Migration patterns of dendritic leukocytes

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Y

INABA,K., SCHULER,G., WITMER,M.D., V~INSKI, J., ATASSI,B. & STEINMAN,R.M. (1986), Immunologic properties of purified epidermal Langerhans cells. Distinct requirements for stimulation of unprimed and sensitized T lymphocytes. J. exp. Med., 164, 605-613. INABA,K., ROMANI,N. & STFINMAN,R.M. (1989), An antigen-independent contact mechanism as an early step in T-cell-proliferative responses to dendritic cells. J. exp. Med., 170, 52%542. MACATONIA,S.E., KNIGHT,S.C., EDWARDS, A . J . , GRIFFITHS, S. • FRYER, P. (1987), Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. J. exp. Med., 166, 1654-1667. ROMANI, N., LENZ, A., GLASSL,H., STOSSEL,H., STANZL,U., MAJDIC, O., FRITSCH,P. & SCHULER,G. (1989a), Cultured human Langerhans cells resemble lymphoid dendritic ceils in phenotype and function. J. invest. Dermatol., 93, 600-609. ROMANI, N., KOIDE, S., CROWLEY, M., WITMER-PACK, M.D., LIVINGSTONE, A.M., FATHMAN,C.G., INABA,K. & STEINMAN,R.M. (1989b), Presentation of exogenous protein antigens by dendritic cells to T-cell clones" intact protein is presented best by immature epidermal Langerhans cells. J. exp. Med., 169, 1169-1178. ROMANI,N., SCHULER,G. ~ FRITSCH,P. (1990), Identification and phenotype of epidermal Langerhans cells, in "Epidermal Langerhans Cells" (G. Schuler). CRC Press, Boca Raton (June, 1990). SCHULER,G. & STEINMAN,R.M. (1985), Murine epidermal Langerhans cells mature into potent immunostimulat.ory dendritic cells in vitro. J. exp. Med., 161,526-546. SCHULER,G., ROMANI, N., STOSSEL,H. & WOLFF,K. (1990), Structure and function of epidermal Langerhans cells, in "Epidermal Langerhans Cells" (G. Schuler). CRC Press, Boca Raton (June, 1990). SnIMAD^, S., CAUGHMAN,S.W., SnARROW,S.O., STEPBANY,D. & KATZ,S.I. (1987), Enhanced antigen-presenting capacity of cultured Langerhans cells is associated with markedly increased expression of la antigen. J. Immunol., 139, 2551-2556. STEINMAN,R.M. & INABA,K. (1989), lmrnunogenicity: role of dendritic cells. 8ioessays, 10, !~5-152. STINGL,G., TSCnACHLER,E., GROH,V., WOLFF,K. & HAUSER,C. (1989), The immune functions of epidermal cells, in "Immune mechanisms in cutaneous disease" (D.A. Norris) (pp. 3-72). Marcel Dekker, Inc., New York, Basel. T~UNISS~N, M.B.M., WORMMEESTER,J., KmEG, S.R., PETERS, P.J., VOGELS, I.M.C., KAVSENnERG,M.L. & Boa, J.D. (1989), Human epidermal Langerhans cells undergo profound morphological changes during in vitro culture. J. invest. Dermatol., 94, 166-173. WITMER-PACK, M.D., OLIVIER, W., VALINSKY,J., SCHULER,G. & STEINMAN,R.M. (1987), . . . . . . . . . .v,~-, ...",.,. v l J n , ~ ~o~oay-~tmlUlatlng factor is essential for the viability and function of cultured murine epidermal Langerhans cells. J. exp. Med., 166, 1484-1498.

MIGRATION PATTERNS OF DENDRITIC LEUKOCYTES

J.M. Austyn Nuffield Department o f Surger,~, John Radcliffe Hospital, Headington, Oxford (UK) Dendritic leukocytes (DL) (reviewed in Austyn, 1989) are distributed throughout the body. They were first isolated from lymphoid tissues (and termed lymphoid DC) but related cells

are present in almost all non-lymphoid tissues, exist in migratory forms in the blood and lymph. The hallmark of what may be termed " m a t u r e " DL, exemplified by lymphoid DC, is their im-

