Mechanisms

of lymphocyte

homing

Louis J. Picker University The initiation

and maintenance

the coordinated system.

Such

lymphocyte

of Texas, Dallas,

function

Current

is

distinct

critically

and recirculation.

progress in our understanding mechanisms,

of an effective immune

of spatially

coordination

homing

dependent

Introduction

physiological

requires

of the immune mechanisms

of

has seen significant

basis of these homing consequences.

1992, 4:277-286

Physiology

The remarkable ability of the immune system to recognize and eliminate external invaders depends on three essential, sequential steps. First, the body must produce a large and varied repertoire of lymphocyte clones bearing unique antigen receptors so that all potential foreign antigens can be distinguished from self Second, those rare lymphocytes bearing a given antigen receptor specificity must be brought together with their specific antigen in an appropriate microenvironment that allows the sub-, sequent expansion of that clone, and differentiation into ’ memory and effector populations. Finally, these resulting effector and memory cells must be efficiently distributed to those parts of the body where they can eliminate the ‘present’ invasion, and later, patrol against the ‘future’ return of the same invaders. For this system to function, nature must either provide all possible sites of antigen entry into the body with a complete representation of the immune repertoire - an improbable, if not impossible, situation - or must compartmentalize the principal functions of the lymphoid system into discrete organs and tissues, and then connect and unify these organs with a system of targeted lymphocyte trafhcking and recirculation. Indeed, a large body of evidence has accumulated over the past three decades documenting the operation of an elaborate system of lymphocyte recirculation in which individual lymphocytes may go from blood to tissue to lymph and back again as often as one or two times per day [ 1,2* 1. In the present review, I will provide a brief update on current advances in this field, focusing on the physiology of lymphocyte homing as it relates to immune function, and on the molecular mechanisms that are thought to control the homing of lymphocyte populations in vim, particularly those adhesive interactions involved in the selective extravasation of lymphocyte populations into different tissues.

on

The past year

in Immunology

response

compartments

of both the molecular

and their potential

Opinion

USA

of lymphocyte

Compartmentalization

homing

of the immune

system

understanding of the roles that lymphocyte homing and recirculation play in the regulation of immunity first requires an appreciation of the functional anatomy of the immune system [2*]. From an immunological perspective, the various tissues of the body can be divided into three basic units, the so-called primary, secondary, and tertiary lymphoid organs. Primary lymphoid tissues (e.g., l&e marrow, thymus, and in some species, specialized Peyer’s patches) represent those sites capable of supporting the production of functionally mature, albeit naive, lymphocytes from non-functional precursors. Secondary lymphoid tissues (e.g. lymph node, Peyer’s patch, spleen) represent the predominant sites of secondary lymphoid differentiation. Exogenous antigen collected and funneled into these tissues via the lymph or blood is presented by a highly organized lymphoid microenvironment that drives the proliferation and differentiation of antigen-specific B and T lymphocytes, particularly naive lymphocytes responding to antigen for the -first time. Tertiary lymphoid tissues (traditionally considered ‘extralymphoid’) can be interpreted to include all other tissues of the body. These tissues represent sites where memory lymphocytes and effector precursor cells can be restimulated by antigen, resulting in further clonal expansion of these previously stimulated populations, or where the terminal effector responses of B and T lymphocytes are carried out, whether these be Ig secretion, cytotoxic or delayed type hypersensitivity (DTH) responses, or immunoregulatory roles. Tertiary lymphoid tissues normally have small or absent constitutive lymphoid components, but ‘import’ such components after inflammatory stimuli. Of course, the functional specialty of any given tissue should not be regarded as a rigid compartmentaization; in some species, or under specific physiologic or

An

Abbreviations

CM-cutaneous lymphocyte antigen; CRP-complement

regulatory protein; DTH4elayed type hypersensitivity; ELAM--endothelial leukocyte adhesion molecule; ECF-epidermal growth factor; HEV-high endothelial venule; ICAM-intercellular adhesion molecule; IL-interleukin; LECAM-leukocyte endothelial cell adhesion molecule; LFA-lymphocyte function antigen; mA&monoclonal antibody; MAd-mucosal addressin; MU-mucosal lymphocyte antigen; PLN-peripheral lymph node; PMLpolymorphonuclear leukocyte; PNAd-peripheral lymph node addressin; VCAIK-vascular cell adhesion molecule; W-very late activation antigen. @

Current

Biology

Ltd ISSN 0952-7915

277

278

Lvmuhocvte

activation

and effector

functions

pathologic conditions there may be overlap of these functions in any given tissue. For example, memory/effecter responses can and do occur in primary and secondary lymphoid tissues, and the production of functional mature lymphocytes may, in certain instances, occur in a va riety of tissue sites.

