Lymphocyte Migration to the Intestinal Mucosa and its Relation to Mucosal Defense

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Lymphocyte Migration to the Intestinal Mucosa and its Relation to

Mucosal Defense

Soichiro Miura

¹

, Yoshikazu Tsuzuki

¹

, Ryota Hokari

¹

, Hiroshi Nagata

²

, and Hiromasa Ishii

²

Summary. Lymphocyte recirculation is a key phenomenon in immunology.

However, in vivo regulation of the homing phenomenon of lymphoid cells to the intestinal mucosa, and its pathophysiological role in intestinal inflamma- tion, has not been clearly understood. In this chapter we summarize the dynamic process of lymphocyte–endothelium recognition in the lymphoid and nonlymphoid area of intestine under an intravital microscope using fluorescence-labeled lymphocytes and discuss how regulation of lymphocyte homing is disturbed or altered under inflammatory conditions. Microvessels of intestinal lamina propria efficiently attracted gut-derived T cells via the a4b7/MAdCAM-1 system, but under tumor necrosis factor-a-induced inflamed conditions vascular cell adhesion molecule (VCAM)-1 was also sig- nificantly involved. Under physiological conditions there was little lympho- cyte adherence to the colonic mucosa, but in inflamed colonic mucosa T-cell migration became significant, comparable to that in the small intestinal mucosa. The chemokine CCL25/TECK may play an important role in T-cell migration to uninflamed as well as inflamed small intestine, but not colon. In an animal model of chronic colitis enhanced upregulation of mucosal addressin cell adhesion molecule (MAdCAM)-1 levels in the colonic mucosa was demonstrated, and administration of anti-MAdCAM-1 antibody signifi- cantly attenuated the colonic injury, suggesting this adhesion molecule as a useful target for inflammatory bowel diseases.

Key words. Lymphocyte migration, Lamina propria lymphocyte, MAdCAM- 1 , Inﬂammatory bowel disease, TECK/CCL25

213

1

Second Department of Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan

2

Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi,

Shinjuku-ku, Tokyo 160-8582, Japan

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Lymphocyte Migration in Peyer’s Patches

Naïve lymphocytes generally can migrate very efficiently from the blood into secondary lymphoid tissues, such as Peyer’s patches, by extravasating through high endothelial venules (HEVs). Bargatze et al. [1] have demonstrated that efficient arrest of naïve cells to Peyer’s patch HEVs requires the sequential engagement of L-selectin to initial contact, a4b7 to slow rolling, and lymphocyte function-associated antigen (LFA)-1 in conjunction with a4b7/MAdCAM-1 to mediate activation-dependent arrest. Our previous data on lymphocyte behavior of nonactivated splenic T cells in Peyer’s patches showed complete block of interaction of splenic lymphocytes with Peyer’s patch HEVs by antibody against either L-selectin, a4-integrin, b7-integrin, or mucosal addressin cell adhesion molecule (MAdCAM)-1 is strong evidence for a multimolecular adhesion cascade in these HEVs [2]. The differential expression of adhesion-triggering chemokines, including CCL21, will control lymphocyte subset trafficking in Peyer’s patches. In our results on rat Peyer’s patches, B-cell arrest occurs in a wider range of vessels than T cells, includ- ing smaller venules observed inside follicles, whereas T cells preferentially accumulate in interfollicular HEVs [3]. It is known that T cells preferentially arrest in segments displaying high levels of luminal CCL21 in PP-HEVs.

After adhesion, the migration of lymphocytes from inside to outside of the venules (transendothelial migration) is observed, and eventually these cells migrate into the interstitium of Peyer’s patches apart from the venular walls.

Subsequently, T cells, especially CD4

⁺

cells, preferentially appear in parafol- licular microlymphatics. This sequential migration process of naïve T cells in Peyer’s patches could be modulated not only by chemokines, but also by various neurohumoral factors, including vasoactive intestinal peptide, nitric oxide, and ingested long-chain fatty acids [4–6].

