By
From * The Center for Blood Research, the Department of Cardiology, and the § Department of
Pathology, Harvard Medical School, Boston, Massachusetts 02115; the
Division of Newborn
Medicine, Department of Pediatrics, Tufts University School of Medicine, Boston, Massachusetts
02111; and the ¶ Department of Pathology, University of Texas Southwestern Medical Center, Dallas,
Texas 75235-9069
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Abstract |
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Peripheral lymph nodes (PLN) are critical for immunologic memory formation in response to
antigens that penetrate the skin. Blood-borne lymphocytes first encounter such antigens after
they home to PLN through a multi-step adhesion process that is normally initiated by L-selectin (CD62L) in high endothelial venules (HEV). Since naive T cells can not enter PLN normally in L-selectin-deficient mice, a delayed type hypersensitivity response to cutaneously applied antigen cannot be mounted. In this study, we report that the administration of activated
platelets into the systemic circulation of L-selectin knockout mice restores lymphocyte trafficking to PLN, and reconstitutes T cell-mediated immunity in response to a cutaneous antigen.
These effects required platelet-expressed P-selectin that allows activated platelets to transiently
form a bridge between lymphocytes and HEV, thereby enabling lymphocytes to undergo subsequent 2 integrin-dependent firm adhesion. These profound effects of platelet-mediated
cell-cell interactions on lymphocyte trafficking and formation of immunologic memory may impact on a variety of autoimmune and inflammatory conditions.
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Introduction |
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Immunocompetent lymphocytes continuously patrol the
body in search of foreign antigen (1). To effectively do
this, a sequence of critical adhesion events that allows these
cells to migrate from the circulation into tissues must occur. In peripheral lymph nodes (PLN), the interaction of
the lymph node homing receptor, L-selectin (CD62L), with
its counterreceptor expressed on high endothelial venules
(HEV), the peripheral node addressin (PNAd), is the first
essential step in the extravasation of immunologically naive
lymphocytes (2). L-selectin-PNAd interaction mediates the tethering and rolling of most lymphocytes in HEV, but
is not sufficient by itself for lymphocyte emigration (5).
The rolling cells must first encounter an activating stimulus
that transmits a G protein-dependent transmembrane signal
and triggers stationary adhesion through functional upregulation of the 2 integrin LFA-1 (6). The importance of
L-selectin as the physiologic initiator of this adhesion cascade is best exemplified by the inability of naive T cells to
home to PLN in L-selectin-deficient mice (7, 8). In fact,
the lack of T cell trafficking through PLN in these animals
was shown to be responsible for the inability to elicit a contact hypersensitivity (CHS) immune response by cutaneous exposure to a hapten antigen (7, 9).
In addition to L-selectin, circulating activated platelets can provide a second mechanism for initiating lymphocyte interactions with the HEV of PLN (10). P-selectin (CD62P) expressed on activated platelets can mediate adhesion to both lymphocytes and HEV via interactions with P-selectin glycoprotein ligand 1 (PSGL-1) and PNAd, respectively, resulting in sustained rolling of lymphocyte-platelet aggregates even in the absence of functional L-selectin. In this study, we have set out to examine the functional significance of this platelet-mediated lymphocyte delivery to HEV. We tested whether rolling promoted by this alternative adhesion pathway is sufficient to reestablish lymphocyte trafficking in L-selectin-deficient mice and whether lymphocytes delivered in this manner to PLN were capable of participating in a CHS immune reaction. We found that lymphocytes delivered by platelets could indeed accumulate and extravasate in PLN-HEV. L-selectin-deficient lymphocytes homed to PLN via a multi-step adhesive process that was initiated by platelet P-selectin and was followed by activation of rolling lymphocytes that arrested via engagement of LFA-1. Importantly, a transfusion of activated platelets 1 d after cutaneous sensitization of L-selectin-deficient mice normalized the CHS response in these animals, suggesting that platelet-delivered T cells were immunocompetent and could efficiently detect and respond to antigen presented in PLN.
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Materials and Methods |
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Antibodies.
Function blocking anti-murine L-selectin mAb Mel-14, anti-murine LFA-1 (CD11a) mAb TIB 213, and anti- human P-selectin mAb WAPS 12.2 were provided by Dr. E.C. Butcher (Stanford University, Stanford, CA). Anti-human CD41 mAb 7E3 was a gift of Dr. B. Coller (Mt. Sinai Medical Center, New York). FITC-conjugated mAbs to murine TCRIntravital Microscopy.
