1 Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada 2 Departments of Pediatrics, Microbiology/Immunology and Pathology, Dalhousie University, Halifax, Nova Scotia B3J 3G9, Canada
Correspondence to: T. B. Issekutz, 8 East Research, IWK Health Centre, 5850 University Avenue, Halifax, Nova Scotia B3J 3G9, Canada. E-mail: Thomas.Issekutz{at}.dal.ca
Transmitting editor: M. Miyasaka
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Abstract |
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Keywords: adhesion molecule, delayed-type hypersensitivity, in vivo animal model, inflammation, recruitment
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Introduction |
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Selectins are a family of calcium-dependent adhesion molecules with an extracellular lectin-like carbohydrate-binding domain. Three members, L-selectin, E-selectin and P-selectin, have been identified. L-selectin is expressed on leukocytes, E-selectin is expressed on activated endothelium, and P-selectin is expressed both on activated endothelium and activated platelets. P-selectin is stored in Weibel-Palade bodies in endothelial cells and -granules in platelets (4,5), and is rapidly expressed on the cell surface upon stimulation with thrombin, histamine, tumor necrosis factor (TNF)-
, IFN-
and IL-1 (68). E-selectin expression requires de novo synthesis, and is therefore delayed (9). TNF-
and IL-1 induce E-selectin synthesis and surface expression on endothelial cells, and IFN-
has been shown to induce E-selectin expression on human dermal microvascular cells in vitro (911).
E-selectin has been proposed to be critical for T lymphocyte migration to skin inflammatory reactions (12). A subset of T cells express a ligand for E-selectin, the cutaneous lymphocyte antigen (CLA) (12,13). These T cells comprise <5% of T lymphocytes in extracutaneous inflammatory sites, but represent 8090% of the T cells found in skin lesions of patients with skin inflammatory conditions (14). Direct evidence for the role of E-selectin in in vivo T lymphocyte migration comes from blocking antibody studies. Anti-E-selectin mAb partially inhibited blood lymphocyte recruitment to delayed-type hypersensitivity (DTH) reactions in pigs (15) and tuberculin DTH reactions in some Macaque monkeys (16). Anti-E-selectin mAb also partially inhibited the migration to contact hypersensitivity reactions in mice of in vivo stimulated mouse CD4+ T cells of the Th1 phenotype (17). Although antibody studies have shown a role for E-selectin in T lymphocyte migration, in E-selectin-deficient mice contact sensitivity reactions were not histologically different from those in wild-type mice (18,19) and leukocyte rolling after TNF- stimulation was not altered (18,19).
The role of P-selectin in mediating in vivo T cell migration is not clear. T cells roll on immobilized P-selectin in flow chambers (20,21). In P-selectin-deficient animals the number of CD4+ T cells in contact hypersensitivity reactions was found to be decreased (22) and anti-P-selectin mAb partially inhibited the migration of cultured CD4+ Th1 cells to contact hypersensitivity reactions in mice (17), demonstrating P-selectin involvement in in vivo T cell migration.
Systematic evaluation of the role of E-selectin and P-selectin in in vivo T lymphocyte migration to various intradermally (i.d.) injected inflammatory agents has not been reported. Our studies were designed to examine the contribution of E-selectin and P-selectin to T lymphocyte infiltration in vivo in cutaneous inflammatory reactions, with the goal of determining the importance of each of these molecules. We used 51Cr-labeled spleen T lymphocytes to quantify T cell migration to i.d. injected IFN-, a major cytokine responsible for T cell migration to DTH reactions (23), TNF-
and concanavalin A (Con A), a mitogen which induces DTH-like reactions in rats and pigs (24).
Our results demonstrate that T lymphocytes use both E-selectin and P-selectin for in vivo migration into cutaneous inflammatory tissues in mice. In general, E-selectin contributes more than P-selectin to the migration of T lymphocytes, but the relative contributions vary depending on the inflammatory stimulus. However, the absence of both E-selectin and P-selectin does not abolish T lymphocyte migration to dermal inflammatory reactions, suggesting alternate adhesion pathways are also involved in the initial interaction with the vascular endothelium.