Differential

homing

properties

of naive

and memory

lymphocytes

Functional unification of the ‘lymphoid’ tissues described above is accomplished by an elaborate system of lym phocyte homing and recirculation (Fig. 1). Conventional naive B and T lymphocytes, exported to the periphery from the primary lymphoid tissues, localize preferentially in organized secondary lymphoid organs. In general, such lymphocytes migrate poorly, if at all, to tertiary sites, even under conditions of inflammation. Their migratory properties appear to be determined ontogenetically as a function of their class (i.e. T cell versus B cell; CD4+ versus CD8+ T cell), but within a class show roughly equivalent migration to the various secondary sites [ 1,2*]. Naive lym phocytes probably traffic continuously between the vanous secondary lymphoid organs until they die or respond to their cognate antigen [ 2*,3], however, there are exceptions to this general pattern [ 2.1. One is the peritoneal localization of the ‘unconventional’ CD5+ B-lineage subset and another is the y6 T-cell receptor bearing T cells that seed different epithelia during ontogeny, prior to exposure to exogenous antigen. Memory and effector lymphocytes, generated in set ondary lymphoid tissues in response to antigen exported back into the circulation, display very different migratory properties to naive cells (Fig. 1). Firstly, these cells can tralfic effectively to tertiary lymphoid tissues particularly when these tissues are inflamed [ 1,2*,4,5*]. Secondly, differentiation into the memory subset is accompanied by the development of tissue-restricted homing capability The tissue(e.g. to the skin or mucosal sites) [i&6]. selective homing properties of memory and effector cell subsets may enhance the efficiency of the immune system by targeting immune surveillance to tissues that are most similar to those where the antigen initially entered the body, and might also be important in reducing opportunities for autoimmune crossreactions with components from unrelated tissues,

Regulation

of lymphocyte

homing

The physiology of lymphocyte homing and recirculation is largely determined by the differential regulation of lym phocyte adhesion and de-adhesion. Lymphocytes have an extraordinary ability to finely regulate their adhesive properties throughout their lifespan [7-Y]. While essentially all adhesive events have some impact on a given lym phocyte’s recirculatory potential by regulating the ability of that cell type to pass through a particular tissue, appear in the lymph and return to the circulation, the adhesive interaction central to the regulation of lymphocyte homing is that between blood-borne lymphocytes and post-capillary venular endothelium [ 1,201. Lymphocyte

Virgin

O@ 0

Virgin

lymphocytes n 4

ce”S ff=?Y

L-

Secondary lymphoid tissues (peripheral lymph node, Peyer’s patch, spleen)

\, (skin,

intestinal

lamina

propria,

synovium,

etc)

Fig. 1. The immune

system is compartmentalized into functionally and spatially distinct units. Virgin lymphocytes of a particular class produced in primary lymphoid tissues migrate with roughly equivalent efficiency to secondary lymphoid tissues. These cells continuously recirculate through these secondary lymphoid tissues until they either die, or are activated by specific antigen. Antigen-driven activation results in conversion to a ‘memory’ phenotype which is associated with the development of heterogeneity with regard to homing potential. Memory subsets exhibit differing, tissue-selective homing behavior, and gain the ability to extravasate in particular tertiary C’extralymphoid’) sites.

binding to endothelium is a requisite first step in the process of extravasation, and this interaction, mediated by lymphocyte ‘homing receptors’ and their endothelial ligands, largely determines the extravasation potential of lymphocytes at various tissue sites.

Leukocyte-endothelial

interaction:

new

copcepts Perhaps the most important recent advance in the understanding of lymphocyte homing is the realization that in many, and perhaps all, instances leukocyte extravasation is an active, multistep process [lo**]. Ironically, this concept has been developed primarily from in vitro and in vivo studies of neutrophil+zndothelial interaction, but is likely to hold true for lymphocytes as well. The ‘multistep’ hypothesis proposes that leukocyte extravasation is the result of the successful completion of at least three successive steps (Fig. 2). In the first

