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The Journal of Immunology, 1998, 160: 6112-6120.
Copyright © 1998 by The American Association of Immunologists

Altered Intestinal Immune System but Normal Antibacterial Resistance in the Absence of P-Selectin and ICAM-1

Ulrich Steinhoff1, Uwe Klemm, Manuela Greiner, Karin Bordasch and Stefan H. E. Kaufmann

Department of Immunology, Max Planck Institute of Infection Biology, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ICAM-1 and P-selectin are adhesion molecules that regulate leukocyte migration, extravasation to inflammatory sites, and other immune cell interactions. T cell-mediated resistance against acute infection with Listeria monocytogenes and chronic infection with Mycobacterium bovis Calmette-Guérin bacillus was investigated in mutant mice lacking P-selectin and/or ICAM-1. Mice deficient in P-selectin (Psel-/-), ICAM-1 (ICAM-/-), or the combination of both (Psel-/- x ICAM-/-) showed normal bacterial clearance, comparable delayed-type hypersensitivity reactions, and equivalent memory T cell responses. Additionally, the distribution of {alpha}ß vs {gamma}{delta} T lymphocyte populations was examined. Normal lymphocyte distributions were noted in thymus, spleen, and blood, whereas mutant mice showed marked alterations in the intestinal intraepithelial (i-IEL) and lamina propria lymphocytes. Differences in i-IEL populations were reflected functionally by differential lytic activities and cytokine productions of i-IEL populations from mutant mice. Despite these changes within the mucosal immune system of mutant mice, their resistance against oral infection with L. monocytogenes was apparently unimpaired. These findings demonstrate that P-selectin and ICAM-1 are critically involved in the shaping of lymphocyte populations of the gut but have only a minor influence on systemic and regional host defense against intracellular bacteria.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immune surveillance depends on the circulation of leukocytes between the bloodstream and the extravascular space where pathogens are encountered and local immune reactions are initiated. Recirculation is not random but, rather, is targeted by active mechanisms of lymphocyte endothelial cell interactions. Leukocytes differ in their traffic routes and organ-specific preference for migration. At sites of microbial deposition, extravasation is increased, and immigrant polymorphonuclear granulocytes (primarily neutrophils) and monocytes induce a prompt inflammatory response that is crucial for early control of microbes and for initiation of local immune responses (1, 2). Subsequent influx of T cells to sites of chronic inflammation is promoted by up-regulation of adhesion molecules on endothelial cells at the site of inflammation (3). Extravasation of leukocytes involves at least four distinct steps: leukocyte rolling along the activated endothelium, leukocyte activation, firm attachment, and transendothelial migration. Different families of adhesion molecules are involved in this process. Selectins and members of the Ig superfamily, comprising different ICAMs, the VCAMs, mucosal addressin, and CD31, together with their respective ligands play a crucial role (4). The selectin family, which is largely responsible for leukocyte rolling, comprises three known members, the L (leukocyte), P (platelet and endothelial cells), and E (endothelial) selectins. Although the three selectin molecules show overlapping functions, their differential expression levels and tissue distributions suggest that they play different roles during the immune response. While L-selectin is constitutively expressed on most circulating leukocytes, E-selectin is expressed on endothelial cells by de novo mRNA synthesis after stimulation with inflammatory agents. In contrast, preformed P-selectin is stored in platelets and endothelial cells and, upon stimulation, is translocated from their storage organelles to the cell surface within minutes (5). Due to its fast expression, P-selectin initiates the earliest step of leukocyte recruitment into inflammatory sites, including extravasation of neutrophils (6) and recruitment of CD4+ T lymphocytes (7). Four intracellular adhesion molecules, ICAM-1, -2, -3, and -4, have been defined to date, two of which, ICAM-1 and ICAM-2, serve as endothelial ligands for leukocytes. ICAM-1 plays a central role in immune cell interactions because it not only interacts with numerous ligands expressed on immune cells such as LFA-1, Mac-1, and CD43, but is also misused by pathogens for host cell invasion (8, 9). ICAM-1 has been further implicated, e.g., in mixed lymphocyte reactions (10), in granuloma formation (11), and in the development of the intestinal mucosal immune system (12). In light of these findings we were interested in the influence of P-selectin and/or ICAM-1 1) on the composition of lymphoid cells in different organs and 2) on resistance against bacterial infections. Acute (Listeria monocytogenes) and chronic (Mycobacterium bovis Calmette-Guérin bacillus (BCG)2) infections that are controlled by both innate and acquired immune responses were investigated. Innate immunity primarily involves the action of monocytes, polymorphonuclear granulocytes, and NK cells, whereas acquired immunity is mainly mediated by Ag-specific T lymphocytes. Because the gut represents the first line of defense against many food-borne bacterial pathogens such as L. monocytogenes, and the intestinal immune system shows high sensitivity to alterations of adhesion molecules (12), we also analyzed oral infections with L. monocytogenes in adhesion molecule-deficient mice.