Mice were anesthetized and catheterized, and the left subiliac LN was microsurgically prepared as previously described (5). Human platelets were isolated from blood of healthy donors following established procedures (11). For microscopic visualization of endogenous white blood cell (WBC) interactions with vascular endothelium, a bolus injection of saline (10 ml/kg body weight) containing 1 mg/ml of the nuclear dye Rhodamine 6G (Molecular Probes, Inc., Eugene, OR) was given intravenously. Interacting and freely flowing blood-borne WBCs were videotaped during their passage through LN HEV under fluorescent stroboscopic epi-illumination and observation through a ×40 Zeiss objective (Achroplan, numerical aperture 0.75Flow Cytometry and Platelet-PBMC Aggregation Assay.
Murine PBMCs were isolated by density gradient centrifugation using lympholyte-M (Accurate Chemical and Science Corp., Westbury, New York), washed in PBS with 1% heat-inactivated human serum albumin (HSA) and 5 mM EDTA, pH 7.4 (PBE) and subsequently resuspended in HBSS containing 10 mM Hepes, 2 mM CaCl2, and 0.2% HSA, pH 7.4 (assay media). Purified PBMCs (2 × 106/ml) in PBE were preincubated at 4°C for 30 min with FITC-conjugated antibodies that recognize murine Mac-1, TCR-Elicitation of CHS Response.
A cutaneous delayed type hypersensitivity immune response was induced by application of 0.5% 2,4-dinitrofluorobenzene (DNFB) in acetone/olive oil (4:1) on the shaved abdomina of mice as previously described (7). 18 h after sensitization, mice were anesthetized, the right external jugular vein was cannulated, and a total volume of 3 ml of TRAP-activated human platelets (108/ml) were administered intravenously over 6 h as repeated small boluses. Activation status of platelets was confirmed by flow cytometry of P-selectin expression. For antibody inhibition studies, platelets and mice were pretreated with function blocking antibodies (50 µg/ml and 100 µg/mouse, respectively) to human P-selectin (mAb WAPS 12.2) or human CD31 (mAb Hec 7). Mice were rechallenged with 0.25% of DNFB on the left pinna (10 µl) on day 5, and ear swelling responses were measured 24 h after elicitation. Subsequently, the animals were killed and lymphocytes pooled from cervical, subiliac, and axillary nodes were counted on a hemocytometer.In Vitro Lymphocyte Proliferation Assay.
For analysis of antigen-specific T cells in the PLN of mice, nodes were harvested 5 d after DNFB sensitization of mice with and without infusion of platelets and antibodies as described above. Isolated lymphocytes (2 × 104-1.6 × 105 cells) were cultured for 36 h in triplicate in the presence or absence of DNBS, the water soluble form of DNFB (50 µg/ml), and then pulsed with 1 µCi/well of [3H]thymidine for 18 h before harvesting cells as previously described (7).Statistical Methods.
Where appropriate, data are presented as the arithmetic mean ± 1 SD. A statistical analysis of the ear swelling response was performed using the Kruskal-Wallis test for one-way analysis of variance followed by multiple comparisons on ranks using the Bonferroni correction of P. Results were considered statistically significant when P <0.05. ![]() |
Results |
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The behavior of rhodamine 6G-labeled circulating WBCs in PLN-HEV of L-selectin- deficient mice was evaluated using intravital microscopy (5). In agreement with previous studies using a neutralizing antibody to L-selectin (10), the rolling fraction of WBCs in mice lacking this homing receptor (12.1 ± 5.3%, mean ± SD) was considerably reduced in comparison to that in wild-type littermates (72.1 ± 12.1%; Figs. 1 and 2 a). Injection of activated, but not resting, human platelets enhanced leukocyte rolling in the HEV of mutant mice (rolling fraction of 48.9 ± 9.6%), approaching a level similar to that seen in wild-type mice. The pivotal role of platelet P-selectin was confirmed by the ability of mAb WAPS 12.2, directed against human, but not murine, P-selectin, to abolish this effect.
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Although selectin binding to PNAd is highly
efficient at initiating rolling interactions in flow, in vitro
studies indicate that engagement of additional adhesion receptors is required for lymphocyte arrest in HEV (12).
