and provision of lymphokines. These different functions might well reflect the activity of different clones or subsets of NK cells, all of which share the NK markers. This concept of clonality of subsets of killer cells is particularly pertinent in considering the possible function of NK cells in the intestinal mucosa. For example, because there are a great number of epithe-

\

I

l-57

Lytic complex I

I

El ss

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-

w

NKCF _----A or -rcep”

$a++ 7

d

Endocytosis Membrane movement

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“,

5 Assembly/ 3 Activation

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1

1

[ KILLER CELL INDEPENDENT LYSIS I Figure

5. Sequential stages of NK cytotoxicity. Natural killer-target cell binding activates or “triggers” the NK cell to release NK-cell lytic factor (NKCF). Binding of NKCF to structures on the target cell programs the target for cell death. The remaining steps proceed even if the NK cell is removed, as shown in steps 4 through 7, and thus are killer cell-independent.

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ELSON ET AL.

lial cells as well as terminally differentiated B cells (plasma and lymphoblastoid B cells) in the mucosal compartment, clones of NK cells interacting with and regulating these targets may be more prevalent and important there than NK cells that are predominantly cytotoxic. There has been much variability in the observations made by different investigators as to the types of “spontaneous” cytotoxic activity in the intestine. Several investigators have found absent spontaneous or NK cytotoxicity among intestinal cells when using standard NK target assay systems (178,179). Other investigators have found a low amount and low efficiency of spontaneous killing among human intestinal LPL (180,181); i.e., there was less cytotoxic activity per potential cytotoxic cell in the intestine than in the peripheral blood. Adding to the complexity of these observations are two recent findings. First, Fiocchi et al. (124) have demonstrated that LPL incubated with IL2 for 72-96 h in vitro express high levels of cytotoxicity toward both K562, a standard NK target, and Daudi cells, an NK resistant target. Such lymphokine-activated killer activity was strictly dependent on and proportional to the amount of IL2 present in the cultures. When LPL were cultured with IFN y alone, no cytotoxicity was induced, and if both IL2 and IFN were added to the cultures the resultant lymphokine-activated killer activity was not stronger than that induced by IL2 alone (124). Whether this was an activated NK vs. an IL2 responsive T cytotoxic cell was not determined. Similar results have been obtained recently by Hogan et al. (182). Second, cells capable of inhibiting peripheral NK activity have been identified within the intestinal mononuclear cell population (183). This suggests that cellular suppression of cytotoxic cells also needs to be considered when measuring lytic activity in intestinal LPL. Treatment regimens may also need to be considered because sulfasalazine and sulfapyridine have been reported to suppress mucosal LPL lytic activity against K562 targets when added to in vitro cultures (184). Spontaneous lysis by LPL against targets other than K562 has been recently demonstrated by two groups. Shorter et al. (185) using a trypan blue dye assay, showed that there were trypsin-sensitive mononuclear cells in normal and IBD mucosa that were capable of lysing autologous epithelial cells. The type and specificity of effector cells and autologous targets, however, were not determined (185). Fiocchi et al. (186) were able to show that LPL mononuclear cells from IBD mucosa had a low level of cytotoxicity toward erythrocytes coated with defined rat epithelial cell components. This suggests the existence of T-cell clones sensitive to antigens of normal gut epithelium in IBD mucosa. The cytotoxic

GASTROENTEROLOGY Vol. 91, No. 3

Table 4. Biologic Functions Associated With NK cells Demonstrated Function

In vitro

In vivo

Tumor surveillance (metastasis) Viral infection Hematopoiesis regulation B-cell proliferation/function Microbial control Release of lymphokines