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Methods |
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Mouse recombinant TNF- was kindly provided by Genentech (South San Francisco, CA). Recombinant rat IFN-
was a generous gift of Dr Peter Van der Meide (TNO Primate Center, Rijswijk, The Netherlands). Lipopolysaccharide contamination was <10 EU/mg in all the cytokines. Con A was obtained from Sigma (St Louis, MO).
mAb
The mAb 9A9E3.F10 (9A9) was a generous gift from Hoffman-La Roche. It is a rat IgG2b which recognizes mouse E-selectin. 9A9 inhibits mouse PMN adhesion to E-selectin transfected COS cells and HL-60 promyelocyte adhesion to stimulated mouse eEnd.2 endothelial cells (25). RMP-1 is a mouse IgG2a mAb which reacts with mouse P-selectin on activated platelets. RMP-1 blocks P-selectin-dependent adhesion of HL-60 cells to recombinant mouse P-selectin and reacts with P-selectin on activated mouse platelets (26). 5H1 (IgG1) is a rat anti-mouse P-selectin mAb. It recognizes P-selectin induced by TNF- on murine eEnd.2 endothelial cells, and blocks P-selectin function in vitro and in vivo (18). Control mAb included SFR3-DR5, a rat anti-HLA-DR5 antibody, as an isotype-matched control for 9A9 and 5H1, and B9 (IgG1), a mouse anti-pertussis toxin antibody (27), as a control for RMP-1. Rat IgG (Sigma) was used as an additional control for 9A9 and 5H1.
Lymphocyte isolation and labeling
Animals were killed by cervical dislocation. The spleens of the animals were removed and passed through a wire mesh to obtain a cell suspension. The red blood cells were lysed with 0.84% Trisammonium chloride. T cells from the spleen were isolated by passing the spleen cells over a nylon wool column for 1 h at 37°C. The isolated spleen T lymphocytes were >95% CD3+, contaminating cells being B cells and <1% other cells, and were > 98% viable by Trypan blue dye exclusion.
For labeling T lymphocytes were suspended at 5 x 107 cells/ml in HBSS, containing 15mM HEPES and 10% FBS, and incubated with 50 µCi Na251CrO4 /ml at 37°C for 45 min. The cells were then washed twice in RPMI 1640 medium and resuspended in RPMI 1640 containing 0.1% human serum albumin for i.v. injection.
Measurement of T lymphocyte migration in vivo
B6, 129Sv wild-type mice, or E-selectin or P-selectin-deficient mice were injected i.v. with 107 radiolabeled T cells carrying 105 c.p.m. in a volume of 200 µl. Immediately afterwards the mice were anesthetized with methoxyfluorane, the backs of the animals were shaved, and 10 µl of lymphocyte recruiting agents and control diluent were injected i.d. in six sites. Two sites were injected with the diluent and four sites with each of the test samples. After 22 h, at a time previously found to be optimal for the measurement of T cell accumulation (23), the animals were anesthetized with methoxyfluorane, and 500 µl of heparinized blood was collected by heart puncture and the mice were killed. The inguinal and axillary lymph nodes, spleen, lung and liver were removed and weighed. Dorsal skin, including the area of the dermal inflammatory reactions was removed, washed and blood squeezed out of any superficial veins. The injected areas were punched out with a 9-mm leather punch. This produced a circular piece of tissue with a standard surface area. The injected skin sites, the resected organs and the remaining mouse parts were counted in an 1480 WIZARD 3
-spectrometer. The blood was centrifuged and the plasma and cell pellet counted separately. About 7080% of the injected radioactivity was recovered. The 51Cr content of all tissues was normalized for 105 c.p.m. recovered after 22 h. The number of labeled T cells in the tissues was determined as follows. The specific activity of the labeled cells, i.e. the number of i.v. injected T cells/51Cr c.p.m., was calculated and used to convert the 51Cr c.p.m. of the tissues to the numbers of labeled T cells/site.