Mechanisms

step, free-flowing leukocytes within the venular lumen interact with endothelial cells via the action of constitutively functional (i.e. activation independent) leukocyte homing receptors with regulated endothelial cell ligands or counter-receptors. This ‘primary’ adhesion is transient and unstable under physiologic shear force, but temporarily slows leukocyte passage, allowing the leukocyte to ‘sample’ the local environment. Transiently adherent cells are released back into-the circulation unless secondaty adhesion mechanisms are brought into play. The best characterized examples of primary adhesion receptors capable of initiating interactions under conditions of flow are members of the selectin family, (L-selectin on leukocytes; E- and P-selectin on the endothelium) and their carbohydrate ligands (see below). If the leukocyte encounters specific activating or chemoattractant factors (which may be expressed on the endothelial cell surface, or as soluble factors produced by the endothelium or surrounding tissues), a second level of activation-dependent adhesion is triggered that mediates firm attachment, stable under physiologic shear force. This stable attachment initiates the subsequent migration of the cell through the venular wall. Both the pl and p2 integrins represent activation-dependent leukocyte adhesion molecules, capable of mediating triggered leukocyte attachment to endothelial cell ligands, including intercellular adhesion molecule (ICAM)-1, ICAM-2, vascular cell adhesion molecule (VCAM)-I, and probably others, after , . appropriate leukocyte stimulation (see below).

of IvmDhocvte

hominrr

Picker

sion, leukocyte activation, or activation-dependent adhesion. In this context, the activating factors responsible for converting transient adhesion to firm, integrin-medi ated attachment may prove to be as important as specific adhesion molecules in determining the specificity of lymphocyteendothelial cell interactions. Such activating factors are also generally chemoattractants and if derived from surrounding tissues may regulate not only adhesion to endothelial cells, but also subsequent extravasation. Relatively little is known about activating factors involved in lymphocyte homing and they will not be discussed further here, except to refer the reader to recent studies characterizing a family of polypeptide chemoattractants related to interleukin (IL)-8 and platelet factor 4. Some of these display selective chemoattractant activity for specific lymphocyte subsets, and thus have the potential to regulate lymphocyte subset tra&king in uivo [11-l.

Molecular

basis of lymphocyte

homing

A number of lymphocyte and endothelial cell adhesion molecules have been identified that are thought to participate in the interaction of lymphocytes with the high endothelial venules (HEV) of lymphoid tissues and/or pvith cytokine-activated, inflamed endothelium. These ‘molecules (listed in Table 1) include members of several distinct adhesion receptor families, including: (a) the selectin family of leukocyte and endothelial cell C-type lectins; (b) oligosaccharide ligands for the selectins; (c) heterodimeric, activation-dependent integrins

One important implication of this hypothesis is that the specificity of leukocyte-endothelial cell recognition can be determined at any of three steps: primary adhe-

, : Flow i Lumen of venule

Primary adhesion: reversible under sheer activation-independent

Fig. 2. The

force, Movement and

to

transmigration

of leuko-

cytes

activation-indepen-

use primary,

dent receptors regulated

to initially

interact

counter-receptors This

initial

leuko-

on

adhesion

with endo-

is unsta-

ble under shear force, and is, therefore, transient

fi

ond

unless

the cell receives a sec-

activating

signal

the endothelial

Tissue

model

Free flowing

thelium.

Post capillary venule endothelium

multistep

cyte extravasation.

Secondary adhesion: (r3 stable under shear force, activation-dependent n

tissues.

Activation

results

ipation of secondary, dent adhesion hesion

under

leads to leukocyte tion

Thus,

requires

cal adhesion the

Chemotaxis

n

V

Secondary

transmigration

be differentially

recipro-

pairs, and

of leukocyte-type factors,

into

extravasa-

and endothe-

molecule-ligand

availability

ad-

shear force and

of two or more

cific activating extravasation

in the partic-

successful

leukocyte

lial expression

from

activation-depen-

systems.

is stable

the tissues.

originating

cell (EC) or surrounding

spe-

all of which may

regulated so as to insure specificity.

279

280

Lvmphocvte

activation

and effector

functions

of the pl and p2 classes, as well as novel pp-p_I integrins; (d) members of the Ig family of cell adhesion molecules (ICAM-1, ICAM-2, VCAM1); (e) CD44, a proteoglycanlike molecule and member of a hyaluronate-binding receptor family; and, (f) several ‘orphan’ receptors which have not had their molecular structures precisely defined. These molecules have been the subject of many recent reviews [7-9,12,13], and will not be discussed here in detail. Instead, 1 will highlight recent advances in the understanding of the function of selected molecules with regard to the physiology of lymphocyte homing.

