Cell, Vol. 67, 1033-1036,
December
20, 1991, Copyright
0 1991 by Cell Press
Leukocyte-Endothelial Cell Recognition: Three (or More) Steps to Specificity and Diversity Eugene C. Butcher Laboratory of immunology and Vascular Biology Department of Pathology Stanford University Medical Center Stanford, California 943056324 Center for Molecular Biology in Medicine Veterans Administration Medical Center Palo Alto, California 94304
The recruitment of leukocytes from the blood is one of the most dramatic cellular responses to tissue damage and inflammation, and is central to the physiologic trafficking of lymphocytes. Leukocyte extravasation is exquisitely regulated in vivo by mechanisms of selective leukocyteendothelial cell (EC) recognition, which can display extraordinaryspecificityin relation to the inflammatorystimulus, the stage of the inflammatory response, and the tissue site or organ involved. Examples include the almost exclusive attachment of eosinophils to venules in allergic reactions, the specific recruitment of neutrophils early in acute inflammation, and the tissue-selective interaction of lymphocyte subsets with high endothelial venules (HEVs) in organized lymphoid tissues. Adhesion receptors (ARs) mediate and help direct leukocyte-EC interactions (Figure 1; reviewed in Springer, 1990; Pober and Cotran, 1990; Berg et al., 1991). Paradoxically, however, individual receptors often participate in multiple leukocyte-EC interactions that are quite independently regulated in vivo. For example, the vascular E-selectin (ELAM-1) binds both neutrophils and skin-homing memory T cells, yet is thought to support selective recruitment of neutrophils during acute inflammation and of cutaneous memory T cells during chronic inflammation in the skin (Picker et al., 1991a, and references therein). Such observations cannot be explained by simple lockand-key models of cell-cell recognition, and seem to require a more complex control of leukocyte-EC interactions in vivo. Here we explore a general model in which leukocyteEC recognition is viewed as an active process requiring at least three sequential events (Figure 2). First, interaction is initiated by binding of constitutively functional leukocyte ARs to EC counterreceptors. In the best characterized examples, such primary adhesion is mediated by loctincarbohydrate interactions involving leukocyte or vascular “selectins” and their cognate oligosaccharide ligands (see Figure 1). This initial adhesion is transient and reversible, unless followed by a second event-activation of the leukocyte by specific chemoattractant or cell contact-mediated signals capable of triggering secondary ARs whose function is activation dependent. Interaction of the activation-dependent AR with its EC counterreceptor, the third step, results in strong, sustained attachment, completing the process of recognition. The best characterized activation-dependent ARs are heterodimeric integrins of the p2 (CD18) or pl (CD29) classes.
Minireview
The model implies that leukocyte-EC recognition and extravasation can be controlled at any one of the three steps involved; it thus provides a combinatorial mechanism for generating both specificity and diversity in leukocyte-EC interactions. Neutrophil-EC Interaction As a Paradigm: Step 1. Reversible “Rolling” Mediated by the LGelectin Each of the three steps-reversible adhesion, leukocyte activation, and activationdependent binding-can be visualized during neutrophil interactions with venules in acute inflammation. Within minutes after tissue injury, neutrophils begin to interact loosely with the walls of venules, “rolling”along affected segments. This rolling is inhibited by antibody or Fab fragments to neutrophil L-selectin (LECAM-1, LAM-l, the MEL-14 antigen) (von Andrian et al., 1991) and byasolublechimeraof L-selectin and immunoglobulin (Ley et al., 1991). Interestingly, in vitro studies suggest that L-selectin mediates rolling in part by pre-
Step 1. Primary
Adhesion
Pathways
LeukoCYle
Lect!n:Carbohydrate L-selectin CLA slalyl L&W x sialyl Lewis x Other ?
Endothelium Lymph node addressln E-selectin (ELAM-1) E-selectin P-selectin (GMP140. CD62)
CL,N. M) (smTL) (N. W (N, W
Mucosal addressin
Step 2. ChemoatIractant/Activating lntercnne Fam~ty Interleukin-8 hMGSAIGROa Platelet factor 4 RANTES HuMIP-la HuMIP-1P l-309 Monocyte chemoattractant Others Lpds Platelet activating factor Leukotrlene 64 Others Other Chemoattractants c5a Formyl pephdes Interleukin-2 Ce// Contact-Medfated E-selectln binding CD44
(N. U W) W W WL. M)
protein-
Step 3. Activation-Dependent &L&&y& tntegrins LFA-1 (al&Z) Mac-l (aMP2) ~150.95 (aXp2) VLA-4 (a4pl)
Figure Factors
Factors
(CDS+ TL, BL) (vTL, ?M) W) CM)
WI (N. W (N) W W @TU N (6 Adhesion
Pathways
Fndothelium (L>N, M) (N, M, sL) (N, M, sL) (M. mL>vL)
ICAM-1, ICAM-P ICAM-1, others ? VCAM-1
1. Adhesion Receptor-Counterreceptor Pairs Known or Likely to Participate in Leukocyte-EC
and Activating Interactions
This is not a comprehensive list; CD44, LPAM-1 (urlpp), and HEBFpp are not listed as ARs, for example, because their role in primary versus activation-dependent adhesion is not known. Indicated in parentheses are the leukocyte subsets known to express an adhesion receptor or respond to an activating factor. N, neutrophils; M, monocytes; L, lymphocytes; EL, B lymphocytes; vTL, virgin T lymphocytes; mTL, memory T lymphocytes; sTL, a subset of T lymphocytes.