Lymphocyte Migration in the Intestinal Mucosa

The intestinal lamina propria is the abundant source of lymphocytes involved in immune effector functions. However, there have been few reports of an in vivo observation study on how lymphocytes from intestinal mucosa migrate into the intestinal lamina propria itself (Fig. 1). The intravital microscopic procedure for monitoring the dynamic process of lymphocyte migration was used to investigate the possible contribution of cell adhesion molecules to the lymphocyte–endothelial cell adhesive interactions in the intestinal mucosa.

Lamina proprial vessels of intestine are known to express MAdCAM-1, yet are

thought to lack L-selectin-binding carbohydrates. Lamina propria lympho-

cytes (LPLs) were shown to express strongly CD69, an activation marker, b7,

(3)

and a combinatorial epitope of a4b7, whereas there was little expression of

L-selectin. Lamina propria lymphocytes accumulated abundantly in the

microvessels of villus tips but not in the submucosal venules or postcapillary

venules of Peyer’s patches [2]. This may not be due to mechanical trapping in

the capillaries but due to G-protein-mediated intracellular pathways, because

the binding of LPLs to the lamina propria vessels was inhibited by the pre-

treatment with pertussis toxin. The accumulation of LPLs in the villus tip was

almost completely blocked by anti-b7-integrin and was signiﬁcantly inhibited

by anti-MAdCAM-1 and anti-a4-integrin, but not by anti-CD11a or anti-L-

selectin antibody [2]. The direct binding of a4b7 to MAdCAM-1 is thought

to help LPLs migrate extremely efﬁciently to the intestinal lamina propria at

least under physiological conditions. A critical role of b7-integrin in lym-

phocyte recruitment for formation of the gut-associated lymphoid tissue has

Fig. 1. Lymphocyte homing in Peyer’s patches vs intestinal mucosa. In Peyer’s patches

three combinations of adhesion molecules (L-selectin–sLe

^x

, a4b7-integrin–MAdCAM-1,

and aLb2-integrin–ICAM-1) are responsible for T-cell migration with help of CC

chemokine receptor 7. On the other hand, little information has been available on how

lymphocyte–endothelial cell interaction occurs in the intestinal mucosal area. Our intra-

vital observation showed that in both small intestinal or colonic mucosa a4b7-

integrin–MAdCAM-1 interaction is crucial for T-lymphocyte migration in uninﬂamed as

well as inﬂamed conditions. MAdCAM, mucosal addressin cell adhesion molecule; LPL,

lamina propria lymphocytes; IEL, intestinal intraepithelial lymphocytes; VCAM, vascu-

lar cell adhesion molecule; ICAM, intracellular adhesion molecule; LFA- 1, lymphocyte

function-associated antigen-1; GlyCAM, glycosylation-dependent cell adhesion molecule

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been proposed. Wagner et al. [7] have reported that the number of CD4

⁺

cells was signiﬁcantly reduced in the lamina propria of mice deﬁcient for the b7- integrin. However, although the expression and function of b7 are major determinants of adhesion to MAdCAM-1 in lamina propria, the level of b7 expression may not exclusively account for lymphocyte preference to MAdCAM-1 or MAdCAM-1 expressed microvessels.

Intestinal intraepithelial lymphocytes (IELs) are a large cell population of cells in the epithelial layer of the small intestine. Although these cells have diverse origins, the normal IEL in the blood probably gain access to the intes- tinal mucosa, because injected peripheral T cells can ﬁll the epithelial com- partments and acquire the characteristics of IEL in adequate conditions.