The peripheral

lymph

the role of L-selectin

node

(PLN) homing

and the peripheral

cyte surface molecule selectively inhibited lymphocyte binding to PLN HEV in vitro and homing to PLN in vivo [ 14-161. Certain carbohydrate structures were able to mimic this selective inhibition, suggesting a possible lectin function for this homing receptor [ 161. Consistent with this hypothesis, structural characterization revealed that, along with endothelial leukocyte adhesion molecule (EIAM)-1 (E-selectin) and GMP-140 (P-selectin), the PLN homing receptor was a member of the selectin family of adhesion molecules (L-selectin) which feature an aminoterminal C-type lectin domain juxtaposed with a domain homologous to epidermal growth factor (EGF), and then variable numbers of tandem repeats related to complement regulatory proteins (CRPs; [7,12]). The function of L-selectin has been further clarified in the past year with the identification of peripheral lymph node addressin (PNAd) as its endothelial ligand(s) [ 18**,1!9*]. A role for this antigen in PLN traflicking was first suggested by the observations that luminal expression of PNAd is preferentially seen on PLN HEV, and that the MECA79 nib, recognizing PNAd, inhibits lymphocyte interaction with PLN, but not mucosal, HEV in vitro and in vim

specificity: lymph

node

addressin

L-selectin (previously referred to as leukocyte endothelial cell adhesion molecule (LECAM)-1, (LAM)-1, the Mel 14 antigen, or the Ieu 8 antigen) was first implicated as a primary mediator of lymphocyte homing specificity to peripheral lymph nodes (PLNs) by studies demonstrating that monoclonal antibodies (mAbs) against this lympho-

Table 1. Leukocyte Lymphocyte adhesion

and endothelial

cell adhesion

molecules

in lymphocyte

homing.

‘homing’ receptors/

molecules

Endothelial

L-selectin

PNAd

(LECAM-1;

involved

Predominant

ligands

tissue

specificity

(MECA-79

antigen)

Peripheral

lymph

(MECA-367

antigen)

Peyer’s patch and other

node

LAM-l,

Mel14 antigen) MAd

?

E-selectin

CLA

(ELAM-1)

Cutaneous

sites of chronic

HEBF pp VCAM-1;

a4 p7 or a4 pp (LPAM-1) uMLA

p7

HML-1

antigen;

Ber ACT8

antigen;

uM290

? Peyer’s patch and other

ICAM-I

endothelial

with

adhesion

(Peyer’s patch); ICAM, antigen;

lymphocyte

antigen,

to bind hyaluronate, HCAM,

adhesion

lymphocyte

PNAd,

endothelial

molecule;

intercellular

LPAM,

antigen;

sites

None

7*

regard to lymphocyte

leukocyte

sites

(inducible); (constitutive)

Pgp-1)

has been demonstrated

interactions

mucosal

87)

ICAM-

(HCAM,

sites;

sites

? Mucosal

p2 integrins

activation

inflammation

inflammatory

? mucosal

?

UL p2 (LFA-I)

function

Certain

fibronectin,

? others

a4 81 (VLA-4)

*CD44

sites

Peyer’s patch

~4 and related integrins

CD44

mucosal

peripheral

collagen type VI and MAd, binding

molecule;

LECAM,

node vascular

but the physiological

is unclear (see f2.1). CLA, cutaneous

homing-associated

Peyer’s patch-specific lymph

?

cell adhesion

leukocyte

adhesion addressin;

molecule;

endothelial

molecule; VCAM,

MAd,

HEBF,

cell adhesion mucosal

vascular

significance

lymphocyte

of each of these

antigen;

high endothelial molecule;

vascular

cell adhesion

ELAM, binding

factor

LFA, lymphocyte

addressin; molecule;

MLA, VLA,

mucosal

very late

Mechanisms [ 171. Several lines of evidence have confirmed that PNAd is the L-selectin ligand: (a) purified PNAd adsorbed to plastic supports the binding of L-selectin bearing lym phocytes; (b) this binding is completely inhibited by L-selectin-specific mAbs; and (c) transfection of a non HEV binding (L-selectin-) pre-B cell line with L-selectin cDNA simultaneously confers the ability to bind both peripheral lymph node HEV and purified PNAd [I@*]. The mAb MECA-79 recognizes multiple glycoproteins ranging in molecular weight from about 65 to 2OOkD, the most prominent species for iodination are 9&l 15 kD, and a prominent sulfate-incorporating species (which labels poorly with iodine) is observed at 50kD. At least two of these species, the sulfate-labeled 50 kD glycoprotein, and the major 1OOkD iodinated glycoprotein, are immunoprecipitated by a L-selectin-Ig chimeric protein [19**]. The interaction of PNAd with L-selectin is dependent on the presence of Ca2+ and intact sialic acid moieties on PNAd [ 18**,19=*] ; these findings support the critical importance of the C-type lectin domain in mediating L-selectin function. Interestingly, however, a number of recent studies examining structure-function relationships of the L-selectin molecule have suggested that the EGF and CRP domains may also be necessary for ligand recognition [2@23]. Thus, it is likely that these domains are either required for the maintenance of a functional conformation of the lectin domain, or they contribute to the formation of one or more binding domains that,‘. directly interact with ligand.