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Figure 2. Model of Leukocyte-Endotheliai Cell Recognition as an Active, Three-Step Process In step 2, a soluble chemoattractant activating factor is depicted, but EC surface molecules may also mediate leukocyte activation.
senting neutrophil carbohydrate ligands (e.g., sialyl Lewis”) to the vascular E- and P-selectins (ELAM-1 and GMP140); L-selectin seems well suited to this role because it is highly glycosylated and uniquely localized to microvillous sites of initial cellular contact (Picker et al., 1991 b). Importantly, neutrophil L-selectin is constitutively functional. It is present at high levels on circulating, resting neutrophils and mediates their attachment to cytokinestimulated ECs under flow conditions and in the absence of neutrophil activation. Rolling greatly slows the transit of neutrophils through inflamed venules; this may allow time for the neutrophil to sample the local environment or the EC surface for activating or chemoattractant signals. Step 2. Leukocyte Activation: Rolling Neutrophik Can Be Activated in the Vascular Lumen Neutrophils activated by chemoattractants undergo dramatic and rapid changes, including the shedding of L-selectin from the cell surface (Kishimoto et al., 1989). Conversely, the function and surface expression of the an activation-dependent leukocyte integrin Mac-l (a&), AR (Figure l), are rapidly up-regulated (reviewed in Springer, 1990). lmmunohistologic studies show that these changes, and hence neutrophil activation, occur during neutrophil interactions with inflamed venular endothelium in vivo (Kishimoto et al., 1989). The specific factor(s) responsible for activation of rolling neutrophils in situ is unknown and may vary with the physiologic setting (see Figure 1). Step 3. Stable Blnding to the Endothelium Involves the Activation-Dependent Integrin, Mac-l In the absence of activating signals, Mac-l and other leukocyte integrins are thought to be largely inactive (e.g., see Lawrence and Springer, 1991). Consistent with this, monoclonal antibodies to the leukocyte integrin 82 chain have no effect on primary neutrophil rolling in inflamed venules. However, in the presence of anti-82 antibodies, neutrophils are released to return to the circulation; the antibodies completely prevent the arrest and stable attachment of rolling cells (Arfors et al., 1987). Thus, in situ studies confirm the involvement of reversible primary adhesion, leukocyte activation, and secondary activationtriggered attachment in neutrophil-EC recognition.
The requirement for each of the three steps has been elegantly modeled in vitro by neutrophil interactions with either activated cultured endothelium (Smith et al., 1991) or purified EC counterreceptors (Lawrence and Springer, 1991). Under flow conditions, neutrophils adhere reversibly and roll on glass surfaces coated with the P-selectin, a primary adhesion ligand. In contrast, moving neutrophils are unable to interact with surfaces bearing ICAM-1, a ligand for the activation-dependent 82 integrins, even if the neutrophil integrins are preactivated. However, the entire recognition process can be modeled on slides coated with both P-selectin and ICAM- : activation of rolling neutrophils with a phorbol ester results in their rapid arrest and firm adhesion through integrin binding to ICAM-1. The findings emphasize the essential and sequential roles of primary adhesion, activation, and activation-dependent adhesion in the recognition process. Generalization to Other Leukocyte-EC Interactions? Like neutrophils, monocytes express primary ARs (e.g., the leukocyte selectin and carbohydrate ligands for the vascular selectins) and activation-dependent integrins, including Mac-l. Moreover, monocyte recruitment from the blood into the inflamed peritoneum, like neutrophil extravasation, is inhibited by both anti-L-selectin and anti-Mac-l antibodies (Jutila et al., 1988). This parallel with neutrophils suggests that monocyte extravasation also involves both activation-independent and -dependent steps. Specificity of neutrophil versus monocyte recruitment may be determined in part by their activating signals: neutrophils and monocytes respond differentially to several chemoattractants (Figure 1). Monocytes also express more 81 integrin WA-4 than neutrophils and thus may be able to bind preferentially to ECs via the inducible EC ligand VCAM-1. Lymphocyte-EC interactions also utilize both activation-independent and dependent Al%. Lymphocyte homing into peripheral lymph nodes is targeted by primary interaction of the L-selectin with carbohydrates of the peripheral node HEV addressin; homing to intestinal Peyer’s patches may involve by primary adhesion to the mucosal vascular addressin (Berg et al., 1991). Trafficking to both these lymphoid tissues involves the activation-dependent integrin LFA-1 (Hamann et al., 1988). a4 integrins also participate in lymphocyte binding to Peyer’s patch HEVs (Holzmann et al., 1989), but it is unknown whether their role is activation dependent or independent. Importantly, in situ observations of lymphocyte-HEV interactions are consistent with reversible primary and stabilizing secondary adhesion events (Bjerknes et al., 1988). Lymphocyte binding in vitro to cytokine-stimulated ECs also involves activation-independent and -dependent mechanisms. Interaction of memory T cells with interleukin-l-stimulated ECs, for example, can involve both the primary carbohydrate:E-selectin pathway and the activation-triggered LFA-1 and VLA4 integrin pathways (Picker et al., 1991a; Shimizu et al., 1991). Finally, a number of cell surface and secreted activating factors act on distinct subsets of mononuclear cells, including lymphocytes, and may contribute to the selectivity of activation (step 2) in lymphocyte-EC recognition (Figure 1). Signaling via engagement or cross-linking of some lym-