AlphaEb7-integrin is expressed on 90% of the IEL and aEb7 may mediate the binding of IEL to epithelial cells; however, how they reach the microvessels of the intestinal villus microcirculation is not known. Using isolated IELs from murine intestine, we demonstrated that IEL time-dependently accumulated in villus microvessels of the small intestine, but not to the postcapillary venules of Peyer’s patches [8]. The accumulation of IEL in villus arcade was signiﬁ- cantly, but partially inhibited by anti-b7-integrin or anti-MAdCAM-1 anti- body, suggesting the possible role of other adhesion mechanisms as well.

Differences Between Small and Large Intestine, and Role of Chemokine

Relatively little is known about how recirculation of lymphocytes through the

inﬂamed intestinal mucosa is regulated. It is becoming increasingly apparent

that inﬂammation is associated with enhanced expression of endothelial cell

adhesion molecules. However, relatively little is known how the different

endothelial cell adhesion molecules contribute to T-lymphocyte recruitment

in the intestinal mucosa under inﬂammatory conditions. Recent studies indi-

cate that immune regulation in the colonic mucosa is different from that in

the small intestine. For example, colonic IELs and LPLs appear to be less acti-

vated than their small intestinal counterparts. The difference may be related

to the luminal environment of the bowel at these anatomic sites. However,

there is relatively little quantitative information related to lymphocyte traf-

ﬁcking, especially for the colonic mucosa. Our recent study was conducted to

investigate the dynamic process of T lymphocyte–endothelial cell adhesion

in murine colonic mucosa and to examine how intestinal inﬂammation affects

on lymphocyte interactions with microvessels [9]. In tumor necrosis factor

(TNF)-a-stimulated colonic venules, and enhanced adhesion of LPLs was

demonstrated. This increase was also signiﬁcantly observed in the small intes-

tinal mucosa, but the magnitude of the increased LPL adhesion was more

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significant in the colon (2.23 times) than in the small intestine (1.73 times). Immunohistochemistry revealed that after TNF-a-treatment, both MAdCAM-1 and vascular cell adhesion molecule (VCAM)-1 expression were increased in small intestinal and colonic mucosa, but there was a dominant increase in MAdCAM-1 in the colonic mucosa, that is consistent with the increased LPL adhesion to the inflamed colon. There was a significant MAdCAM-1 expression in the lamina propria and VCAM-1 expression in the submucosa of TNF-a-treated colon. The T-lymphocyte interaction in the colonic mucosa was significantly reduced by blocking monoclonal antibodies against either MAdCAM-1, a4-integrin, b7-integrin, or VCAM-1, but not by anti-intercellular adhesion molecule (ICAM)-1. From this observation we can conclude that MAdCAM-1 and VCAM-1, but not ICAM-1, mediate T- lymphocyte recruitment in the inflamed colon. Spatial heterogeneity of MAdCAM-1 and VCAM-1 activation following TNF-a challenge may enhance specific T-lymphocyte recruitment (a4b7 plus a4b1-integrin positive cells, and a4b1-integrin positive cells, respectively) in the inflamed colonic mucosa.

Rolling T cells activate integrins when they receive signals from chemokines on endothelial cell surfaces. Chemokines are secreted poly- peptides that bind to speciﬁc surface receptors, which transmit signals through G proteins. Recently some chemokines have been shown to trigger intravascular adhesion, whereas others direct the migration of leukocytes into and within the extravascular space. Campbell et al. reported that stromal cell- derived facor-1 (CXCL12), 6-C-kine (CCL21), and macrophage inﬂammatory protein (MIP)-3b (CCL19) could induce adhesion of most circulating lym- phocytes, including most CD4

⁺

T cells, and to induce arrest of rolling cells within 1 s under ﬂow conditions similar to those of blood [10]. They also showed that MIP-3a (CCL20) triggered adhesion of memory, but not naïve CD4

⁺

T cells. Inflammatory cytokines such as RANTES, MIP-1a, and MIP1-b bind a distinct chemokine receptor on Th1 cells, CCR5, but these chemokines may mainly induce these cells to infiltrate into the inflammatory tissues. In Peyer’s patches chemotactic stimulation of rolling T cells is known to rely on the pathway involving CCR7, which activates a4b7 integrin and aLb2 integrin [11].