Our findings demonstrate that the most prominent consequences of a lack of P-selectin and/or ICAM-1 are within the lymphocyte composition of the intestinal immune system. We extend recent observations (12) that, in addition to ICAM-1 and ß2 integrins, P-selectin is involved in the establishment of the normal intestinal immune system. However, despite distinct alterations of the gut lymphoid environment in these KO mice, resistance against intestinal and systemic infection is apparently not impaired.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Male and female 6- to 9-wk-old C57BL/6J (B6), C57BL/6J-Icam1tm1Bay (ICAM-/-), C57BL/6J Selptm1Bay (Psel-/-), and C57BL/6J-Icam1tm1Bay x Selptm1Bay (ICAM-/- x Psel-/-) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred under SPF conditions in our animal breeding colony at the bgvv (Berlin, Germany). All experiments were performed with age-matched animals.

Bacteria and bacterial Ags

L. monocytogenes strain EGD Sv1/2a was originally obtained from G. B. Mackaness and grown in tryptic soy broth (Difco, Detroit, MI). M. bovis BCG, strain Chicago (American Type Culture Collection, Rockville, MD; ATCC 27289), was grown in Dubos Middlebrook Medium (Difco) supplemented with albumin and Tween-80. Bacterial numbers were determined by plating serial dilutions on Middlebrook agar plates (Difco). For soluble antigens of L. monocytogenes, bacteria were cultured for 3 days in an ultrafiltrate (<=10,000 m.w.) of tryptic soy broth. The culture medium was centrifuged at 10,000 x g, and the supernatant was concentrated 200-fold (>=10,000 m.w.) with an Amicon concentrator (Amicon, Lexington, MA). The concentrate was dialyzed against PBS, sterile filtered, and stored at -70°C.

Antibodies

The following hybridomas were used for mAb preparations: anti-TCR-{alpha}ß (H57-597), anti-TCR-{gamma}{delta} (GL3), anti-CD8{alpha} (YTS 169.4), anti-CD8ß (H35-17.2), anti-IFN-{gamma} (R4-6A2 and AN 18.17.24), and anti-IL-4 (24G2.7 and 11B11). Hybridoma cells were cultured in medium containing 2% FCS, and supernatants were collected. mAbs were then concentrated by ammonium sulfate precipitation and purified by affinity chromatography on protein G-Sepharose (Pharmacia, Freiburg, Germany). Abs were conjugated with biotin or FITC using conventional methods. Normal hamster Ig was purchased from Dianova (Hamburg, Germany).

Delayed-type hypersensitivity reactions (DTH)

Listeria-immune mice (days 6–7) were challenged s.c. with 30 µl of culture filtrate (>=10,000 mol wt) from L. monocytogenes into the hind footpad. Specificity control of the DTH reaction was performed by injecting similarly treated tryptic soy broth. Footpad thickness was measured 24 and 36 h later using a dial gauge caliper (Kröplin, Schluchtern, Germany).

Cell preparations

The i-IEL were prepared by modifications of a method previously described (13). Briefly, the small intestines were inverted and placed into a bottle containing complete Click/RPMI 1640 medium, and the bottle was agitated on an orbital shaker at 37°C for 30 min. Supernatants were collected, and the gut tissue was transferred to a 50-ml tube containing 15 ml of medium and shaken vigorously for 15 s. This procedure was repeated three times; the supernatants were collected each time and replaced with fresh medium. Cells were pooled and then passed through a 10-ml syringe column packed loosely with siliconized glass wool to remove cell debris and adherent cells. To enrich for the lymphocyte fraction, discontinuous Percoll density gradients were prepared in 10-ml centrifuge tubes by layering from the bottom cells containing 70% Percoll (1 ml) and then 40% Percoll (2 ml). The tubes were then centrifuged at 20°C for 20 min at 600 x g. The interface between the 70% and the 40% Percoll layers contained lymphocytes with >95% cell viability. Approximately 6 to 9 x 106 cells/mouse were obtained by this method.