phocyte adhesion receptors may be important in this context (e.g., CD44; Koopman, 1990). Of particular interest is the intercrine family of chemoattractants (reviewed by Schall, 1991; Oppenheim et al., 1991). Some members of this family are chemotactic for memory T cells, others for virgin T cells, CD8+ T cells, or B cells. Some attract neutrophils or monocytes preferentially (Figure 1). Many remain to be tested on lymphocyte subsets. Interestingly, many intercrines bind heparin, which suggests that they may be presented to leukocytes either as soluble chemoattractants or as immobilized molecules bound to glycosaminoglycans of the EC surface. Although more direct tests of the model will be required in each case, these considerations suggest that reversible adhesion, activation, and activation-dependent binding may prove to be common to many or all leukocyte-EC recognition events. lmpiications of the Mode/: Combinatorial Diversity in Leukocyte-EC Recognition If, as the model implies, the specificity of leukocyte-EC recognition is determined by unique combinations of ARligand or activating receptor-ligand pairs, the maximum number of specific recognition events is the product of the number of possible receptor-ligand pairs at each step, For example, if each of the molecules in Figure 1 were expressed on unique subsets of leukocytes or ECs, there could be as many as 300 (5 x 15 x 4) independent recognition events. Such highly restricted expression is not typical in vivo (see below); but the calculation emphasizes the importance of a multistep mechanism for generating diversity in cellular recognition. Successful Receptor-Llgand Pairs Can Be Used in Multiple Specific Leukocyte-EC Recognition Events The multistep model of recognition permits a given receptor to be involved in more than one recognition event without sacrificing specificity. The ability to use highly effective receptors such as selectins or leukocyte integrins in multiple settings may in fact be one of the most important evolutionary advantages of the multistep recognition process. This concept is supported by the observation that the known receptor-ligand pairs including those listed in Figure 1 are rarely (perhaps never) limited to a single leukocyte-EC recognition event. Involvement of the vascular E-selectin in both T cell and neutrophil traffic was mentioned previously. Similarly, L-selectin supports both lymphocyte and neutrophil binding to peripheral lymph node HEVs, yet only lymphocytes adhere stably to HEVs and enter lymph nodes from the blood (reviewed in Jutila et al., 1988). The utilization of the activation-dependent ARs and their ligands is even more widespread. In fact, leukocyte integrins mediate a wide variety of cell-cell and cell-substrate adhesion events (reviewed in Springer, 1990). Specificity of the Recognition Process Can Exceed That of its Component Steps Although expression of receptors or ligands on leukocyte subsets and on ECs in different vascular beds can be very restricted, it just as often differs quantitatively more than qualitatively. Furthermore, cells can express several primary or secondary ARs at the same time (Figure 1). Such
patterns of expression may reflect an evolutionary balance between the needs for specificity and redundancy in cellcell recognition mechanisms. In this context, a predicted strength of the multistep process is that the specificity of leukocyte-EC recognition and extravasation can exceed that of any of its components. For example, if in a given inflamed vessel, leukocyte A had four times the probability of interacting effectively at each of the three steps compared with leukocyte B, then leukocyte A would extravasate 84 times more often. The model also predicts that recognition is inherently resilient. If, for example, selectivity for leukocyte A versus B were somehow lost at one step (i.e., if leukocyte B now expressed a receptor previously restricted to leukocyte A), leukocyte A could still home 18 times better than leukocyte B. New Homing Specificities from Novel Combinations of Existing ARs and Chemoattractant-Receptor Pairs Another implication of the combinatorial determination of specificity is that novel recognition events can be generated without evolving new adhesion receptors. Instead, new recognition specificities can be selected merely by expressing existing receptor pairs in novel combinations. Comments and Caveats In the simplest form of the model, receptor-ligand pairs in each step of the recognition process are distinct. In some instances, however, molecules may serve more than one role in the process. For example, engagement or crosslinking of primary adhesion receptors on the leukocyte could itself deliver an integrin-activating signal, as proposed for activation of neutrophils by the vascular E-selectin (Lo et al., 1991). Furthermore, certain adhesion receptors can apparently alter their affinity (e.g., the L-selectin; Spertini et al., 1991) and even their ligand specificity upon activation of the cells expressing them. For example, the integrin GPllb-llla binds immobilized fibronectin constitutively but displays induced adhesiveness for other ligands upon platelet activation (Phillips et al., 1991). Thus, some molecules may participate in leukocyte-EC recognition as both primary and secondary adhesion receptors, with overlapping or distinct EC ligands at each step. There may also be additional steps in EC recognition beyond the three emphasized here. For example, specific