Indeed, recent in vivo experiments have shown that L-selectin-dependent lymphocyte rolling is followed by an activation-dependent arrest mediated by LFA-1 (6). We hypothesized that in order for L-selectin-deficient lymphocytes to
accumulate in tissues where PNAd is expressed, a similar
sequence of events must occur to stabilize dynamic interactions initiated by platelet P-selectin. Infusion of activated
platelets in L-selectin-deficient mice not only restored rolling, but >50% of interacting WBCs became stably adherent to HEV, and a large fraction of stuck cells eventually transmigrated (Fig. 2, b and c). Since both WBCs and platelets express several integrin receptors that are capable of
strengthening adhesive interactions with vascular endothelial cells, mutant mice or platelets were treated with function-blocking mAb to LFA-1 (TIB 213) or to CD41 (7E3)
before platelet injection. MAb TIB 213, but not mAb 7E3,
reduced leukocyte sticking by ~90%; neither had any effect on rolling (Figs. 1 and 2 d and data not shown). Since
human platelets do not express 2-integrins and TIB 213 binds only murine, but not human LFA-1 (reference 13 and
our unpublished data), these results demonstrate that the
sticking of WBC-platelet aggregates was mediated by LFA-1
on WBCs. Therefore, the cellular bridge between lymphocytes and HEV that was initially created by platelets allowed for a critical period of exposure of lymphocytes to
chemoattractant signals on HEV that was sufficient to activate and engage LFA-1. Moreover, in intravital microscopy experiments where WBC and platelets were labeled with
different fluorescent dyes, we observed that platelets frequently returned to the circulation after the WBCs they
had delivered became firmly adherent on the surface of an
HEV (Fig. 3). This suggests that platelets may be able to
participate repeatedly in the recruitment of leukocytes.
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Although
in situ labeling of circulating WBCs with rhodamine 6G allows adequate visualization of nucleated cells in HEV, it
does not enable us to differentiate between the various leukocyte populations that may participate in antigen-specific
immune responses. To identify the specific subsets of murine lymphocytes capable of interacting with human platelets, PBMCs from wild-type or L-selectin-deficient mice
were preincubated with fluoresceinated mAbs detecting
specific antigens for either /
T cells or B cells. Cells
were then incubated with activated human platelets prelabeled with a nonblocking PE-conjugated P-selectin antibody, and platelet adhesion was quantified using two-color
flow cytometry (Fig. 4 a). For reference, platelet binding to
monocytes was also analyzed. Approximately half of all B
and T cells from both wild-type and L-selectin-deficient
mice formed aggregates with activated platelets (Fig. 4 b).
Over 98% of monocytes from either genotype also interacted with platelets. The critical role of P-selectin in mediating these interactions was determined by the ability of
mAb WAPS 12.2, but not by the nonblocking P-selectin mAb S12, to significantly reduce the percentage of all
PBMC subsets bound by platelets. These data suggest that a
substantial fraction of circulating B and T lymphocytes can
associate with activated platelets in the blood stream.
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Our results indicate that lymphocytes can use the platelet delivery mechanism to enter the LN interstitium where they may encounter their cognate antigen. Since this intravascular adhesion cascade does not mandate a contribution by L-selectin, we tested whether activated platelets in the circulation of L-selectin-deficient mice can reconstitute the defect in CHS that has been previously described in these animals (7, 9). To address this issue, the abdominal skin of mutant mice was exposed to DNFB. Topically applied antigens are known to be taken up primarily by dermal Langerhans' cells which then migrate to draining PLN where they present antigen to homed lymphocytes. Our analysis has indicated that a peak in the number of Langerhans' cells is achieved 18-24 h after sensitization (Catalina, M.D., and M.H. Siegelman, unpublished data). To ensure optimal lymphocyte delivery to PLN, mutant mice were repeatedly injected during this 6 h time interval with a total number of 3 × 108 activated platelets.