CTL assay

CTL activities of i-IEL were measured in a redirected 51Cr release assay using P815 cells as targets as described previously (14, 15). The i-IEL were incubated with 2 x 103 51Cr-labeled P815 cells for 4 h at 37°C in 7% CO2 at various E:T cell ratios. Assays were performed in the presence or the absence of 2 µg of anti-CD3, anti-{alpha}ß TCR, anti-{gamma}{delta} TCR mAb, or normal hamster IgG. After 4 h, 100 µl of supernatant was removed, and released label was measured in a gamma counter. The percent specific lysis was calculated as follows: [(experimental 51Cr release - spontaneous 51Cr release)/(maximum 51Cr release - spontaneous 51Cr release)] x 100.

ELISPOT assay for IFN-{gamma} and IL-4

We used a modified ELISPOT method described by Taguchi et al. (16). Briefly, ELISPOT plates (Millipore, Bedford, MA; STHA 09619) were coated with 1 µg/ml of anti-IFN-{gamma} mAb (R4-6A2) or anti-IL-4 mAb (24G2.7) in 0.05 M carbonate buffer, pH 9.6 (100 µl/well), at 4°C overnight. Plates were washed once with PBS and blocked with 1% BSA/PBS for 2 h at 37°C. After two washes with PBS, appropriate numbers of i-IEL in RPMI 1640 medium with 10% FCS were added and incubated for 16 h at 37°C in 5% CO2. The plates were washed extensively with PBS containing 0.05% Tween-20, and 0.25 µg/ml biotinylated anti-IFN-{gamma} mAb (AN-18.17.24) was added. After incubation at 37°C for 2 h, the plates were washed several times with washing buffer, and alkaline phosphatase-conjugated streptavidin was added. After 1 h of incubation at 37°C, plates were washed eight times with washing buffer and once with alkaline phosphatase buffer, pH 9.5. Then, 50 µl of freshly prepared substrate (nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate in 70% dimethylformamide) was added. The plates were incubated at 37°C in the dark for 15 min, and spots were counted under a dissecting microscope. Results are expressed as the frequency of cytokine-producing cells per 105 i-IEL.

Immunohistology

Parts of the small intestine were shock-frozen in liquid nitrogen and subsequently kept at -70°C. For immunostaining, cryostat frozen sections were cut to 5 to 8 µm, air dried, and then fixed in acetone for 10 min. After blocking endogenous peroxidase activity (Dakopatts, Glostrup, Denmark) cryostat sections were preincubated with normal mouse serum from Dianova and then incubated with the primary mAb for 60 min before visualizing with horseradish peroxidase using 3-amino-9-ethylcarbazole (Dakopatts) as substrate. For primary incubation, the following mAb were applied: anti-TCR-{alpha}ß FITC (H57-597) and anti-TCR-{gamma}{delta} FITC (GL3). Secondary mAb incubation was performed with rat anti-FITC peroxidase conjugated mAb (Boehringer Mannheim, Mannheim, Germany). PBS was used for washing steps between incubations. Finally, sections were counterstained with hematoxylin.

Flow cytometry

Single-cell suspensions were stained with FITC-conjugated mAb, phycoerythrin-conjugated mAb, and biotin-conjugated mAb, followed by staining with streptavidin-conjugated Red 670. All incubation steps were performed at 4°C for 30 min, and all washing steps were performed with PBS containing 2% FCS and 0.1% sodium azide. After staining, the cells were analyzed with a FACScan (Becton Dickinson, Sunnyvale, CA) equipped with LYSIS II software. Live cells were gated by forward and side scatter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lack of P-selectin and ICAM-1 results in altered lymphocyte populations in the gut