signal transduction linkages could exist between activating receptors and activation-dependent ARs, allowing independent triggering of particular integrins by different activating signals. Such mechanisms would permit even more combinatorial diversity in leukocyte-EC recognition. Events following EC recognition may also regulate leukocyte recruitment. Activation-dependent adhesion via leukocyte integrins, although stable under physiologic shear forces and sustained for minutes, is eventually reversible. For example, neutrophils bound by Mac-lmediated adhesion to cultured ECs are released spontaneously after lo-15 min (Lo et al., 1989). This reversibility suggests that extravasation need not obligatorily follow EC recognition, although these events could be linked in some settings. For example, a common chemoattractant arriving in the vessel from an extravascular source could be the signal both for activation-dependent adhesion to ECs and for diapedesis and migration into the tissues. If,
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instead, the activating signal of step 2 in EC recognition were provided by the endothelium itself, then independent tissue-derived chemoattractant gradients might be required for extravasation. Thus, in some settings extravascular signals may determine whether a leukocyte bound to the vascular endothelium eventually extravasates or instead is freed to return to the circulation. This adds another potential step to the control of leukocyte recruitment. Finally, although this discussion has been limited to those events that follow initial leukocyte encounter with vascular endothelium, the regulation of leukocyte extravasation and recruitment in vivo begins earlier. The properties of the endothelium and the array of local leukocyteactivating factors that control the recognition process must be determined in advance as a function of the local tissue microenvironment and of inflammatory stimuli (reviewed in Pober and Cotran, 1990). Concluding Remarks In conclusion, a model has been presented of leukocyteEC recognition as an active process involving three or more sequential events leading from the initial encounter to stable attachment. It is likely that many variations on this theme exist, only some of which have been considered. The most important implications of the model, however, derive less from the specifics of the individual steps than from the concept that recognition and stable attachment require several sequential events, each representing a go/no go decision point. It is the involvement of several steps, with multiple alternative receptor-ligand pairs available at each one, that provides a combinatorial mechanism for generating both diversity and specificity in leukocyte-EC recognition, that permits molecular conservatism by allowing individual receptor-ligand pairs to participate in more than one leukocyte homing event, and that implies that new homing specificities can be generated during evolution through the use of existing receptor-ligand pairs in novel combinations. References Arfors, K. E., Lundberg, C., Lindborm, L., Lundberg, K., Beatty, P. G., and Harlan, J. M. (1967). Blood 69, 336-346. Berg, E. L., Picker, L. J., Robinson, M. K., Streeter, P. Ft., and Butcher, E. C. (1991). In Vascular Adhesion Molecules, Cellular and Molecular Mechanisms of Inflammation, Volume 2, C. Cochrane and M. A. Gimbrone, Jr., eds. (San Diego: Academic Press), pp. 111-129. Bjerknes, M., Cheng, H.. and Ottaway, C. (1966). Science 23, 402405. Hamann, A., Jablonski-Westrich, D., Duijvestijn, A., Butcher, E. C., Baisch, H., Harder, R., and Thiele, H.-G. (1966). J. Immunol. 140,693699. Holzmann, B., McIntyre, B. W., and Weissman. I. L. (1969). Cell 56, 37-46. Jutila, M. A., Lewinsohn, D. M., Berg, E., and Butcher, E. C. (1988). In Leukocyte Adhesion Molecules: Structure, Function, and Regulation, T. A. Springer, ed. (New York: Springer-Verlag), pp. 227-235. Kishimoto, T. K., Jutila, M. A., Berg, E. L., and Butcher, E C. (1969). Science 245,1236-1241. Koopman, G., van Kooyk, Y., de Graaff, M., Meyers, C. J. L. M., Figdor, C. G., and Pals, S. T. (1999). J. Immunol. 145, 3569-3593. Lawrence, M. B., and Springer, T. A. (1991). Cell 65, 659-673. Ley, K., Gaethgens, P., Fennie, C., Singer, M. S., Lasky, L. A., and Rosen, S. D. (1991). Blood 77, 2553-2555.
Lo, S. K., Detmers, P. A., Levin, S. M., and Wright, S. D. (1969). J. Exp. Med. 169, 1779-1793. Lo, S. K., Lee, S., Ramos, R. A., Lobb, R., Rosa, M., Chi-Rosso. G., and Wright, S. D. (1991). J. Exp. Med. 773, 1493-1566. Oppenheim, J. J., Zachariae, C. 0. C., Mukaida, N., and Matsushima. K. (1991). Annu. Rev. Immunol. 9, 617-646. Phillips, D. R., Charo, I. F., and Scarborough, R. M. (1991). Cell 65. 359362. Picker, L. J., Kishimoto, T. K., Smith, C. W., Warnock, R. A., and Butcher, E. C. (1991a). Nature 349, 796-799. Picker, L. J., Warnock, R. A., Burns, A. Ft., Doerschuk, C. M., Berg, E. L., and Butcher, E. C. (1991b). Cell 66, 921-933. Pober, J. S., and Cotran, R. S. (1996). Transplantation 50, 537-544. Schall, T. J. (1991). Cytokine 3, 1-16. Shimizu, Y., Newman, W., Gopal, T. V., Horgan, K. J., Graber. N., Beall, L. D., van Seventer, G. A., and Shaw, S. (1991). J. Cell Biol. 7 13, 1203-1212. Smith, C. W., Kishimoto, T. K., Abbass, O., Hughes, B., Rothlein, FL, Mclntire, L. V., Butcher, E. C., and Anderson, D. C. (1991). J. Clin. Invest. 87,669-618. Spertini, O., Kansas, G. S., Munro, J. M., Griffin, J. D., and Tedder, T. F. (1991). Nature 349. 691-694 Springer, T. A. (1996). Nature 348, 426-433. von Andrian, U. H., Chambers, J. D., McEvoy, L., Bargatze, R. F., Arfors, K.-E., and Butcher, E. C. (1991). Proc. Natl. Acad. Sci. USA 87, 7536-7542.