mAb treatment
The effect of the mAb on T lymphocyte migration in vivo was assessed by giving animals an i.v. injection of 200 µg 9A9, 100 µg RMP-1, 200 µg 5H1 or 100200 µg of the control antibodies in 100 µl of sterile saline immediately before an i.v. injection of the labeled lymphocytes. These doses of the mAb produced blood levels >20 µg/ml after 22 h, as assessed by an ELISA of the plasma samples. None of the mAb treatments produced leukopenia or clearance of the 51Cr lymphocytes from the circulation. The T cell migration in animals receiving the control antibodies was not different from mice receiving no antibody, so the results of these two groups were pooled.
Measurement of plasma mAb concentration
The plasma concentration of 9A9 was measured by ELISA using the following protocol. MaxiSorb Immunoplates (Nunc, Roskilde, Denmark) were coated with a 1 µg/ml goat anti-rat IgG antibody, adsorbed to remove cross-reactivity to mouse IgG (Caltag, Burlingame, CA), at 4°C overnight. The wells were blocked with 10% normal goat serum and incubated for 1 h at 37°C with dilutions of the plasma samples. After washing, alkaline phosphatase-conjugated goat anti-rat IgG (Caltag) was added and incubated for 1 h. After washing, the reaction was developed with p-nitrophenylphosphate (Sigma) and the absorbency read at 405 nm on a Titertek Multiscan microplate reader.
The plasma concentration of the mouse mAb RMP-1, was measured by ELISA. Briefly, rat platelets (5 x 106 in 100 µl) were added to wells in 96-well tissue culture plates (Nunc, Gibco/BRL, Mississagua, Ontario, Canada) and spun down at 4°C to form a monolayer. The platelets were activated by adding 1 U/ml bovine -thrombin for 5 min at 37°C and fixed with 1% paraformaldehyde in PBS for 45 min. Fixation was stopped with 0.05 M Tris/0.1 M glycine for 15 min. The wells were blocked with 10% NGS followed by the incubation with the plasma samples. Horseradish peroxidase-conjugated goat anti-mouse IgG was added to the samples and incubated for 1 h at 37°C. After washing the reaction was developed with the o-phenylenediamine and the absorbance read at 492 nm.
Statistical analysis
Results are expressed as means ± SEM of multiple animals. Students unpaired t-test was used to compare the difference between means.
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Results |
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The effect of mAb to E-selectin on T lymphocyte migration
In order to determine the role of E-selectin in in vivo T lymphocyte migration, B6 mice were injected i.v. with 51Cr T lymphocytes and treated with a control mAb or a blocking anti-E-selectin mAb. The administration of the control antibodies had no effect on T cell migration. Figure 2(A) shows that the i.v. injection of anti-E-selectin mAb significantly inhibited T cell accumulation in response to IFN- 60% (P < 0.05), to the combination of IFN-
+ TNF-
by 45% (P < 0.05) and to Con A by 65% (P < 0.01). E-selectin blockade had no effect on the migration of T lymphocytes to TNF-
alone.
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T cell migration in E-selectin-deficient mice
The role of E-selectin in T cell migration to skin inflammation was also investigated using E-selectin-deficient mice. Figure 2(A) shows that in E-selectin-deficient B6 mice the migration of labeled T cells to IFN- induced accumulation was inhibited by 65% (P < 0.001), to IFN-
+ TNF-
by 50% (P < 0.05) and to Con A by 60% (P < 0.01). Analogous to the animals treated with the anti-E-selectin mAb, T cell migration to the TNF-
induced lesion in E-selectin-deficient mice was not affected.