Recently we found that a CC chemokine, TECK/CCL25, also plays an impor- tant role in the adherence of mucosal lymphocytes to the microvessels of the small intestine [12]. TECK/CCL25, which had initially been reported to be produced by thymic cells, is highly expressed at the message level in the small intestine, and has recently been localized to the crypt epithelium.

TECK/CCL25 is also known to be expressed by endothelial cells and a subset

of cells in the intestinal lamina propria. Interestingly, the only known recep-

tor for TECK/CCL25, CCR9, is expressed by discrete subsets of circulatory

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memory CD4

⁺

and CD8

⁺

lymphocytes expressing intestinal homing receptor a4b7, suggesting that CCR9 may be a receptor used preferentially by lym- phocytes involved in gut immunity. Lamina propria lymphocytes and IELs adhered to the mucosal microvessels of both small intestine and colon, but the blocking of the TECK/CCL25–CCR9 system by desensitization of CCR9 with TECK/CCL25 or anti-TECK/CCL25 antibody treatment significantly inhibited these adhesions only in the small intestine. Desensitization of CCR9 or anti-TECK/CCL25 antibody also attenuated the TNF-a-induced LPL migration in the small intestinal mucosa. These results suggest that the TECK/CCL25–CCR9 system may play a significant role in LPL and IEL adhe- sion to the microvessels of the small intestine, but not colon, under control as well as inflamed conditions. We speculate that chemokines other than TECK/CCL25 may function in lymphocyte migration in the inflamed colonic mucosa after the administration of TNF-a.

Monocyte Migration in Peyer’s Patches and Intestinal Mucosa

Monocyte adhesion to the vascular endothelium and subset diapedesis are important events that occur during chronic inflammation, immune mediated reactions and atherosclerosis. Several monocyte–endothelial adhesion pathways have been described, including adhesion molecules such as L-selectin–peripheral node addressin (PNAd), b2-integrin–ICAM-1, a4-integrin–VCAM-1,and P-selectin glycoprotein-1 (PSGL-1)–P-selectin [13], and subsequent monocyte migration into tissues is regulated by activation of vascular endothelial cells by cytokines such as interleukin (IL)-1, TNF-a, or interferon (IFN)-g, and production of chemotactic factors including interferon-inducible protein (IP)-10 or monocyte chemoattractant protein (MCP)-1. Although intestinal mucosa monocytes play a pivotal role in specific immunological protection against enteric pathogens, and in chronic inflam- matory changes such as Crohn’s disease, it has not been clearly understood how circulatory monocytes actually migrate to the intestinal mucosa.

We monitored the migration of ﬂuorescence-labeled monocytes derived

from spleen in intestinal microvessels with or without LPS (lipopoly-

saccharide) treatment to investigate the role of various adhesion molecules

(P-selectin, VCAM-1, and ICAM-1) [14]. We found that in control mice

without LPS, interactions between infused monocytes and endothelium of

small intestinal microvessels were negligible; on the other hand, signiﬁcant

monocyte–endothelial interactions were observed after LPS treatment in

postcapillary venules of Peyer’s patches, submucosal venules, and micro-

vessels of intestinal villi. Anti-P-selectin monoclonal antibody (mAb) signiﬁ-

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cantly suppressed the LPS-induced increase in monocyte rolling in Peyer’s patches and submucosal venules. Anti-VCAM-1 mAb significantly suppressed the LPS-induced monocyte adherent to each site. But anti-ICAM-1 mAb sig- nificantly inhibited monocyte adhesion only in postcapillary venules of Peyer’s patches, suggesting the more dependency on VCAM-1 in nonlymphoid regions than Peyer’s patches at inflammatory conditions.