December
20, 1991, Copyright
0 1991 by Cell Press
Leukocyte-Endothelial Cell Recognition: Three (or More) Steps to Specificity and Diversity Eugene C. Butcher Laboratory of immunology and Vascular Biology Department of Pathology Stanford University Medical Center Stanford, California 943056324 Center for Molecular Biology in Medicine Veterans Administration Medical Center Palo Alto, California 94304
The recruitment of leukocytes from the blood is one of the most dramatic cellular responses to tissue damage and inflammation, and is central to the physiologic trafficking of lymphocytes. Leukocyte extravasation is exquisitely regulated in vivo by mechanisms of selective leukocyteendothelial cell (EC) recognition, which can display extraordinaryspecificityin relation to the inflammatorystimulus, the stage of the inflammatory response, and the tissue site or organ involved. Examples include the almost exclusive attachment of eosinophils to venules in allergic reactions, the specific recruitment of neutrophils early in acute inflammation, and the tissue-selective interaction of lymphocyte subsets with high endothelial venules (HEVs) in organized lymphoid tissues. Adhesion receptors (ARs) mediate and help direct leukocyte-EC interactions (Figure 1; reviewed in Springer, 1990; Pober and Cotran, 1990; Berg et al., 1991). Paradoxically, however, individual receptors often participate in multiple leukocyte-EC interactions that are quite independently regulated in vivo. For example, the vascular E-selectin (ELAM-1) binds both neutrophils and skin-homing memory T cells, yet is thought to support selective recruitment of neutrophils during acute inflammation and of cutaneous memory T cells during chronic inflammation in the skin (Picker et al., 1991a, and references therein). Such observations cannot be explained by simple lockand-key models of cell-cell recognition, and seem to require a more complex control of leukocyte-EC interactions in vivo. Here we explore a general model in which leukocyteEC recognition is viewed as an active process requiring at least three sequential events (Figure 2). First, interaction is initiated by binding of constitutively functional leukocyte ARs to EC counterreceptors. In the best characterized examples, such primary adhesion is mediated by loctincarbohydrate interactions involving leukocyte or vascular “selectins” and their cognate oligosaccharide ligands (see Figure 1). This initial adhesion is transient and reversible, unless followed by a second event-activation of the leukocyte by specific chemoattractant or cell contact-mediated signals capable of triggering secondary ARs whose function is activation dependent. Interaction of the activation-dependent AR with its EC counterreceptor, the third step, results in strong, sustained attachment, completing the process of recognition. The best characterized activation-dependent ARs are heterodimeric integrins of the p2 (CD18) or pl (CD29) classes.
Minireview
The model implies that leukocyte-EC recognition and extravasation can be controlled at any one of the three steps involved; it thus provides a combinatorial mechanism for generating both specificity and diversity in leukocyte-EC interactions. Neutrophil-EC Interaction As a Paradigm: Step 1. Reversible “Rolling” Mediated by the LGelectin Each of the three steps-reversible adhesion, leukocyte activation, and activationdependent binding-can be visualized during neutrophil interactions with venules in acute inflammation. Within minutes after tissue injury, neutrophils begin to interact loosely with the walls of venules, “rolling”along affected segments. This rolling is inhibited by antibody or Fab fragments to neutrophil L-selectin (LECAM-1, LAM-l, the MEL-14 antigen) (von Andrian et al., 1991) and byasolublechimeraof L-selectin and immunoglobulin (Ley et al., 1991). Interestingly, in vitro studies suggest that L-selectin mediates rolling in part by pre-
Step 1. Primary
Adhesion
Pathways
LeukoCYle
Lect!n:Carbohydrate L-selectin CLA slalyl L&W x sialyl Lewis x Other ?
Endothelium Lymph node addressln E-selectin (ELAM-1) E-selectin P-selectin (GMP140. CD62)
CL,N. M) (smTL) (N. W (N, W
Mucosal addressin
Step 2. ChemoatIractant/Activating lntercnne Fam~ty Interleukin-8 hMGSAIGROa Platelet factor 4 RANTES HuMIP-la HuMIP-1P l-309 Monocyte chemoattractant Others Lpds Platelet activating factor Leukotrlene 64 Others Other Chemoattractants c5a Formyl pephdes Interleukin-2 Ce// Contact-Medfated E-selectln binding CD44
(N. U W) W W WL. M)
protein-
Step 3. Activation-Dependent &L&&y& tntegrins LFA-1 (al&Z) Mac-l (aMP2) ~150.95 (aXp2) VLA-4 (a4pl)
Figure Factors
Factors
(CDS+ TL, BL) (vTL, ?M) W) CM)
WI (N. W (N) W W @TU N (6 Adhesion
Pathways
Fndothelium (L>N, M) (N, M, sL) (N, M, sL) (M. mL>vL)
ICAM-1, ICAM-P ICAM-1, others ? VCAM-1
1. Adhesion Receptor-Counterreceptor Pairs Known or Likely to Participate in Leukocyte-EC
and Activating Interactions
This is not a comprehensive list; CD44, LPAM-1 (urlpp), and HEBFpp are not listed as ARs, for example, because their role in primary versus activation-dependent adhesion is not known. Indicated in parentheses are the leukocyte subsets known to express an adhesion receptor or respond to an activating factor. N, neutrophils; M, monocytes; L, lymphocytes; EL, B lymphocytes; vTL, virgin T lymphocytes; mTL, memory T lymphocytes; sTL, a subset of T lymphocytes.