In keeping with the physiological evidence that essentially all naive B and T lymphocytes home to secondary lymphoid organs, including lymph nodes, the vast majority of naive T lymphocytes in human peripheral blood, or naive T cells in human appendix and tonsil, express L-selectin (L Picker, unpublished data) [6]. In contrast, circulating memory T cells are divided into discrete L-selectin+ and L-selectin- populations consistent with the heterogeneity in their tissue-specific homing patterns Several lines of evidence suggest the L-selectin- T-subset in peripheral blood represent the immediate precursors of human lamina propria lymphocytes, whereas the the L-selectin+ subset represent T-cells selectively recirculating through peripheral tissues [6,24]. For example, memory T-cells expressing the skin homing receptor cutaneous lymphocyte antigen (CIA) (the skin being a ‘peripheral’ tissue intimately associated with PLNs) are largely included in the L-selectin+ subset [6]. In the multistep model of leukocyteepidermal cell recognition, primary adhesion via the L-selectin-PNAd interaction would have to be followed by activating signals, and by activation-dependent adhesion, to stabilize the 1ymphocyteHEV interaction and to permit subsequent diapedesis. Indeed, the activation-dependent leukocyte integrin, lymphocyte function antigen (LFA)-1, has also been found to participate in lymphocyte interactions with lymph node HEV. Anti-LFA-1 antibodies can inhibit lym phocyte binding to HEV in vitro by 4&50%, and also significantly reduce lymphocyte extravasation into lymph nodes in viva [ 25,261.

ClAE-selectin: mediating

of lymphocyte

a tissue-selective

memory

sites of chronic

T-cell

receptor

extravasation

homing

Picker

ligand pair

at cutaneous

inflammation

E-selectin (ELAM-1) was originally described as an inducible endothelial cell selectin adhesive for neutrophils. However, immunohistological studies revealed intense E-selectin expression on venules not only in acute inflam mation, but also in settings dominated by mononuclear cell influx [27**]. Indeed, in the context of chronic inflammation, E-selectin appeared to be preferentially expressed in cutaneous sites. (Although E-selectin can be found on venules in some other sites of chronic inflam mation, the degree of expression is substantially less than that observed in the majority of skin lesions.) The significance of this tissue-selective expression is revealed by observations that E-selectin binds not only neutrophils, but also a unique skin-associated population of memory T cells, characterized by the expression of CLA (recognized by mAb HECA-452) [ 27**]. CLA+ memory T-cells are found selectively in cutaneous inflammatory sites and as a circulating memory T-cell subset in blood [ 281. Moreover, CIA is expressed by the epidermotropic malignant T-cells of the skin-associated lymphoma, mycosis fungoides. Thus, E-selectin appears to be a cuta neous vascular addressin for these unique skin-homing memory T cells. We have further shown that CIA itself is a carbohydrate-bearing ligand for E-selectin, and is, therefore, a skin lymphocyte homing receptor [29**]. The precise structure of the lymphocyte CLA determinant remains to be elucidated, but preliminary evidence suggests that CIA is closely related to the major neutrophil oligosaccharide ligand for E-selectin, sialyl Lewis X [29”]. CLA is a constitutively functional, activationindependent homing receptor that operates well under shear force [ 27**,3Q**,31**]. Its ability to mediate selective memory T-cell homing to cut&aeous sites of chronic inllammation may also depend on lymphocyte-selective chemoattractants, and secondary adhesion structures such as LFA-l-ICAMor very late activation antigen (VLA~; 4pI integrin)-VCAMI (see below).

l32 integrins: molecules

non-tissue-selective involved

in adhesion

secondary strengthening

adhesion and

transmigration As

described above, the l32 integrins, including the predominant lymphocyte integrin LFA-1 (aLp2; CDllaCD18), provide the paradigm for activation-dependent, secondary adhesion structures that provide stable adhesion after initial leukocyteendothelial binding [7,8,10**]. These molecules are also critical for the last step of the extravasation process, i.e. transmigration through the venular wall [ 7,8,32]. A wide variety of stimuli have been shown to upregulate j32 integrin function on both lymphocytes and phagocytes [7,8,1000], but the precise mechanism of activation-induced avidity increases is still obscure. However, recent studies indicate that the molecular changes associated with functional activation of LFA-1 result in the appearance of new antigenic epitopes, appear to require conserved regions within the cytoplasmic domain of the P-chain, and may involve