The results obtained from E-selectin-deficient 129Sv mice were somewhat different than the E/ B6 mice. Figure 3 shows that although B6 and 129Sv wild-type control mice did not differ in the magnitude of the T cell responses to the inflammatory agents tested, E-selectin-deficient B6 and 129Sv mice did. In E//129Sv mice, the migration of labeled T cells was inhibited to the Con A inflammatory site by 50% (P < 0.05), but not to the other inflammatory agents tested.
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Effect of treatment with anti-E-selectin and anti-P-selectin on T lymphocyte migration
Mice were also treated with the combination of the anti-E-selectin and anti-P-selectin mAb. Figure 5 shows that the combined mAb treatment inhibited T cell recruitment to all four types of inflammatory reactions, but to a varying extent. The inhibitory effect was greatest (80%) for Con A (P < 0.001), 55% for IFN- (P < 0.01), 50% for the IFN-
+ TNF-
combination (P < 0.001) and 40% for TNF-
(P < 0.01). The migration of T lymphocytes to Con A and to TNF-
was inhibited significantly more in animals treated with both anti-E-selectin plus anti-P-selectin than in those treated with anti-E-selectin mAb alone (Fig. 5). The combined anti-E-selectin and anti-P-selectin treatment did not alter the migration of T lymphocytes to the spleen or lymph nodes (data not shown).
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Figure 6(B) shows that the i.v. injection of anti-E-selectin mAb in P-selectin-deficient mice also significantly inhibited T cell accumulation in all four types of lesions. Similar to animals given the combination of anti-E-selectin and anti-P-selectin treatment and in E-selectin-deficient mice treated with anti-P-selectin mAb, the inhibitory effect was greatest (87%) for Con A (P < 0.001), and somewhat less for IFN- (60%) (P < 0.01) and for TNF-
(40%) alone or the combination of IFN-
+ TNF-
(P < 0.05).
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Discussion |
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We chose mice to study the mechanisms of lymphocyte migration to take advantage of E-selectin and P-selectin-deficient mice. In vivo studies of leukocyte migration have frequently been carried out using mAb to block the function of adhesion receptors. A common criticism of this method is that mAb may be exerting non-specific effects, either by activating cells through the receptor they bind to or by mediating Fc receptor interactions. Adhesion molecule-deficient mice offer an alternative to mAb, but may also be problematic since these mice may have developmental alterations in the expression of adhesion molecules as compared to the wild-type. To avoid these potential problems, we investigated T cell migration in both antibody-treated and selectin-deficient mice, and compared the results.
Our results suggest several novel findings with regard to the utilization of E-selectin and P-selectin by T lymphocytes for migration to various inflammatory stimuli in the skin. Anti-E-selectin treatment significantly inhibited T cell migration to all of the stimuli except TNF- (Fig. 2). In E-selectin-deficient C57BL/6 mice, T lymphocyte migration was also impaired to all stimuli except TNF-
(Fig. 2). The inhibition of T cell migration in the absence of functional E-selectin suggests that T cells require E-selectin for migration to a variety of inflammatory reactions in the skin. These observations suggest that mouse T cells have functional ligands for E-selectin. CLA, which was described on skin homing human T lymphocytes, is a possible ligand, but a mouse homologue has not yet been identified. L-selectin is another possible ligand. L-selectin, which is expressed on the majority of blood leukocytes, was shown to mediate human neutrophil adhesion to E-selectin (29). Yet another E-selectin ligand has been identified on bovine
T cells (30). The inhibition of T cell migration observed in our model, however, is unlikely to be due to the inhibition of
T cells, since these comprise <1% of the T cells in mouse spleen (31).