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Figure 2. Model of Leukocyte-Endotheliai Cell Recognition as an Active, Three-Step Process In step 2, a soluble chemoattractant activating factor is depicted, but EC surface molecules may also mediate leukocyte activation.
senting neutrophil carbohydrate ligands (e.g., sialyl Lewis”) to the vascular E- and P-selectins (ELAM-1 and GMP140); L-selectin seems well suited to this role because it is highly glycosylated and uniquely localized to microvillous sites of initial cellular contact (Picker et al., 1991 b). Importantly, neutrophil L-selectin is constitutively functional. It is present at high levels on circulating, resting neutrophils and mediates their attachment to cytokinestimulated ECs under flow conditions and in the absence of neutrophil activation. Rolling greatly slows the transit of neutrophils through inflamed venules; this may allow time for the neutrophil to sample the local environment or the EC surface for activating or chemoattractant signals. Step 2. Leukocyte Activation: Rolling Neutrophik Can Be Activated in the Vascular Lumen Neutrophils activated by chemoattractants undergo dramatic and rapid changes, including the shedding of L-selectin from the cell surface (Kishimoto et al., 1989). Conversely, the function and surface expression of the an activation-dependent leukocyte integrin Mac-l (a&), AR (Figure l), are rapidly up-regulated (reviewed in Springer, 1990). lmmunohistologic studies show that these changes, and hence neutrophil activation, occur during neutrophil interactions with inflamed venular endothelium in vivo (Kishimoto et al., 1989). The specific factor(s) responsible for activation of rolling neutrophils in situ is unknown and may vary with the physiologic setting (see Figure 1). Step 3. Stable Blnding to the Endothelium Involves the Activation-Dependent Integrin, Mac-l In the absence of activating signals, Mac-l and other leukocyte integrins are thought to be largely inactive (e.g., see Lawrence and Springer, 1991). Consistent with this, monoclonal antibodies to the leukocyte integrin 82 chain have no effect on primary neutrophil rolling in inflamed venules. However, in the presence of anti-82 antibodies, neutrophils are released to return to the circulation; the antibodies completely prevent the arrest and stable attachment of rolling cells (Arfors et al., 1987). Thus, in situ studies confirm the involvement of reversible primary adhesion, leukocyte activation, and secondary activationtriggered attachment in neutrophil-EC recognition.
The requirement for each of the three steps has been elegantly modeled in vitro by neutrophil interactions with either activated cultured endothelium (Smith et al., 1991) or purified EC counterreceptors (Lawrence and Springer, 1991). Under flow conditions, neutrophils adhere reversibly and roll on glass surfaces coated with the P-selectin, a primary adhesion ligand. In contrast, moving neutrophils are unable to interact with surfaces bearing ICAM-1, a ligand for the activation-dependent 82 integrins, even if the neutrophil integrins are preactivated. However, the entire recognition process can be modeled on slides coated with both P-selectin and ICAM- : activation of rolling neutrophils with a phorbol ester results in their rapid arrest and firm adhesion through integrin binding to ICAM-1. The findings emphasize the essential and sequential roles of primary adhesion, activation, and activation-dependent adhesion in the recognition process. Generalization to Other Leukocyte-EC Interactions? Like neutrophils, monocytes express primary ARs (e.g., the leukocyte selectin and carbohydrate ligands for the vascular selectins) and activation-dependent integrins, including Mac-l. Moreover, monocyte recruitment from the blood into the inflamed peritoneum, like neutrophil extravasation, is inhibited by both anti-L-selectin and anti-Mac-l antibodies (Jutila et al., 1988). This parallel with neutrophils suggests that monocyte extravasation also involves both activation-independent and -dependent steps. Specificity of neutrophil versus monocyte recruitment may be determined in part by their activating signals: neutrophils and monocytes respond differentially to several chemoattractants (Figure 1). Monocytes also express more 81 integrin WA-4 than neutrophils and thus may be able to bind preferentially to ECs via the inducible EC ligand VCAM-1. Lymphocyte-EC interactions also utilize both activation-independent and dependent Al%. Lymphocyte homing into peripheral lymph nodes is targeted by primary interaction of the L-selectin with carbohydrates of the peripheral node HEV addressin; homing to intestinal Peyer’s patches may involve by primary adhesion to the mucosal vascular addressin (Berg et al., 1991). Trafficking to both these lymphoid tissues involves the activation-dependent integrin LFA-1 (Hamann et al., 1988). a4 integrins also participate in lymphocyte binding to Peyer’s patch HEVs (Holzmann et al., 1989), but it is unknown whether their role is activation dependent or independent. Importantly, in situ observations of lymphocyte-HEV interactions are consistent with reversible primary and stabilizing secondary adhesion events (Bjerknes et al., 1988). Lymphocyte binding in vitro to cytokine-stimulated ECs also involves activation-independent and -dependent mechanisms. Interaction of memory T cells with interleukin-l-stimulated ECs, for example, can involve both the primary carbohydrate:E-selectin pathway and the activation-triggered LFA-1 and VLA4 integrin pathways (Picker et al., 1991a; Shimizu et al., 1991). Finally, a number of cell surface and secreted activating factors act on distinct subsets of mononuclear cells, including lymphocytes, and may contribute to the selectivity of activation (step 2) in lymphocyte-EC recognition (Figure 1). Signaling via engagement or cross-linking of some lym-