281

282

Lvmphocvte

activation

and effector

functions

displacement of Ca*+ from extracellular domains [33,34*,35-l.

cation-binding

One ligand for LFA-1 is ICAM-I, a member of the Ig superfamily with five Ig-like domains [7]. ICAM- is broadly expressed in vivo, on both hematolymphoid and nonhematolymphoid cell types [ 36.1. It is weakly expressed on resting endothelium, but expression is markedly increased after inflammatory stimuli [7,36*]. More recently, a second endothelial ligand for LFA-1 has been characterized [7,36*,37]. This molecule, ICAM-2, has only two Ig-like domains, and differs considerably from ICAMwith regard to both its tissue distribution (expression is more restricted to hematolymphoid cells and endothelium) and its ability to be induced [36*,37]. ICAM2 is the predominant LFA-1 ligand on resting endothelial cells, but does not appear to be subject to inflammationinduced upregulation. In addition, unlike ICAM-1, ICAMshows no detectable binding to the leukocyte integrin Mac-l (aMP2 [36-l). Thus, the physiological role of ICAM- may be to selectively facilitate lymphocyte recirculation through ‘resting’ vascular beds, for example the low-level recirculation of memory T-cells through normal tertiary tissues; whereas ICAM- would have a broader physiologic role in the recruitment of multiple leukocyte types to sites of inflammation. Interestingly, preliminary data suggest that additional LPA-1 ligands exist, and that the ICAMs may have ligands other than LFA-1. With regard to the latter point, one study has suggested that CD43 (sialophorin or leukosialin, a molecule defective in Wiscott-Aldrich syndrome), is an alternative ligand for ICAfv-1 [38].

crrl-heterodimers receptors

and related

involved

integrins:

in lymphocyte

multifunctional

homing

The understanding of the physiological role of the ~14. heterodimers and related integrins in lymphocyte homing has been complicated by several factors: (a) their structural diversity; (b) their multiple and seemingly overlapping binding specificities; (c) the observation that at least some adhesive functions of these integrins are subject to activation-induced upregulation; and (d) the fact that most of the available data on these molecules have been obtained in vitro. With regard to the first point, at least two and possibly three distinct ai-heterodimers have been identified, ~14pl (VLA4; CD49dCD29); clipp (LPAMl), and ~44p7 [3+44] (a4fip and a4p7, which have been identified by different laboratories, are either very closely related or identical; they may represent the product of a single gene, or of closely related duplicated genes; they may exist in variant forms characterized by differences in amino-terminal sequences and/or in cytoplasmic domains). Moreover, a novel integrin bearing a P7-chain associated with a unique a-chain, mucosal lymphocyte antigen (MLA)a, has been recently characterized [44-46]. This novel integrin has been previously described as a MIA because of its restricted in vivo expression on mucosal T cells, both intraepithelial and in the lamina propria. a4pl has been to shown to bind both the inducible endothelial molecule VCAM-1 and fibronectin [41], and is

likely to participate in lymphocyteendothelial interaction at sites of chronic inflammation (VCAM1 is present on activated endothelial cells at many, but not all, such sites; L Picker, unpublished data). c~4bp has been implicated in the binding of lymphocytes to Peyer’s patch HEV [ 39,401, although the endothelial counter-receptor involved in this interaction is not yet known, It is not likely to be VCAMI since this molecule is not usually present on resting mucosal HEV [47] ; however, the mucosal addressin (MAd) remains a potential candidate [48]. The extent to which these integrins overlap in mediating these interactions is at present unclear, especially under physiological conditions. Recent in viva studies in a rat model indicate that an &specific mAb (TA-2) can efficiently inhibit all lym phocyte migration to the Peyer’s patch and memory lym phocyte migration to cutaneous inflammatory reactions, partially inhibit memory lymphocyte homing to PLN, but have little or no effect on the migration of putative naive cells to PLN [49*]. The mechanism(s) by which TA-2 mediates its effects are unclear. It is not known which particular c&ntegrins and endothelial ligands are involved, whether the required cl4-mediated interaction is a secondary, activation-dependent interaction, like that of p2integrins, a primary interaction, or a combination of both depending on the particular molecules involved. It is of interest to note that the migration of putative memory Tcells to cutaneous inflammatory sites in the mouse can be inhibited by synthetic peptides that mimic integrin-binding domains of fibronectin; this suggests that lymphocyte interactions with extracellular matrix are a critical component of migrational capabilities [ 50* 1, Adhesive capabili ties have not yet been reported for MI& although given the participation of a4 and p_IIpp integrins in lymphocyte binding to Peyer’s patch HEV and the restricted distribution of lymphocytes bearing this novel integrin, it is reasonable to hypothesize a role for MLA in lymphocyte homing to the gut and/or other adhesive interactions with components of mucosal tissues.