FACS analyses of leukocytes from untreated control and KO mice were performed to assess the relative lymphocyte subset distribution in different tissues. Deficiency of either adhesion molecule did not lead to apparent changes in lymphocyte subsets in thymus, spleen, or blood. A twofold increase in the percentage of neutrophils was found in spleen and blood of Psel-/- and ICAM-/- mice, and a threefold increase was found in double deficient animals (data not shown). Pronounced changes, however, were observed in the i-IEL compartment of KO mice. While Psel-/- mice showed ~20 to 30% higher proportions of {alpha}ß T cells and a concomitant decrease in {gamma}{delta} T cells, ICAM-/- and ICAM-/- x Psel-/- animals exhibited consistently increased proportions of {gamma}{delta} T cells together with a lowered proportion of the TCR-{alpha}ß i-IEL. In accordance with and in extension of recent work by Lefrancois (12), we found that in ICAM-/- mice and to an even greater degree in ICAM-/- x Psel-/- mice, {alpha}ß T cells bearing the heterodimeric CD8 {alpha}ß molecule were preferentially reduced (Table IGo). Interestingly, despite the inverse distribution pattern of {alpha}ß and {gamma}{delta} T cells in Psel-/- and ICAM-/- single KO mice, the combined deficiency of both adhesion molecules in ICAM-/- x Psel-/- double KO mice lead to a phenotype resembling that of ICAM-/- mice. These findings emphasize 1) that P-selectin and ICAM-1 are both important for physiologic lymphocyte homeostasis in the gut; 2) that P-selectin and ICAM-1 exert differential effects; and 3) that in double deficiencies, the ICAM-/- phenotype dominates over the Psel-/- phenotype. Further, immunohistologic analysis of the small intestines of mutant mice revealed that the distribution of LPL was affected by the lack of the adhesion molecules in a manner similar to that of i-IEL. Increased numbers of TCR-{gamma}{delta} LPL were found in ICAM-1-/- and ICAM-1-/- x Psel-/- mice, whereas TCR-{alpha}ß LPL were elevated in Psel-/- mice (Fig. 1Go).


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  		Table I. Phenotypes of i-IEL from normal and mutant mice1

 


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  		FIGURE 1. Immunohistology of LPL and i-IEL in the small intestine of normal and mutant mice. Frozen sections (8 µm) of C57BL/6 (A and B), Psel-/- (C and D), ICAM-/- (E and F), and Psel-/- x ICAM-/- mice (G andH) were stained with mAb against TCR-{alpha}ß (A, C, E, andG) or TCR-{gamma}{delta} (B, D,F, and H). Stained lymphocytes are dark red. i-IEL are indicated by the closed arrow; LPL are indicated by the open arrow. Original magnification, x400.

 
Ab-induced cytolytic activities of i-IEL

Next, we investigated whether the lack of adhesion molecules had any influence on cytolytic activities of i-IEL as measured in a redirected lysis assay using mAb. The {alpha}ß and the {gamma}{delta} i-IEL populations of mice kept under conventional housing conditions express spontaneous killer activity upon TCR engagement (17, 18, 19). The i-IEL from control and KO mice exhibited a clear, but strain-dependent, cytolytic activity against P815 target cells in the presence of anti-CD3, anti-TCR-{alpha}ß, and anti-TCR-{gamma}{delta} mAb. Figure 2GoA shows that anti-TCR-{alpha}ß mAb-induced lytic activity was decreased approximately 10-fold in ICAM-/- x Psel-/- mice and 3- to 5-fold in ICAM-/- mice. In contrast, i-IEL of Psel-/- mice exhibited slightly increased lytic activity. Furthermore, when i-IEL were stimulated via anti-TCR-{gamma}{delta} mAb (Fig. 2GoB), ICAM-/- and ICAM-/- x Psel-/- mice lysed P815 cells more efficiently than C57BL/6 controls, while Psel-/- mice exhibited greatly reduced CTL activity. This difference probably reflects the increased proportion of TCR-{gamma}{delta} i-IEL in ICAM-/- mutants and double deficient animals, while this population is markedly decreased in Psel-/- mice. Oral infection of mutant mice with L. monocytogenes did not influence the lytic activities of i-IEL (data not shown).



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  		FIGURE 2. Cytolytic activities of i-IEL from normal and mutant mice. Cytolytic activities of i-IEL were assayed on 51Cr-labeled P815 target cells in the presence of anti-TCR-{alpha}ß (A), anti-TCR-{gamma}{delta} (B), anti-CD3 (C), and normal hamster Ig (D). Symbols represent i-IEL from C57BL/6 ({square}), ICAM-/- ({blacktriangleup}), Psel-/- ({circ}), and Psel-/- x ICAM-/- ({diamondsuit}). Results are plotted as the percent specific lysis at various E:T cell ratios. Representative results for a total of four mice per group are shown.