phocyte adhesion receptors may be important in this context (e.g., CD44; Koopman, 1990). Of particular interest is the intercrine family of chemoattractants (reviewed by Schall, 1991; Oppenheim et al., 1991). Some members of this family are chemotactic for memory T cells, others for virgin T cells, CD8+ T cells, or B cells. Some attract neutrophils or monocytes preferentially (Figure 1). Many remain to be tested on lymphocyte subsets. Interestingly, many intercrines bind heparin, which suggests that they may be presented to leukocytes either as soluble chemoattractants or as immobilized molecules bound to glycosaminoglycans of the EC surface. Although more direct tests of the model will be required in each case, these considerations suggest that reversible adhesion, activation, and activation-dependent binding may prove to be common to many or all leukocyte-EC recognition events. lmpiications of the Mode/: Combinatorial Diversity in Leukocyte-EC Recognition If, as the model implies, the specificity of leukocyte-EC recognition is determined by unique combinations of ARligand or activating receptor-ligand pairs, the maximum number of specific recognition events is the product of the number of possible receptor-ligand pairs at each step, For example, if each of the molecules in Figure 1 were expressed on unique subsets of leukocytes or ECs, there could be as many as 300 (5 x 15 x 4) independent recognition events. Such highly restricted expression is not typical in vivo (see below); but the calculation emphasizes the importance of a multistep mechanism for generating diversity in cellular recognition. Successful Receptor-Llgand Pairs Can Be Used in Multiple Specific Leukocyte-EC Recognition Events The multistep model of recognition permits a given receptor to be involved in more than one recognition event without sacrificing specificity. The ability to use highly effective receptors such as selectins or leukocyte integrins in multiple settings may in fact be one of the most important evolutionary advantages of the multistep recognition process. This concept is supported by the observation that the known receptor-ligand pairs including those listed in Figure 1 are rarely (perhaps never) limited to a single leukocyte-EC recognition event. Involvement of the vascular E-selectin in both T cell and neutrophil traffic was mentioned previously. Similarly, L-selectin supports both lymphocyte and neutrophil binding to peripheral lymph node HEVs, yet only lymphocytes adhere stably to HEVs and enter lymph nodes from the blood (reviewed in Jutila et al., 1988). The utilization of the activation-dependent ARs and their ligands is even more widespread. In fact, leukocyte integrins mediate a wide variety of cell-cell and cell-substrate adhesion events (reviewed in Springer, 1990). Specificity of the Recognition Process Can Exceed That of its Component Steps Although expression of receptors or ligands on leukocyte subsets and on ECs in different vascular beds can be very restricted, it just as often differs quantitatively more than qualitatively. Furthermore, cells can express several primary or secondary ARs at the same time (Figure 1). Such
patterns of expression may reflect an evolutionary balance between the needs for specificity and redundancy in cellcell recognition mechanisms. In this context, a predicted strength of the multistep process is that the specificity of leukocyte-EC recognition and extravasation can exceed that of any of its components. For example, if in a given inflamed vessel, leukocyte A had four times the probability of interacting effectively at each of the three steps compared with leukocyte B, then leukocyte A would extravasate 84 times more often. The model also predicts that recognition is inherently resilient. If, for example, selectivity for leukocyte A versus B were somehow lost at one step (i.e., if leukocyte B now expressed a receptor previously restricted to leukocyte A), leukocyte A could still home 18 times better than leukocyte B. New Homing Specificities from Novel Combinations of Existing ARs and Chemoattractant-Receptor Pairs Another implication of the combinatorial determination of specificity is that novel recognition events can be generated without evolving new adhesion receptors. Instead, new recognition specificities can be selected merely by expressing existing receptor pairs in novel combinations. Comments and Caveats In the simplest form of the model, receptor-ligand pairs in each step of the recognition process are distinct. In some instances, however, molecules may serve more than one role in the process. For example, engagement or crosslinking of primary adhesion receptors on the leukocyte could itself deliver an integrin-activating signal, as proposed for activation of neutrophils by the vascular E-selectin (Lo et al., 1991). Furthermore, certain adhesion receptors can apparently alter their affinity (e.g., the L-selectin; Spertini et al., 1991) and even their ligand specificity upon activation of the cells expressing them. For example, the integrin GPllb-llla binds immobilized fibronectin constitutively but displays induced adhesiveness for other ligands upon platelet activation (Phillips et al., 1991). Thus, some molecules may participate in leukocyte-EC recognition as both primary and secondary adhesion receptors, with overlapping or distinct EC ligands at each step. There may also be additional steps in EC recognition beyond the three emphasized here. For example, specific