Role of Adhesion Molecule in Inﬂammatory Bowel Disease (IBD) and its Animal Models

There is now growing evidence that IBD is associated with altered lymphocyte trafficking in the intestinal mucosa. MAdCAM-1 has been implicated in the selective recruitment of lymphocytes to the gut,and in human ulcerative colitis and Crohn’s disease, the expression of MAdCAM-1 is upregulated in factor VIII-positive vessels in inflamed colonic mucosa [15]. Although other adhe- sion molecules such as VCAM-1 are upregulated in the intestinal mucosa of inflammatory bowel disease,a possible involvement of VCAM-1 in lymphocyte recruitment into the inflamed gut in IBD is still controversial, and the relative functional importance of MAdCAM-1 and VCAM-1 in altered lymphocyte trafficking should be elucidated, especially in the chronic type of colitis.

We investigated this issue using two different models for colitis; one is dextran sulfate sodium (DSS)-induced colitis in mice [16], the other is gran- ulomatous colitis in rats induced by peptidoglycan-polysaccharide (PG-PS) [17]. In DSS colitis BALB/c mice that received 5% DSS solution for 14 days had significant histological damage with shortening of crypts observed in colonic mucosa. Before mucosal inflammation had become significant, expression of MAdCAM-1 was already increased in the microvessels of the lamina propria, preceding the lymphocyte infiltration and VCAM-1 upregu- lation. Administration of anti-MAdCAM-1 antibody significantly reduced colonic injury as well as the infiltration of b7-integrin-postitive lymphocytes, and this antibody also was effective when given 7 days after the start of DSS treatment. Peptidoglycan-polysaccharide is a structural component of the cell walls of bacteria that has well-described proinflammatory properties. After subserosal intestinal injection, PG-PS induces chronic relapsing inflammation in a susceptible strain (Lewis) of rat. The characteristic inflammatory hall- marks of this model are the development of transmural, granulomatous enterocolitis, which shares several histological features of Crohn’s disease.

In our study the colonic submucosa was drastically thickened after PG-PS

injection with inﬁltration of CD4-positive lymphocytes and ED-1-positive

macrophages. Enhanced MAdCAM-1 expression was demonstrated in venular

endothelium of the submucosal venules in inﬂamed colon. Administration of

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anti-MAdCAM-1 antibody (OST-2) signiﬁcantly attenuated the PG-PS- induced colonic damage and cell inﬁltration, while nonblocking antibody (OST-20) did not. The attenuating effect of anti-MAdCAM-1 suggests the importance of the MAdCAM-1 dependent process in the formation of chronic granulomatous colitis.

MAdCAM-1 is induced on a murine endothelial cell line by TNF-a and IL- 1 -mediated nuclear factor kB protein in vitro. Because MAdCAM-1 is an adhesion molecule more unique to the gut mucosa than other adhesion mol- ecules such as VCAM-1 or ICAM-1, attenuation of MAdCAM-1 may be more speciﬁc therapy for colitis. However, as shown in our studies, MAdCAM-1 is also essential for lymphocyte trafﬁcking to gut-associated lymphoid tissues as well as to the intestinal mucosa under physiological conditions. It was also suggested that MAdCAM-1 functions as a vascular addressin in not only mucosal, but also nonmucosal lymphoid tissues during ontogeny [18].

Although it has not been clinically applied yet, it should be taken into account not to oversuppress MAdCAM-1 function if anti-MAdCAM-1 blocking is used for treatment of IBD. Recently the European study group described results of a double-blinded, randomized trial comparing natalizumab, a recombinant humanized monoclonal antibody, against a4 integrin, with placebo in the treatment of moderate-to-severe Crohn’s disease [19]. In this trial, natal- izumab induced remissions and responses roughly twice as often as placebo.

The authors concluded that in the short-term study natalizumab showed efﬁ- cacy for reducing signs and symptoms at least similar to that of inﬂiximab.

Treatment with selective adhesion-molecule inhibitors may become a poten- tial strategy for management of intestinal inﬂammation in future.

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