 
It has been shown that CD3-mediated signaling differs in TCR-{gamma}{delta} vs TCR-{alpha}ß i-IEL (12, 20). We therefore investigated whether a similar dichotomy in signal transduction could be seen in Psel-/- and Psel-/- x ICAM-/- mice. ICAM-/- and Psel-/- x ICAM-/- mice exhibited normal cytolytic activity after ligation of TCR-{gamma}{delta}, yet CD3-mediated signaling was greatly reduced in these mutant mice (Fig. 2GoC). In contrast, i-IEL from Psel-/- animals were only marginally affected. The inability of CD3 to efficiently mediate TCR triggering in ICAM-/- and ICAM-/- x Psel-/- mice correlated with increased proportions of {gamma}{delta} T cells within the intestine. Thus, the lytic activity of TCR-{alpha}ß i-IEL in mice with reduced numbers of {gamma}{delta} T cells (Psel-/-) could be equally induced by anti-TCR-{alpha}ß and anti-CD3 mAb. In contrast, the cytolytic potential of {gamma}{delta} i-IEL from mice with diminished {alpha}ß T cells (ICAM-/- and ICAM-/- x Psel-/-) could be induced by mAb against TCR-{gamma}{delta}, but was drastically reduced by mAb against CD3.

Influence of P-selectin and ICAM-1 on secretion of IFN-{gamma} and IL-4 by i-IEL

To assess whether the frequency of IFN-{gamma} and IL-4 producers among i-IEL exhibits a similar strain dependency as the cytolytic activity, we determined the frequencies of cytokine-producing cells by ELISPOT assay. Anti-CD3 mAb, anti-TCR-{alpha}ß mAb, or anti-TCR-{gamma}{delta} mAb was used for stimulation of i-IEL. While TCR-{alpha}ß i-IEL were the main source of intestinal IFN-{gamma} secretion (Table IIGo), low IL-4 secretion was observed in TCR-{alpha}ß and TCR-{gamma}{delta} i-IEL (Table IIIGo). Mutations in P-selectin and/or ICAM-1 had only a marginal influence on the intestinal cytokine profile; Psel-/- mice exhibited slightly increased numbers of IFN-{gamma}- and IL-4-secreting cells in the uninfected gut. Intestinal listeriosis increased the frequencies of IFN-{gamma}-secreting cells in mutant animals by a factor of 2 to 4, leading to comparable numbers of IFN-{gamma}-secreting cells in all strains (Table IIGo). IL-4 production by i-IEL although above background was still low in all animals (Table IIIGo).


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  		Table II. IFN-{gamma}-producing cells among i-IEL from different mice and influence of intestinal L. monocytogenesinfection1

 

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  		Table III. IL-4-producing cells among i-IEL from different mice and influence of intestinal L. monocytogenesinfection1

 
Effect of P-selectin and/or ICAM-1 deficiency on resistance against infection with L. monocytogenes and M. bovis BCG

Bacterial counts in mutant mice (Psel-/-, ICAM-/-, and Psel-/- x ICAM-/-) and controls systemically infected with L. monocytogenes (5 x 103 CFU) were performed at different time points to evaluate early (neutrophils, monocytes, and NK cells) and late (specific T cells) resistance mechanisms. Figure 3Go shows that bacterial counts in livers, but not in spleens, of Psel-/- mice and Psel-/- x ICAM-/- mice were slightly increased 3 days after infection. At all other time points bacterial numbers in spleen and liver were comparable between mutant and control mice. Next, we determined whether the observed alterations in lymphocyte subsets of mutant mice influenced resistance against intestinal infection with L. monocytogenes. Interestingly, spread of listeriae from the gut to the spleen and the liver was comparable in all animals after oral infection with 6 x 107 CFU (Fig. 4Go). Similar results were obtained with higher oral infection doses of L. monocytogenes (2 x 108 CFU; data not shown). Thus, alterations in i-IEL distribution in mutant mice did not have an apparent influence on regional defense. To further examine resistance mechanisms against a chronic bacterial infection, mutant and control mice were infected i.v. with M. bovis BCG (5 x 106 CFU). Deficiency of P-selectin and/or ICAM-1 did not impair immunity against chronic mycobacterial infection (Fig. 5Go).