signal transduction linkages could exist between activating receptors and activation-dependent ARs, allowing independent triggering of particular integrins by different activating signals. Such mechanisms would permit even more combinatorial diversity in leukocyte-EC recognition. Events following EC recognition may also regulate leukocyte recruitment. Activation-dependent adhesion via leukocyte integrins, although stable under physiologic shear forces and sustained for minutes, is eventually reversible. For example, neutrophils bound by Mac-lmediated adhesion to cultured ECs are released spontaneously after lo-15 min (Lo et al., 1989). This reversibility suggests that extravasation need not obligatorily follow EC recognition, although these events could be linked in some settings. For example, a common chemoattractant arriving in the vessel from an extravascular source could be the signal both for activation-dependent adhesion to ECs and for diapedesis and migration into the tissues. If,
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instead, the activating signal of step 2 in EC recognition were provided by the endothelium itself, then independent tissue-derived chemoattractant gradients might be required for extravasation. Thus, in some settings extravascular signals may determine whether a leukocyte bound to the vascular endothelium eventually extravasates or instead is freed to return to the circulation. This adds another potential step to the control of leukocyte recruitment. Finally, although this discussion has been limited to those events that follow initial leukocyte encounter with vascular endothelium, the regulation of leukocyte extravasation and recruitment in vivo begins earlier. The properties of the endothelium and the array of local leukocyteactivating factors that control the recognition process must be determined in advance as a function of the local tissue microenvironment and of inflammatory stimuli (reviewed in Pober and Cotran, 1990). Concluding Remarks In conclusion, a model has been presented of leukocyteEC recognition as an active process involving three or more sequential events leading from the initial encounter to stable attachment. It is likely that many variations on this theme exist, only some of which have been considered. The most important implications of the model, however, derive less from the specifics of the individual steps than from the concept that recognition and stable attachment require several sequential events, each representing a go/no go decision point. It is the involvement of several steps, with multiple alternative receptor-ligand pairs available at each one, that provides a combinatorial mechanism for generating both diversity and specificity in leukocyte-EC recognition, that permits molecular conservatism by allowing individual receptor-ligand pairs to participate in more than one leukocyte homing event, and that implies that new homing specificities can be generated during evolution through the use of existing receptor-ligand pairs in novel combinations. References Arfors, K. E., Lundberg, C., Lindborm, L., Lundberg, K., Beatty, P. G., and Harlan, J. M. (1967). Blood 69, 336-346. Berg, E. L., Picker, L. J., Robinson, M. K., Streeter, P. Ft., and Butcher, E. C. (1991). In Vascular Adhesion Molecules, Cellular and Molecular Mechanisms of Inflammation, Volume 2, C. Cochrane and M. A. Gimbrone, Jr., eds. (San Diego: Academic Press), pp. 111-129. Bjerknes, M., Cheng, H.. and Ottaway, C. (1966). Science 23, 402405. Hamann, A., Jablonski-Westrich, D., Duijvestijn, A., Butcher, E. C., Baisch, H., Harder, R., and Thiele, H.-G. (1966). J. Immunol. 140,693699. Holzmann, B., McIntyre, B. W., and Weissman. I. L. (1969). Cell 56, 37-46. Jutila, M. A., Lewinsohn, D. M., Berg, E., and Butcher, E. C. (1988). In Leukocyte Adhesion Molecules: Structure, Function, and Regulation, T. A. Springer, ed. (New York: Springer-Verlag), pp. 227-235. Kishimoto, T. K., Jutila, M. A., Berg, E. L., and Butcher, E C. (1969). Science 245,1236-1241. Koopman, G., van Kooyk, Y., de Graaff, M., Meyers, C. J. L. M., Figdor, C. G., and Pals, S. T. (1999). J. Immunol. 145, 3569-3593. Lawrence, M. B., and Springer, T. A. (1991). Cell 65, 659-673. Ley, K., Gaethgens, P., Fennie, C., Singer, M. S., Lasky, L. A., and Rosen, S. D. (1991). Blood 77, 2553-2555.
Lo, S. K., Detmers, P. A., Levin, S. M., and Wright, S. D. (1969). J. Exp. Med. 169, 1779-1793. Lo, S. K., Lee, S., Ramos, R. A., Lobb, R., Rosa, M., Chi-Rosso. G., and Wright, S. D. (1991). J. Exp. Med. 773, 1493-1566. Oppenheim, J. J., Zachariae, C. 0. C., Mukaida, N., and Matsushima. K. (1991). Annu. Rev. Immunol. 9, 617-646. Phillips, D. R., Charo, I. F., and Scarborough, R. M. (1991). Cell 65. 359362. Picker, L. J., Kishimoto, T. K., Smith, C. W., Warnock, R. A., and Butcher, E. C. (1991a). Nature 349, 796-799. Picker, L. J., Warnock, R. A., Burns, A. Ft., Doerschuk, C. M., Berg, E. L., and Butcher, E. C. (1991b). Cell 66, 921-933. Pober, J. S., and Cotran, R. S. (1996). Transplantation 50, 537-544. Schall, T. J. (1991). Cytokine 3, 1-16. Shimizu, Y., Newman, W., Gopal, T. V., Horgan, K. J., Graber. N., Beall, L. D., van Seventer, G. A., and Shaw, S. (1991). J. Cell Biol. 7 13, 1203-1212. Smith, C. W., Kishimoto, T. K., Abbass, O., Hughes, B., Rothlein, FL, Mclntire, L. V., Butcher, E. C., and Anderson, D. C. (1991). J. Clin. Invest. 87,669-618. Spertini, O., Kansas, G. S., Munro, J. M., Griffin, J. D., and Tedder, T. F. (1991). Nature 349. 691-694 Springer, T. A. (1996). Nature 348, 426-433. von Andrian, U. H., Chambers, J. D., McEvoy, L., Bargatze, R. F., Arfors, K.-E., and Butcher, E. C. (1991). Proc. Natl. Acad. Sci. USA 87, 7536-7542.