The results indicate that E-selectin is important for mediating T lymphocyte migration in this model and its function could not be substituted by other adhesion molecules. Nevertheless, in both anti-E-selectin mAb-treated and E-selectin-deficient mice, T cell migration to skin lesions was only partially decreased, indicating that either only a subpopulation of T lymphocytes utilize E-selectin for entry into inflamed skin or T cells can use E-selectin-independent mechanisms for migration to skin. In contrast to E-selectin, P-selectin by itself appeared to play a relatively minor role in T lymphocyte migration to skin inflammation in this model. Two different function-blocking mAb to mouse P-selectin had no effect on in vivo T cell migration to the four i.d. injected inflammatory agents (Fig. 3). This lack of inhibition was not due to low antibody levels in the mAb-treated animals, since higher doses of anti-P-selectin mAb also did not affect T cell migration and blocking levels (>20 µg/ml) of anti-P-selectin mAb remained in the serum after 22 h (data not shown). The lack of effect of anti-P-selectin treatment was not the result of failure of the mAb to block the receptor. Both 5H1 and RMP-1 have been shown in vitro to block P-selectin-dependent adhesion of HL-60 cells (18,26), and in vivo 5H1 blocked neutrophil migration to peritonitis in E-selectin-deficient mice (18), confirming that these antibodies block P-selectin-mediated adhesion and migration. In our studies, anti-P-selectin mAb further suppressed T lymphocyte migration in vivo when used in conjunction with anti-E-selectin mAb, or in E-selectin-deficient mice (Figs 5 and 6), confirming that the anti-P-selectin mAb are indeed function blocking. Furthermore, T cell migration in P-selectin-deficient mice was also unaffected, except to one lesion. Thus, in conclusion, the mAb to P-selectin are effective blockers, but P-selectin, although involved, is not essential for T cell migration in this model.
The minor role for P-selectin in T cell migration is not due to absence of P-selectin ligands on T cells. The P-selectin glycoprotein ligand (PSGL-1), a high affinity P-selectin ligand on myeloid cells (32,33), has also been shown to be expressed on T cells, and can mediate T cell adhesion and rolling in vitro (20,21,34). Furthermore, blocking P-selectin in addition to E-selectin further suppressed T cell recruitment in the Con A lesion and was required to observe inhibition of T cell migration to TNF- (Fig. 5), indicating that T cells have ligands for P-selectin, and that both E-selectin and P-selectin contribute to T lymphocyte recruitment to skin inflammation in this model.
Blocking both E-selectin and P-selectin did not completely inhibit T lymphocyte migration to inflamed skin, indicating that at least one other receptor must be capable of mediating T cell tethering and rolling. Even in the Con A lesion, where blocking both endothelial selectins resulted in the greatest inhibition of T lymphocyte migration, T cell accumulation was suppressed by only 80%. This residual T cell migration may be mediated by L-selectin, since it can mediate T cell tethering in vivo (35), and L-selectin has been shown to be involved in the migration of mononuclear cells to peritonitis (36). Alternatively, the T cell tethering may be mediated by VLA-4. VLA-4, an integrin expressed on the majority of lymphocytes, was reported to support T lymphocyte tethering and rolling in vitro (3739) and rolling in vivo (35,40) via the counter-receptor, vascular cell adhesion molecule (VCAM)-1. Recent studies using a rat model of dermal inflammation and exclusively a mAb blocking approach, suggest that VLA-4 may be mediating the E-selectin- and P-selectin-independent migration (41). Another possibility is a yet unidentified receptor capable of initiating tethering and rolling of T cells, independent of E-selectin and P-selectin.
Our results regarding E-selectin utilization by T lymphocytes for migration to skin inflammation agree with those of Binns et al. (15), who found that anti-E-selectin mAb partially inhibited blood lymphocyte recruitment to cutaneous DTH in sensitized pigs, and with those of Silber et al., who showed that anti-E-selectin treatment can partially inhibit the intensity of T cell infiltrates in the skin in some Macaque monkeys (16). Our results also agree with those of Austrup et al. (17), who demonstrated partial inhibition of cultured Th1 cell migration to contact sensitivity reactions with anti-E-selectin treatment.
In contrast to these results, contact sensitivity reactions in E-selectin-deficient mice, as measured by ear thickness measurements and histological observations, were not different from wild-type (18,19). However, contact sensitivity reactions in mice are heavily infiltrated by neutrophils, which may not depend on E-selectin for their migration (42).