+ + + + + +

+ + + + -

cell type and actual sensitivity and specificity of this lysis, however, remains to be defined. These recent studies illustrate the difficulties in defining mucosal cytotoxic cell function and in demonstrating a direct role for epithelial cell “autodestruction” by a cytolytic mechanism in IBD. Clearly, the biology of cytotoxic cells in normal mucosa needs first to be more specifically investigated. Then the following elements need to be defined in investigations of cytotoxic cell function in IBD: (a) a clear separation and evaluation of normal, ulcerative colitis, and Crohn’s disease intestinal mononuclear cell populations; (b) a broader search for target cell types sensitive to mucosal cytotoxic cells; (c) accurate definition of target cell specificities of mucosal mononuclear cells; (d) delineation of surface marker phenotypes and the lytic mechanism of the effector cell types; (e) examination for additional functional roles of “cytotoxic” cells; and (f) production of viable long-term epithelial cell cultures to better study the interactions of these target cell types and their cell surface target antigens with the appropriate effector cell populations present in the intestine. In studies underscoring these points, James and colleagues (187) have reported the first attempt in nonhuman primates to analyze the presence of cytotoxic cells in the mucosa. Using human K562 targets as an indicator system in 18-h assays, they were able to identify mucosal lymphocytes that would lyse these targets. The phenotype of these cells, using monoclonal antibodies directed against human cell surface antigens, was determined to be Leu-11, the same as human peripheral blood NK cells, although the percentage of these LPL expressing Leu-11 was much lower than that in peripheral blood. Using fluorescence-activated-cell-sorter-enriched cells, they demonstrated that on a per cell basis the peripheral blood Leu-ll+ cells in these animals were much more lytically efficient than the intestinal Leu-ll+ cells. To study the changes in the activity or number of these cells during inflammation, or both, James et al. produced a self-limited intestinal inflammation by intrarectal innoculation

September

INTESTINAL IMMUNITY

1986

of trachomas of the lymphogranuloma venereum serotype. These monkeys developed a localized granulomatous proctitis which resolved spontaneisolated from the ously in 6-8 wk. Lymphocytes lesions at 2 wk had diminished cytolytic activity against K562 targets with the same number of Leullf lymphocytes present. This suggested that the cells became less efficient and that the inflammatory process per se was capable of inhibiting the cytolytic activity of these cells. These results demonstrate in an animal model that the cytotoxic activity of NKlike cells can be inhibited by the inflammatory process. These studies reemphasize the need to specifically define conditions when studying cytotoxic effector cells and to examine both the ability of cells to bind to targets as well as to lyse them, because cells capable of binding but not of lysis have been identified (176,177). In summary, several important considerations need to be incorporated into future studies analyzing the role of cytotoxicity in the intestinal mucosa: (a) many cytotoxic cell types with different lytic mechanisms can kill the same target; (b) in defining which cell is cytotoxic one must consider that the surface marker phenotype of the cells in peripheral blood can differ from that in the mucosa; (c) peripheral blood and mucosal lytic efficiency of the same effector cell against a common target can differ secondary to an exogenous regulation of the lytic mechanism by cells, soluble products, or differential maturation; (d) the primary target cell system of the same effector cell types in peripheral blood and mucosa may differ owing to the possibility of differential localization of effector cell clones in various lymphoid compartments; and (e) the cytotoxic function of the mucosal NK cell may be secondary to its primary function in this compartment, i.e., regulation of differentiated B cells.

Summary The previous sections illustrate that we are still defining (a] which sets of lymphoid cells are present in the intestine and which are not, (b) which sets are peculiar to the intestine, and (c) how the sets that are there function in the intestinal microenvironment. An understanding of the latter point is going to require knowledge of how these sets communicate with and regulate one another via cell surface molecules such as MHC class I and class II molecules, and via soluble mediators or lymphokines. The recent advances in various technologies make this a particularly exciting time in this field because the tools are now available to address and answer some of these basic and important questions in mucosal immunology. At the same time these

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advances hold great promise for our eventual understanding of chronic inflammatory diseases of the intestine. As was mentioned at the outset, the immune system has considerable power for both protection and destruction. It remains a puzzle how this latter potential is contained and controlled in the intestine of most individuals, such that they do not have inflammatory disease even in the setting of intense stimulation by substances, such as endotoxin, that are phlogistic elsewhere in the body. An answer to the question of why everyone does not have intestinal inflammation could provide new insights into the mechanisms involved in chronic intestinal inflammatory diseases. The recent advances just detailed, as well as others sure to come, suggest that it is only a matter of time before such questions are answered.

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