Microenvironmental

regulation

of lymphocyte

homing There are at least three broad mechanisms by which local microenvironments can regulate patterns of lymphocyte homing and extravasation: (a) the induction and maintenance of endothelial adhesion molecules on local vascular beds; (b) the regulation and maintenance of homing receptor expression patterns on lymphocytes, especially primary, tissue-selective homing receptors; and (c) the local availability and expression .of chemoattractamactivating factors capable of initiating secondary adhesive mechanisms and vessel transmigration. As mentioned above, characterization of the physiologically relevant chemoattractant/activating factors involved in lymphocyte homing is in its infancy. Similarly, little is known regarding the in vivo mechanisms responsible for the regulation of lymphocyte homing receptor expression. However, it is clear that differential, microenvironment-dependent regulation of tissue-selec-

Mechanisms

tive homing receptors occurs in vivo. In recent studies, we have demonstrated that human T-cells undergoing the naive to memory cell transition in PLN and appendix differentially up- or downregulate the PLN homing receptor L-selectin and skin-selective homing receptor CL4. Memory cells generated in the appendix are generally L-selectin- and CLAnegative, whereas those generated in PLN are more often L-selectin and CIApositive (L Picker, unpublished data). At least two components are required for the regulation of endothelial adhesion molecule expression: regulation by immune cytokines and regulation by non-immune factors that maintain site-specific endothelial cell differentiation. With regard to the first component, a variety of studies with cultured endothelial cells indicate that specific endothelial adhesion molecules (ICAMI, VCAMl, E-selectin), and endothelial adhesiveness in general can be dramatically modulated by particular immune cy tokines, alone or in combination [51-53,54=,55-l. For example, tumor necrosis factor-a in combination with either IL-4 or interferon specifically enhances lymphocyteendothelial binding [ 521, whereas transforming growth factor fl has an inhibitory effect [ 531. Such cytokines can also upregulate tissue-selective lymphocyte adhesion to cultured HEV cells, although in these experiments, the observed increased adhesion may be due to secondary mechanisms (i.e. enhancement by up-regulation ‘of activation-dependent secondary receptors, or endotheliai production of activating factors) rather than upregulation of endothelial ligands for tissue-selective primary homing receptors [54*,55*]. However, the observation that PNAd and MAd can be induced at some sites of chronic inflammation indicates that immune cytokines can influ ence expression of tissue-selective adhesion molecules (L Picker, unpublished data) [56]. Thus, it is possible that certain endothelial adhesion molecule phenotypes, including tissue-selective phenotypes, are dependent on the relative, local availability of immune cytokines. For example, it is possible that the preferential expression of E-selectin on cutaneous venules in the context of chronic inflammation is due to their proximity to epidermal cells and dermal mast cells, cell types that together are capable of producing large amounts of the E-selectin-inducing IL-1 [ 57,581. The second level of regulation of endothelial adhesiveness concerns the factors which maintain the sitespecific, constitutive endothelial differentiation. Endothelial cells at particular sites display tissue-specific differ ences in phenotype that are thought to be related to specific components of the extracellular matrix, or to factors produced by the local, differentiated epithelial or mesenchymal cells [59,60~]. With regard to lymphocyte homing, the constitutive expression of PNAd and MAd on PLN and mucosal HEV, respectively, is, at least in part, determined by stable factors present during ontogeny (reviewed in [2-l). In late fetal and early postnatal mice, MAd is widely expressed in lymphoid tissues, including presumptive HEV (or their precursors) in PLN (PNAd is

of lymphocyte

homing

Picker

absent). In PLN, MAd expression is lost over the first 3_4 weeks of postnatal development, in association with increased levels of expression of PNAd (MAd expression is retained on Peyer’s patch and mesenteric lymph node HEV). Interestingly, adult explants of intestines or mesenteric lymph nodes transplanted to non-mucosal sites retain MAd expression, whereas neonatal mesenteric lymph nodes transplanted to the popliteal fossa sometimes lose MAd expression, displaying only PNAd. Maintenance of PNAd expression on PLN HEV in adult mice appears to require factors present in alferent peripheral lymph. Interrupting afferent lymph results in loss of HEV morphology and luminal PNAd expression, as well as the ability to mediate lymphocyte influx; restoration of afferent lym phatic connections reverses these effects [61*],