When ears of sham-treated mutant mice were rechallenged with DNFB 5 d after sensitization, there was no detectable CHS response (Fig. 5 a). Moreover, activated platelet treatment of sensitized wild-type mice did not enhance the CHS response, which, in fact, was slightly lower in this group (P <0.05 versus sensitized wild-type without platelets). In contrast, a dramatic CHS immune reaction was elicited in sensitized mutant animals that had received activated platelets. The extent of ear swelling elicited with DNFB in platelet-treated L-selectin-deficient mice was not different from that elicited in sensitized wild-type mice irrespective of whether or not the latter had received activated platelets. Pretreatment with P-selectin function-blocking mAb WAPS 12.2, but not control mAb Hec 7 to the human platelet antigen CD31, abolished the CHS response in L-selectin-deficient mice. Since WAPS 12.2 neutralizes human, but not murine, P-selectin, its inhibitory effect was most likely due to blocking of platelet-mediated lymphocyte delivery to PLN during the sensitization phase, and not a result of the inhibition of endothelial P-selectin- mediated trafficking of effector cells during the elicitation phase.
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To address whether the effect of activated platelets on the CHS response was in the afferent phase of the response, the presence of antigen-specific T cells within draining PLN of sensitized mice was determined by the ability of DNBS to induce in vitro lymphocyte proliferation (Fig. 5 b). LN T cells isolated from sensitized platelet-treated mutant mice proliferated in response to the antigen comparably to those of wild-type animals, whereas T cells from sensitized untreated L-selectin-deficient animals did not proliferate at all. Furthermore, DNFB-reactive T cells resided overwhelmingly in the PLN of both L-selectin-deficient and wild-type mice after contact sensitization, as no detectable proliferation was found in either mesenteric LNs or spleens of these animals (reference 7 and data not shown). The magnitude of the CHS response in platelet-treated mutant animals directly correlated with the number of resident lymphocytes in PLN, suggesting that the number of antigen-specific T cell clones generated during the sensitization phase determined the extent of the cutaneous reaction (Fig. 5 c).
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Discussion |
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The observation that circulating, activated platelets can promote not only adhesion of lymphocytes to HEV but also reconstitute cell-mediated immunity in L-selectin- deficient mice is important in several ways. It emphasizes the mandatory requirement for lymphocyte localization to PLN in a T cell-dependent immune response to a cutaneous antigen challenge. Moreover, it shows that L-selectin per se is not the exclusive mechanism for lymphocyte homing to PLN, at least in situations that lead to platelet activation in the blood stream. Platelets, through surface P-selectin, can provide a significant compensatory mechanism for initiating lymphocyte-HEV interactions without perturbing subsequent adhesion steps or later events such as antigen recognition or lymphocyte migration and differentiation, which are requisite for activation and clonal expansion of immunocompetent T cells.
Our intravital microscopy observations of PLN-HEV in L-selectin-deficient mice also suggest a second constitutive adhesion pathway that can mediate rolling and perhaps lymphocyte recruitment to HEV. Although impaired in their ability to interact with HEV, a minority of circulating L-selectin-deficient WBCs (~12%) were still capable of rolling, even when no activated platelets were injected. This low level interaction was also observed in wild-type mice treated with an L-selectin function-blocking antibody (10). Interestingly, treatment of L-selectin-deficient animals with a P-selectin function-blocking antibody reduced these residual lymphocyte-HEV interactions by ~75% (Diacovo, T.G., and U.H. von Andrian, unpublished data). However, it remains to be determined whether this L-selectin-independent component of rolling is mediated by endogenous P-selectin expressed on platelets, on endothelial cells, or on both.
Recent intravital microscopy studies of PLN-HEV in wild-type mice have shown that stationary adhesion of lymphocytes requires that L-selectin-mediated rolling is followed by a G protein-coupled transmembrane signal that results in the upregulation and subsequent engagement of LFA-1 (6). Consistent with these observations, our present results indicate that lymphocytes that become tethered to HEV by platelets must also activate LFA-1 before they can arrest. Although the precise mechanism(s) that control(s) the physiologic modulation of LFA-1 activity remain(s) to be defined, previous in vitro studies have implicated a role for L-selectin (14). In fact, cross-linking of L-selectin on naive, but not memory, T cells with either antibodies or the HEV-derived L-selectin ligand GlyCAM-1, has been reported to promote binding of LFA-1 to one of its counterreceptors, ICAM (intercellular adhesion molecule)-1 (17). Signal transduction through PSGL-1 has been suggested to mediate similar interactions in nonlymphoid leukocytes (18). However, platelet-mediated cross-linking of PSGL-1 on T cells did not appear to directly induce integrin activation; platelets bound to purified human peripheral blood T cells failed to convert rolling interactions to firm adherence on PNAd coimmobilized with ICAM-1 in flow assays (Diacovo, T.G., and U.H. von Andrian, unpublished data). Similarly, surface-adherent platelet monolayers supported P-selectin-dependent T cell rolling, but not firm adhesion, which has been shown to occur during platelet interactions with neutrophils (21, 22). Thus, the predominant contribution of platelets to lymphocyte trafficking appears to be the facilitation of lymphocyte margination. Although our data do not exclude more subtle platelet-mediated effects that could potentially affect integrin function on lymphocytes, it appears likely that additional direct communication events between the passively tethered lymphocyte and the vascular wall must occur to initiate the necessary downstream events that are required for lymphocyte accumulation in lymphoid or chronically inflamed tissues.