Our results regarding P-selectin utilization by T cells migrating to skin inflammation contrast with those of Austrup et al. (17), who demonstrated partial inhibition of cultured Th1 cell migration to a contact sensitivity reaction with anti-P-selectin treatment. However, these T cells were cultured in vitro and stimulated to develop a Th1 or Th2 cytokine profile, before being injected in vivo. Therefore their requirement for P-selectin may be different from that of resting T cells.
Subramaniam et al. demonstrated decreased CD4+ T cell numbers in contact hypersensitivity reactions in P-selectin-deficient animals (22). This observation is in contrast with our results, which show that P-selectin is not essential for T cell migration to skin lesions within the first 24 h. The observed difference in P-selectin utilization by T cells may be related to the inflammatory agent used to elicit skin inflammation: the contact sensitizing agent oxazolone versus i.d. injected cytokines and Con A. Their studies demonstrate that P-selectin-deficient mice had decreased neutrophil accumulation in contact hypersensitivity reactions at 24 h as compared to the wild-type, whereas several other studies report no decrease in neutrophil accumulation in contact hypersensitivity reactions, in either anti-P-selectin-treated or in P-selectin-deficient mice at 24 h (18,19). Our results agree with those of Tang et al., showing that in P-selectin-deficient mice, mononuclear cell accumulation in the inflamed cerebrospinal fluid is not inhibited at 24 h (43), and to those of Tang et al., showing that P-selectin-deficient mice reject allogeneic skin grafts normally (44).
Another interesting finding was that, in contrast to the other stimuli used, T cell migration to TNF- was not inhibited in anti-E-selectin mAb-treated or E-selectin-deficient mice. TNF-
has been shown to be a potent inducer of E-selectin expression on dermal vascular endothelial cells both in vitro and in vivo (911,4548). TNF-
also induces considerable T lymphocyte recruitment when injected i.d. in mice, as shown here, as well as in other species (46,4850). Furthermore, Binns et al. have shown a correlation between E-selectin expression and T cell migration to TNF-
lesions (46). Together, these findings strongly suggest an essential role for E-selectin in T cell migration to TNF-
. However, our results indicate that while E-selectin is involved, it is not essential for T cell migration to TNF-
-induced dermal inflammation in the mouse; P-selectin appears able to substitute for E-selectin function, as functional absence (either by mAb treatment or in selectin-deficient mice) of either receptor alone did not affect T cell migration to TNF-
(Figs 24), whereas eliminating both E-selectin and P-selectin function resulted in significant inhibition (Figs 5 and 6). The explanation for these observations may lie in the ability of another receptor to substitute for E-selectin or P-selectin function in mediating T cell migration to TNF-
. One possible candidate is VCAM-1, an activation inducible endothelial adhesion molecule. TNF-
induces VCAM-1 expression on endothelial cells (51,52), and VCAM-1, as mentioned earlier, supports tethering and rolling of T cells in vitro via VLA-4 (3739). Some in vivo studies, however, suggest that this interaction supports rolling, but not tethering, of leukocytes (35,40). It remains to be determined which adhesion molecules are involved in T cell migration to TNF-
.
The finding that anti-E-selectin mAb treatment inhibited T cell migration to IFN- suggests that E-selectin is present on dermal vascular endothelium in mice. Literature disagrees on this issue because IFN-
has been shown to induce E-selectin expression on human dermal microvascular endothelial cells (10), but not on cultured human umbilical vein endothelium or in vivo in mouse skin (11). Our results suggest that IFN-
induces E-selectin expression on vascular endothelial cells when injected i.d. in mice.
The observation that combined E-selectin and P-selectin blockade did not inhibit T cell migration to IFN- more than E-selectin blockade alone (Fig. 5) suggests that P-selectin may have minimal involvement in T cell migration to IFN-
. This is in keeping with lack of effect on T cell migration to IFN-
of P-selectin blockade or genetic deletion (Fig. 4).