Conclusions In this review, I have outlined recent advances in our understanding of the physiology of lymphocyte trafhcking, emphasizing the importance of analyzing these mechanisms in the context of the overall function of the immune system. Although the pace of discovery has been rapid, our understanding of the molecular mechanisms regulating lymphocyte homing, and of the physiological consequences of these mechanisms is clearly still in the formative stages. A number of important areas for future investigation can be stressed. Firstly, physiological studies of the tissue-selective expression and function of known adhesion elements, particularly those that have as yet been studied almost exclusively in vitro. Secondly, identification of the molecular mechanisms of lymphocyte targeting to primary, secondary andternary lymphoid tissues that have not yet been systematically examined, including the bone marrow, thymus, spleen, synovium, the central nervous system, the lung, as well as other: specialized tissue sites. The extent to which unique homing mechanisms exist in each of these sites is as yet unclear, but preliminary s&dies support the concept of specific homing receptors and vascular addressins in additional tissues. For example, the physiological role of the recently identified lung endothelial cell-specific adhesion molecule involved in the mediation of lung metastasis may be lungselective lymphocyte homing [GO-]. Thirdly, determination of the factors regulating the expression of homing receptors and their ligands on lymphocytes and endothelial cells, respectively; and finally determination of the role of lymphocyte subset-specific chemoattractant/activating factors in lymphocyte-endothelial cell recognition and in lymphocyte extravasation. Further analysis of the available repertoire of adhesion molecules and chemoattractant/activating factors involved in lymphocyte homing and their physiological regulation may allow fuller understanding of the pathophysiology of normal and abnormal immune or inllammatory responses, and lead to novel strategies for combating diseases such as rheumatoid arthritis and multiple sclerosis.

283

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and effector

functions

Acknowledgments

(h) leukocyte activation hy local factors, and (c) secondav, activationdependent binding leads to the extravasation specificities seen in ~iiz’o.

I am thankful to J Treer

and M Siegelman for critical review of this thoughtful discussions, manuscript, and to E Butcher for continuing This work was supported by NIH grant AI31545, a research grant from the Texas Higher Education Coordinating Board, and an award from the President’s Research Council of the University of Texas Southwestern Medical Center ar Dallas.

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6.

PICKERLJ, TERSTAP~EN LWMM, Rorr LS, S?‘KEE’I’EK PR, STEINI-I, B~JTCHEREC: Differential Expression of Homing-associated Adhesion Molecules by T-Cell Subsets in Man. J Immunol 1990, 145:3247-3255.

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BUTCHERE: Leukocyte-EndotheIiaI Ceil Recognition: Three (or More) Steps to Specificity and Diversity. Cell 1991, 67:1033-1036. This paper synthesizes a wide range of observations in the literature into the ‘multistep’ hypothesis of leukocyte extravasation, the hypothesis that exrravasation requires successful complerion of at ieast three sequential steps which can he regulated independently. It is proposed that differential regulation of (a) initial, activation-independent binding,

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49. .

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erogeneity: Interactions with Leukocytes and Tumor Cells. Cancer Metastasis Rev 1989/90, 8~353-389 ZH~J D, CHENG CF, PALIU BU: Mediation of Lung Metastasis of Murine Melanomas by a Lung-specific Endothelial CelJ Adhesion Molecule. Proc Nat1 Acud Sci USA 1991, 88:956%9573. Endothelial cells within the lung selectively express a novel cell adhesion molecule that promotes the selective attachment of a lungmetastatic melanoma cell line. Although the subject of lymphocyte homing is not directly addressed in this paper, these findings support the concept of tissue~specilic endothelial differentiation, and suggest the possibility that the physiologic function of this molecule may be to promote lung~selective leukocyte homing. 60. .

MEBIUS RE, STREETERPR, BREVE J, DUI.JVESTIJN AM, KRAALG: The ltiuence of merent Lymphatic Vessel-interruption on Vascular Addressin Expression. J Cell Biol 1991, 115:85-95. The ‘high endothelial’ phenotype of murine PLN HEV, including PNAd expression and the ability to mediate lymphocyte influx, is dependent on ‘factors’ entering the PLN via tierent lymphatics. Ablation of tierent lymph intlux results in progressive loss of high endothelial differentiation over a period of one week, a process that is reversible with lymphatic ‘reconnection.’ 61. .

LJ Picker, Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75235, USA.