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munostimulatory capacity: these cells present antigens and deliver potent activation signals to resting T cells, but they may be unable to process native antigens. On the other hand, "immature" DL, such as Langerhans cells from skin, seem able to process native antigens, but have weak immunostimulatory activity; recent evidence from our laboratory (JMA, Larsen et al., in preparation) indicates that freshly isolated DL from murine hearts and kidneys are also relatively immature. The apparently reciprocal distribution of immature and mature DL respectively in nonlymphoid (peripheral) and lymphoid (central) tissues, and their traffic in lymph, suggests that DL in the periphery may internalize and process antigens from their environment before migrating into lymph nodes where they present processed forms of those antigens to T cells and initiate immune responses. Our studies on the migration patterns of DL in normal animals were prompted by the observation that " m a t u r e " DC could be isolated from human peripheral blood. Although DL from afferent lymph (veiled cells) had been studied for some years, and there was evidence that they originated from peripheral sites such as skin epithelium and migrated into T areas of lymph nodes, the traffic of DL in blood was somewhat enigmatic, the key questions perhaps being "Why, Whence and Whit!~er?" (*). DL were known to be bone-marrow-derived and are presumably transported to peripheral sites in the blood, so one possibility was that "blood D C " , as isolated, were precursors to DL in non-lymphoid tissues. Alternatively, they could have an entirely different origin and destination.

(*) Into this Universe, and Why not knowing Nor Whence, like water willy-nilly flowing" And out of it, as Wind along the Waste, I know not whither, willy-nilly blowing. Ttte Rubaiyat of Omar Khayyam (Ed. 1. xxix)

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In our initial studies, mature DC were purified from mouse spleens, radiolabeUed with indium-ll I and injected intravenously into normal mice (Kupiec-Weglinski et al., 1988). We found that they migrated out of the blood into the spleen, but they were not detectable in any peripheral sites except the liver (DC from lymph nodes behaved similarly, implying this was not simply homing back to their site of origin). The next step was to label DC with a fluorochrome so they could be localized in frozen sections of spleens in relation to areas that were defined by antibodies labelled with a contrasting fluorochrome against various antigens, e.g. T-cell markers (Austyn et al., 1988). This revealed that DC homed into the central white pulp of the spleen where the bulk of T cells were located together with interdigitating cells (IDC). This study strongly indicated that lymphoid DC could become IDC, although for reasons outlined elsewhere (Austyn et al., 1988) we do not think that IDC per se can be obtained by conventional isolation techniques. Rather, we believe lymphoid DC are usually resident in other areas of the spleen, perhaps the marginal zone to which they were found to bind in a froz6n section assay (o19ciO. who,°,,,~,. . . . . . . . . . . . . ,h.. . ,, case, ~h~o~ ,u,,~ experiments showed that the destination of mature DC in the blood of normal animals is the spleen (an answer to the question " W h i t h e r ? " ) and that these cells are most probably not the precursors to DL in normal non-lymphoid organs. Our discovery of the origin of these cells came, in a rather round-about way, from studies on the migration of DL in mice bearing allografts. A long-standing problem in transplantation is precisely where host sensitization and the initiation of rejection occurs. As discussed elsewhere (Austyn and Steinman, 1988; Austyn and Larsen, 1990), it has been thought that sensitization to skin grafts occurs "centrally" in the draining lymph nodes, while that to fully vascularized organs occurs "peripherally" in the graft itself. (An additional problem is whether host T cells are sensitized against donor DL from the graft "passenger leukocytes" - - o r against host DL bearing graft antigens.) As will

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be seen, our work to examine the possibility of DL migration into and out of grafts has led us to propose that sensitization probably occurs centrally for all grafts. In earlier studies (above), we had found that DC traffic from the blood into the spleen was dependent on the presence of T cells at this site. This raised the possibility that mature DL might be recruited from the blood into peripheral sites where T cells had accumulated, such a3 allografts. Therefore, C. Larsen and others asked whether mature DL of the host strain (radiolabeUed and injected intravenously) could migrate into fully vascularized heterotopic heart grafts or skin grafts m the mouse (Larsen et al., 1990a). However, in an extensive series of experiments, we were unable to detect migration into isografts or allografts at any time after transplantation even though entry of T cells was evident. Instead, the DL continued to migrate into the spleen as in normal animals. This makes it highly unlikely that mature DC from the blood of the host can enter allografts and sensitize T cells peripherally, and for reasons detailed elsewhere (op cit.), it seems most unlikely that donor DL within the graft are involved. So, could donor DL migrate out of the graft into the spleen (via the blood) and sensitize host T cells centrally? (Perhaps surprisingly, at least in retrospect, the spleen does not seem to have been regarded as a potential site of sensitization in early studies in the transplantation literature.) Studies to address this question have, in fact, defined an important route for initiation of graft rejection and provided an answer to the question of where the mature DC in blood originate. As before, hearts were transplanted into allogeneic mice, but this time the heart grafts were examined for changes in the density of DL and the recipient's spleens were examined for the presence of cells bearing donor alloantigens (Larsen et al., 1990b). Soon after transplantation, a dramatic reduction in the numbers of DL in the grafted hearts occurred and, at the same time, donor cells appeared