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  		FIGURE 3. Bacterial growth in spleen and liver after systemic infection with L. monocytogenes in mutant mice. Animals were infected i.v. with 5 x 103 CFU L. monocytogenes, and bacterial growth was assessed at the indicated time points. Data shown are representative of three separate experiments and are expressed as log10 of individual titers and the median (bar) of each group.

 


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  		FIGURE 4. Bacterial growth in spleen and liver after oral infection with L. monocytogenes in mutant and control animals. Animals were infected orally with 6 x 107 CFU, and bacterial growth was tested 3 days later in liver and spleen. Data shown are representative of two independent experiments and are expressed as log10 of individual titers and the median (bar) of each group.

 


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  		FIGURE 5. Growth of M. bovis BCG in mutant and control animals. Animals were infected i.v. with 5 x 106 CFU of M. bovis BCG, and 31 days later bacterial growth in the lung, liver, and spleen was determined. Data shown are representative of two separate experiments. Data are expressed as the log10 of individual titers and the median (bar) of each group.

 
Listeria-specific DTH and memory T cell responses are independent of P-selectin and ICAM-1 expression

To obtain further information about the roles of P-selectin and ICAM-1 in T cell functions in vivo, we compared Listeria-specific T cell-mediated DTH reactions and Listeria-specific memory T cell responses of control and mutant mice. DTH reactions were tested in day 6 Listeria-immune mice by challenge with a culture filtrate of L. monocytogenes. Figure 6Go shows that footpad swelling was similarly pronounced in KO and control mice. The capacity of mounting a functional T cell memory was tested in Listeria-immune mice that were challenged with an otherwise lethal dose of L. monocytogenes (2 x 105 CFU) 5 wk later. Figure 7Go illustrates that lack of P-selectin and/or ICAM-1 did not impair the formation of a memory T cell response against secondary infection with L. monocytogenes.



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  		FIGURE 6. DTH reactions to listerial culture filtrate in L. monocytogenes-immune mice. Day 6 Listeria-immune mice received 30 µl of culture filtrate into the footpads, and footpad swelling was measured 36 h later. Each animal is represented by an individual symbol, and the median (bar) is shown.

 


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  		FIGURE 7. Memory T cell responses to secondary infection with L. monocytogenes. Mutant and control animals were infected i.v. with 5 x 102 CFU of L. monocytogenes and challenged 35 days later with a normally lethal dose (2 x 105 bacteria). Bacterial counts were performed 3 days later in spleen and liver. Two independent experiments with similar results were performed. Data are expressed as the log10 of individual titers and the mean (bar) of each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that ICAM-1 and P-selectin play important roles in the establishment of normal lymphocyte populations in the gut, but not in thymus, spleen, or blood. While the deficiency in P-selectin resulted in increased proportions of TCR-{alpha}ß i-IEL, lack of ICAM-1 or of both adhesion molecules increased the proportion of i-IEL bearing TCR-{gamma}{delta}. Interestingly, T cell effector functions against acute or chronic infections caused by the intracellular bacteria, L. monocytogenes and M. bovis BCG, respectively, were not influenced dramatically by the lack of ICAM-1, P-selectin, or both adhesion molecules.

Lymphocytes of the gut can be divided at least into three distinct populations according to their localization. These are lymphocytes that reside within the epithelial layer of the gut, in the lamina propria or in specialized lymphoid organs, the Peyer’s patches. The function of i-IEL is still obscure because these cells differ in many aspects from T cells in central lymphoid organs (21, 22, 23). Murine i-IEL can be almost equally divided into TCR-{alpha}ß and TCR-{gamma}{delta} cells, most of which expresses the CD8 coreceptor. In contrast to the peripheral T cell populations expressing the CD8{alpha}ß heterodimer, the majority of the TCR-{alpha}ß i-IEL population expresses a CD8{alpha}{alpha} homodimer, and all TCR-{gamma}{delta} i-IEL express the CD8{alpha}{alpha} homodimer. Generally, it is accepted that the TCR-{gamma}{delta} i-IEL and all TCR-{alpha}ß CD8{alpha}{alpha} i-IEL develop in the gut independently from the thymus (24, 25).