P-selectin is stored within the Weibel-Pallade bodies of endothelial cells and is rapidly (<30 min) up-regulated after cytokine activation of the endothelium (48). E-selectin expression is thought to require de novo synthesis by the endothelium and takes 23 h to be up-regulated (911). This suggests that there may be a greater reliance for leukocyte migration on P-selectin early during an inflammatory reaction. Unfortunately, it was not possible to test differences in the kinetics of T cell migration in the absence of E-selectin or P-selectin in the mouse model used here, because the numbers of T cells accumulating are relatively small in response to the inflammatory lesions studied; so that measurements over short time periods to determine the kinetics of migration are not possible. However, lymphocyte migration to the dermal inflammatory sites induced with cytokines, based on previous studies, takes several hours, so it is unlikely that significant T cell accumulation occurs prior to both P-selectin and E-selectin being up-regulated on the endothelium (23).
This study also sheds some light on the mechanisms of T cell migration to lymph nodes and spleen. The fact that blocking either E-selectin or P-selectin or both had no effect on T cell migration to lymph nodes and spleen suggests that neither of these molecules are required for T cell homing to these sites. Both E-selectin and P-selectin are expressed in human tonsil (53), but their functional significance in mediating T cell migration to lymphoid tissues has not yet been assessed. L-selectin, on the other hand, appears to be essential for lymphocyte migration to peripheral lymph nodes (54,55). This demonstrates that neither E-selectin nor P-selectin are involved in T lymphocyte migration to peripheral lymph nodes and spleen.
Our results indicate that 129Sv E-selectin-deficient mice are different from B6 E-selectin-deficient mice in the utilization of E-selectin for T cell migration. 129Sv mice and B6 mice have similar accumulation of labeled T lymphocytes in skin lesions, but T cell migration is partially decreased to lesions induced by IFN-, IFN-
+ TNF-
and Con A in E-selectin-deficient B6 mice, but only to Con A in E-selectin-deficient 129Sv mice (Fig. 3). This lack of effect of E-selectin deletion in 129Sv mice was not due to the presence of E-selectin, because T cell migration to Con A in these mice was decreased. In addition, P-selectin mAb treatment in these mice inhibited T cell migration to all the lesions tested to the same extent as in E/ B6 mice (Fig. 6). Two reasons may account for the difference in T cell migration in E-selectin-deficient mice of 129Sv and B6 backgrounds. First, there could be a strain difference such that 129Sv mice do not utilize E-selectin for T cell migration as much as do B6 mice. Ramos et al. (56) have reported differences in the effect of E-selectin blockade between BALB/c and C57BL/6 mice. Second, knocking out a gene may yield different compensatory mechanisms for the use of adhesion molecules on various backgrounds.
Our results suggest several important conclusions. (i) They demonstrate that both E-selectin and P-selectin mediate part of the T lymphocyte migration to various inflammatory stimuli, since the absence of both selectins suppressed T cell migration to some of the stimuli further than the blockade of either of the selectins alone. (ii) There is an alternate adhesion pathway capable of initiating T cell migration to skin inflammation in the mouse, since the absence of both endothelial selectins did not completely inhibit T cell migration to any of the agents tested. (iii) P-selectin function in T cell migration can be substituted by E-selectin or by another adhesion molecule, since the absence of P-selectin alone did not affect T cell migration. (iv) E-selectin function in T cell migration cannot be replaced by P-selectin or any other adhesion molecule, since a lack of E-selectin alone partially inhibited T cell migration to most stimuli tested.
In conclusion, these results show the complexity of in vivo T lymphocyte migration to inflammation and demonstrate the importance of studying adhesion molecules in the context of leukocyte migration to numerous stimuli.
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Acknowledgements |
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Abbreviations |
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CLAcutaneous lymphocyte antigen
Con Aconcanavalin A
DTHdelayed-type hypersensitivity
i.d.intradermal
TNFtumor necrosis factor
VCAMvascular cell adhesion molecule
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References |
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