in the hosts' spleens. (Control experiments established they were not derived from donor blood carried over in the grafts.) The precise localization of donor DL within recipient spleens was then determined by two-colour immunofluorescence and immunoperoxidaseimmunogold staining. The cells were found in the peripheral white pulp, which contains the majority of B cells, but they were tightly associated with CD4+ but not CD8 + T cells. The close proximity of donor DL expressing high levels of alloant,_'gens with host CD4+ T cells strongly suggests that this is the site of sensitization prior to rejection of a fully vascularized organ allograft. Why graft-derived DL should localize differently from lymphoid DC administered intravenously (peripheral vs. central white pulp) is intriguing, and under investigation. Conceivably the migration of allogeneic DL deeper into the white pulp is inhibited when they encounter alloreactive T cells in the peripheral area, or they may represent somewhat different stages of maturation and behave differently. It seems likely that in physiological circumstances, mature DC in the blood originate from peripheral non-lymphoid tissues (thus answering the question "Whence ?"). Presumably another cell in blood is the precursor to DL in nonlymphoid tissues. In summary, so far, it seems there are two parallel pathways for DL migration: non-lymphoid tissues ~ blood ~ spleen; and non-lymphoid tissues --, lymph -, lymph nodes. In answer to the earlier question " W h y ?", these routes may be required to ensure that immune responses are only ever initiated centrally (i.e. in lymphoid tissues) where there is the greatest chance of specific T cells meeting DL bearing the appropriate antigenic peptides. We have also started to examine the migration of DL within a tissue, the skin, during an immune response. It has generally been assumed that Langerhans

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cells from the epidermis of skin directly give rise to veiled cells in afferent lymph, particularly after s o m e , ~tigenic stimulus (e.g. application of a contact sensitizer). However, this belief is not ubiquitous, and others have suggested instead that epidermal Langerhans cells may be sloughed-off and that the cell that actually enters the lymphatics is derived from a precursor in the dermis. We (Larsen et al., in preparation) have studied the distribution of Langerhans cells after skin grafting and in an organ culture model. Soon after transplantation of full-thickness skin grafts to allogeneic or syngeneic mice, the Langerhans cells became much larger in size and were then found in progressively reduced numbers within the epm,.r,.as." "" "~" These changes were also seen in organ culture where we have, in addition, been able to observe the dermis. Concomitant with the above changes, the number of Langerhans cells in the dermis decreased until they assembled into long "strands", somewhat resembling a "traffic jam ~', which we believe are cells within lymphatics. These observations strongly support the idea that veiled cells do in fact originate as Langerhans cells (some cells were even seen apparently crossing the junction between epidermis and dermis) and this is also consistent with much other data on changes in the phenotype and immunostimulatory function of these cells as they mature in situ (Larsen et al., in preparation). T a k e n t o g e t h e r , our f i n d i n g s demonstrate the remarkable extent to

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which DL are migratory cells. This is further reinforced by the studies of P. Fairchild (in preparation) on DL in another lymphoid tissue, the thymus. DL are believed to exist in the thymus like IDC in the medulla and there is also evidence for the existence of an Ia-DL precursor. In essence, the question we are addressing (although our data will not be discussed here) is what is the function of thymic DL in Tcell ontogeny ? To approach this issue, embryonic thymus lobes were cultured in deoxyguanosine to kill presumptive DL and thymocytes, leaving an epithelial cell framework, before reconstitution with mature DC and thymocytes. We found that DC and thymocytes migrated into complementary regions of dGuo-treated thymus lobes in organ culture. Although there were areas where both cell types were present, DC migrated predominantly into the medulla (defined by a monoclonal antibody kindly provided by M. Ritter) while thymocytes moved to the developing cortex. There are several important issues raised by these observations alone. For example, what attracts DC into the medulla? Why should they migrate into the thymus in vitro when we found no evidence for their migration from the blood into the thymus in vivo ? Do DL precursors have access only at certain stages of thymic development ? And so forth. Thus we once again find ourselves searching for answers to the questions " W h y , Whence and Whither?".