Our results demonstrate that activated platelets can have a profound effect on the formation of immunologic memory because they can significantly impact on the trafficking pattern of lymphocytes. In particular, activated platelets are likely to affect the migratory behavior of lymphocyte subsets that express little or no L-selectin. This would include populations such as activated and/or memory T cells, which play a vital role in the initiation and maintenance of inflammatory conditions. It is interesting in this context that Th1, but not Th2, cells were recently found to acquire the ability to interact with P-selectin, and this differential ability was shown to be responsible for the preferential recruitment of Th1 cells into inflamed skin and joints (23). However, it appears that P-selectin binding activity is probably not restricted to this specific subset of T cells, as our studies indicate that a much larger proportion (40-50%) of both T and B cells can bind activated platelets, at least in the murine system.
In humans, platelets may not only participate in hemostasis but may similarly modulate immune function by virtue of their ability to interact with both vascular endothelium and circulating leukocytes. This dual function of platelets may contribute to vascular pathology associated with chronic inflammation. For instance, it is conceivable that platelets may play a role in exacerbations of inflammatory bowel disease, a pathological condition associated with an increase in activated platelets in the systemic circulation (24), and endothelial cell expression of PNAd in inflamed tissues (25). Circulating, activated platelets may not only occlude the intestinal microvasculature resulting in tissue infarction, but also alter the homing pattern of T cells that play a pivotal role in the pathogenesis of this and other chronic inflammatory diseases (26).
Clinical studies suggest that intravascular platelet activation may be a frequent occurrence in other settings as well. For instance, elevated numbers of P-selectin-expressing platelets have been detected in patients with diabetes mellitus, and in healthy subjects at increased risk for developing insulin-dependent diabetes mellitus (29, 30). In these settings as well as in inflammatory bowel disease, activated platelets may be encountered in the blood stream for extended periods of time. However, our experiments indicate that even a single episode during which large numbers of activated platelets are present in the systemic circulation can affect lymphocyte trafficking. The total number of platelets that were infused into L-selectin-deficient mice during a 6-h period corresponded to ~40-50% of the circulating platelet pool in each animal. This dose of activated platelets may be clinically relevant since similar levels may be achieved as a result of platelet transfusions to individuals with thrombocytopenia. At the time of transfusion, 15-50% of platelets stored under blood bank conditions have been reported to express P-selectin, depending on the method of isolation and concentration in the transfusate (31, 32). Acute activation of circulating platelets can also occur as a consequence of surgical procedures requiring cardiopulmonary bypass or hemodialysis with bioincompatible filter membranes (33, 34). Thus, our studies simulate an extremely common clinical condition that has not been examined so far with regards to its consequences for a patient's immune system.
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Footnotes |
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Received for publication 22 July 1997 and in revised form 6 November 1997.
Address corrrespondence to Dr. Ulrich H. von Andrian, The Center for Blood Research, 200 Longwood Ave., Boston, MA 02115. Phone: 617-278-3130; Fax: 617-278-3190; E-mail: uva{at}cbr.med.harvard.eduWe wish to thank Drs. E.C. Butcher, B. Coller, R.P. McEver, and W. Muller for supplying antibodies.
This work was supported by National Institutes of Health grants K08 HL-03361 (to T.G. Diacovo); HL-54936, HL-48675, and HL-56949 (to U.H. von Andrian); and CA-56746 and HL-56746 (to M.H. Siegelman); and a grant from the Arthritis Foundation (to M.H. Siegelman). M.H. Siegelman is an Established Investigator of the American Heart Association.
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