Here we show that the establishment of a normal intestinal immune system requires the expression of ICAM-1 and P-selectin. Interestingly, proportions of lymphocyte subsets in thymus, spleen, and blood were apparently normal in mutant mice (data not shown). This is in contrast to earlier experiments showing that addition of anti-ICAM-1 mAb to fetal organ culture blocked thymocyte maturation from the double-negative CD4- CD8- to the double-positive CD4+ CD8+ stage (26). Although we cannot exclude that the ICAM-/- mice used here show only a partial functional deletion (27), recent reports employing another mutant ICAM-1 strain (28) also observed unimpaired T cell development.

Disruption of ICAM-1 or ß2 integrins leads to partial loss of Thy1+ TCR-{alpha}ß CD8{alpha}ß lymphocytes within the intestinal epithelium and lamina propria (12). Elegant studies with bone marrow chimeras from the same group further indicate that activation and expansion of TCR-{alpha}ß i-IEL are dependent on bone marrow-derived ICAM-1+ cells, most likely dendritic cells (12, 29). Our data are consistent with and further extend these findings, in that we observed an inverse proportion of TCR-{alpha}ß and TCR-{gamma}{delta} i-IEL between Psel-/- and ICAM-/- mice. While lack of P-selectin resulted in a significant increase in TCR-{alpha}ß i-IEL and a concomitant decrease in TCR-{gamma}{delta} i-IEL, lack of ICAM-1 and ICAM-1 x P-selectin showed the opposite effect. The fact that double KO mice lacking both adhesion molecules exhibited an even more pronounced phenotype than ICAM-/- mice could be explained by the assumption that the establishment of a normal gut mucosal immune system requires P-selectin and ICAM-1 sequentially, with a dominance of the latter adhesion molecule. Interestingly, the effects of these adhesion molecules are not restricted to the i-IEL compartment but also encompass lymphocyte distribution within the lamina propria. Whether P-selectin and ICAM-1 are involved in lymphocyte migration from the bloodstream into the mucosa or in the development of intestinal T cells, or mediate selective regional expansion of i-IEL and LPL cannot be decided as yet.

The i-IEL from normal mice exhibit strong cytolytic activities and produce IFN-{gamma} either spontaneously or after TCR cross-linking (30). Intestinal infection with L. monocytogenes did not increase the lytic activity of i-IEL, but up-regulated the frequency of IFN-{gamma}-producing i-IEL, suggesting that these lymphocytes contribute to regional protection (31). To date, the role of ICAM-1 in T cell activation remains controversial (28, 32), and in vivo data concerning the requirement for ICAM-1 and/or P-selectin in regional defense against intestinal infection do not exist.

We observed that bacterial transmigration from the intestinal lumen to the liver and spleen was comparable in KO strains and controls regardless of the expression of ICAM-1 and P-selectin. The cytolytic potential as well as IFN-{gamma} secretion of TCR-{gamma}{delta} i-IEL were genetically controlled (13). Since all the mouse strains tested here are of a genetic responder background (C57BL/6), the influence of adhesion molecules on effector functions of TCR-{alpha}ß and TCR-{gamma}{delta} i-IEL was examined. Results of Ab-mediated cytotoxicity and IFN-{gamma} secretion do not support direct involvement of P-selectin and ICAM-1 in triggering i-IEL effector functions. Rather, the differences observed are most likely due to the numerical increase in TCR-{alpha}ß or TCR-{gamma}{delta} i-IEL in Psel-/- or ICAM-/- mice. However, in accordance with previous findings (16, 12), we observed that triggering of TCR-{gamma}{delta} i-IEL is different from that of TCR-{alpha}ß i-IEL. While the lytic activity of the latter population was similarly stimulated by CD3 and TCR-{alpha}ß ligation, the TCR-{gamma}{delta} i-IEL responded to TCR-{gamma}{delta}, but less well to CD3 cross-linking. The reason for this difference is unclear, but could be due to the fact that the Fc{epsilon}R1 {gamma}-chain is engaged in the CD3 complex of TCR-{gamma}{delta} i-IEL, but not in that of TCR-{alpha}ß i-IEL (20).