References.

AUSTVN, J.M. (1989), Antigen-presenting cells, in "Focus series" (Male D.). IRL Press, Oxford. AUSTVN,J.M., KUPIEC-WF.Gt.INSKI,J.W., HANKINS,D.F. & MORRIS,P.J. (1988), Migration patterns of dendritic cells in the mouse. Homing to T-cell-dependent areas of spleen, and binding within marginal zone. J. exp. Med., 167, 646-651. AtJSTVN,J.M. & LARSEN,C.P.L. (1990), Migration patterns of dendritic leukocytes: implications for transplantation. Transplantation (in press). AUSTYN,J.M. & STEINMAN,R.M. (1988), The passenger leukocyte: a fresh look. Transplant. Rev., 2, 139-176. LARSEN,C.P.L., BARKER,H., MORRIS,P.J. & AUSTVN,J.M. (1990a), Dendritic leukocytes of host strain fail to migrate into cardiac or skin allografts. Transplantation (submitted).

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LARSEN,C.P.L., MORRIS,P.J. & AUSTYN,J.M. (1990b), Migration of dendritic leukocytes from cardiac allografts into host spleens: a novel route for initiation of rejection J. exp. Med. (in press). KUPIEC-WEGLINSKI,J.W., AUSTYN,J.M. & MORRIS,P.J. (1988), Migratioll patterns of dendritic cells in the mouse. Traffic from the blood, and T-cell-dependent and -independent entry to lymphoid tissues. J. exp. Med., 167, 632-645.

DENDRITIC CELLS AS SPECIALIZED

ANTIGEN-PRESENTING CELLS C.J.M. Melief Division o f Immunology, The Netherlands Cancer Institute, A n t o n i van Leeuwenhoek Huis, Plesmanlaan 121, 1066 C X Amsterdam

Dendritic cells (DC) isolated from lymphoid organs have a mysterious ontogeny and controversy surrounds their kinship with other cells, notably phagocytic cells of the monocyte/macrophage lineage. Nevertheless, from tile functional point of view, DC are clearly not capable of ingesting corpuscular matteL lack Fc receptors for immunoglobulins and have only one o~Jtstanding known function in which they excel, presentation of antigen to T ceils in the context of class I or class II MHC molecules. Obviously, this implies that DC are capable of endocytosis of exogenous proteins and of breakdown of microbial (viral, bacterial) proteins and processing of these proteins for presentation of peptides in the MHC groove. Below we shall review the evidence that DC are supremely equipped for this task by a number of criteria. Other aspects, notably the fate of endocytosed antigen in DC, need more study. Dendritic cells are very efficient antigenpresenting cells (APC).

In comparison with other types of antigen-presenting cells, DC are highly

effective in stimulating allogeneic or syngeneic mixed lymphocyte reactions (Steinman and Witmer, 1978; Nussenzweig and Steinman, 1989; Sunshine et al., 1982), proliferative T-cell reactions against soluble antigen (Guidos et al., 1984; Erb et al., 1985; Inaba and Steinman, 1985; Chain et al., 1986; .,,W~llU~l s et al., ~oo~ a~lU lymphokine production by T cells (ROllinghoff et al., 1982; Inabaetal., 1983). DC also support antibody responses to SRBC (Inaba et al., 1983) and induce a T-cell-dependent antibody response against tobacco mosaic virus (Francotte and Urbain, 1985). Finally, DC are highly efficient APC in the generation of cytotoxic T-cell (CTL) responses against viruses (Kast et al., 1988) and haptens (Inaba and Steinman, 1985; R611inghoff et al., 1982; Steinman, 1981). Recently virus-specific CTL were even induced in cultures of unprimed T cells with DC pulsed with a potent CTL epitope-bearing peptide of the influenza NP protein (Macatonia et ai., 1989). It was not possible to generate a primary in vitro antiviral CTL response with other types of APC. Generally, much fewer DC are required to induce a specific T-cell response than other types of: A~P._ (e.g. macrophages, spleen