In uninfected animals, TCR-{alpha}ß i-IEL represent the majority of IFN-{gamma}-secreting lymphocytes, whereas during intestinal listeriosis, TCR-{gamma}{delta} i-IEL are also primed for IFN-{gamma} secretion (33). The high frequency of IFN-{gamma}-producing cells in Psel-/- and ICAM-/- mice emphasizes the importance of IFN-{gamma} as an essential regulator of the intestinal immune response. Less information is available about the synthesis of the Th2 cytokine, IL-4, by i-IEL. A very low frequency of IL-4-secreting i-IEL residing equally within the TCR-{alpha}ß and TCR-{gamma}{delta} populations was detected in all strains. However, in comparison with the high frequency of IFN-{gamma}-producing cells, we consider the observed differences in IL-4-secreting cells in mutant and control mice to be of minor biologic significance. Thus, a Th1 cytokine milieu predominates in normal and infected gut that is not significantly disturbed by the lack of ICAM-1 and/or P-selectin.

In all mutant mice, bacterial clearance of L. monocytogenes and M. bovis BCG was virtually normal. Only animals lacking P-selectin exhibited transiently elevated listerial titers in the liver but not in the spleen. This can be explained by the observations that 1) early resistance to L. monocytogenes in the liver, but not in the spleen, is mainly neutrophil dependent (1); and 2) neutrophil extravasation from the bloodstream is controlled by P-selectin (6). T cells are pivotal for acquired immunity against L. monocytogenes and M. bovis BCG, and they mediate granuloma formation and DTH reactions. Granulomas represent complex tissue reactions involving activation and recruitment of monocytes and T cells to the sites of microbial replication. Granulomas promote containment of bacilli to discrete tissue sites and in this way contribute to protection against chronic infections. Accordingly, granuloma formation is critical for control of M. bovis BCG (34). In contrast, it is of little if any importance in defense against L. monocytogenes because these bacteria are so rapidly eradicated that highly structured granulomas do not develop (35, 36). DTH reactions develop in response to soluble Ags and are mainly mediated by emigrant CD4 T cells and monocytes. Since bacterial numbers as well as DTH and memory T cell responses were normal in mutant mice, we suggest that T cell activation and migration in response to these infections are not critically dependent on P-selectin and/or ICAM-1, most likely because other adhesion molecules compensate for their activities.

Analogous to the Th1 immune response during intracellular bacterial infection, chemically induced inflammation results in migration of Th1, but not Th2, cells into sensitized tissue sites (37). Despite the lack of P-selectin and/or ICAM-1, we observed normal bacterial Ag-specific DTH responses as measured by footpad swelling. Previous experiments using radiolabeled cells have shown that Psel-/- mice exhibit a reduced cellular infiltration into sites of chemical sensitization (7, 38). However, parallel experiments using ear-swelling measurements revealed no differences in ear thickness between wild-type and Psel-/- mice (7). This observation conforms with our data. In an attempt to explain this discrepancy the authors assume that despite reduced numbers of mononuclear cells and neutrophils in Psel-/- mice, either the number of phagocytes was still sufficiently high to cause vascular leakage (i.e., ear swelling) or, alternatively, cellular infiltration and vascular leakage are controlled by separate mechanisms, differentially dependent on the expression of P-selectin.

The results presented here indicate that lack of P-selectin and/or ICAM-1 does not dramatically influence antimicrobial T cell functions, as measured by clearance of bacterial pathogens, formation of Ag-specific DTH reactions, and T cell memory. However, the establishment of the intestinal T cell system requires both P-selectin and ICAM-1, thus emphasizing its uniqueness from other lymphoid compartments. Additional experiments will more clearly characterize the roles of these adhesion molecules in the development, migration, and activation of defined T cell population within the intestine.


   Acknowledgments
 
We thank Drs. H. Collins and M. Munk for critically reading the manuscript and for many helpful suggestions. We greatly appreciate the technical help of M. Stäber and D. Oberbeck-Müller, and express our thanks to Dr. D. Wolff, M. Primke, and D. Gollub for excellent maintenance of the animal facilities.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Ulrich Steinhoff, Department of Immunology, Max-Planck-Institute for Infection Biology, Monbijoustr. 2, 10117 Berlin, Germany. E-mail address: Back

2 Abbreviations used in this paper: BCG, Calmette-Guérin bacillus; KO mice, knockout mice; DTH, delayed-type hypersensitivity reaction; i-IEL, intraepethelial lymphocytes; ELISPOT, enzyme-linked immunospot; LPL, lamina propria lymphocytes; Psel-/-, P-selectin knockout; ICAM-/-, intercellular adhesion molecule-1 knockout; Psel-/- x ICAM-/-, P-selectin x intercellular adhesion molecule-1 double knockout. Back

Received for publication November 13, 1997. Accepted for publication February 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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