Death, destruction, danger and dendritic cells.

Dendritic cells are known to be involved in recognition of foreign antigens and initiation of specific T-cell responses. The 'danger hypothesis' suggests that the immune system can also respond to endogenous signals of distress. New data indicate that dendritic cells are the first to respond to these signals, although the mechanisms involved in their activation are unclear (pages 1249-1255).

Expression of the macrophage-specific antigen F4/80 during differentiation of mouse bone marrow cells in culture.

We have defined the expression of the macrophages (m phi)-specific antigen (Ag) F4/80 during differentiation in culture. The progenitor cells-the colony-forming unit in culture and cluster-forming cell-lacked Ag F4/80 but gave rise to colonies of F4/80-positive adherent m phi, as shown by fluorescence-activated cell sorting and clonal assays with L cell-conditioned medium as the source of growth factor. Ag F4/80 first appeared on a nonadherent precursor found in mass liquid BM cultures after 3 d. Once adherent, m phi expressed high levels of Ag F4/80 and other markers. The role of L cell-conditioned medium and of adherence on expression of Ag F4/80 was also examined. Clonal analysis of F4/80 and other Ag, Mac-1, and 2.4G2 (FcR) showed that all cells in all independent colonies come to express these markers. These studies establish that F4/80 is a marker for the more mature stages of m phi development and that Ag expression increases progressively during maturation in vitro. Heterogeneity of Ag expression can be ascribed to variation in development and not to independent subsets of the m phi.

Migration patterns of dendritic cells in the mouse. Traffic from the blood, and T cell-dependent and -independent entry to lymphoid tissues.

Dendritic cells (DC) are critical accessory cells for primary immune responses and they may be important stimulators of transplantation reactions, but little is known of their traffic into the tissues. We have studied the migration of purified splenic DC and T lymphocytes, labeled with 111Indium-tropolone, in syngeneic and allogeneic mice. First we demonstrate that DC can migrate from the blood into some lymphoid and nonlymphoid tissues. Immediately after intravenous administration, radio-labeled DC were sequestered in the lungs, but they actively migrated into the liver and spleen and reached equilibrium levels between 3 and 24 h after transfer. At least half of the radiolabel accumulated in the liver, but the spleen was the principal site of DC localization in terms of specific activity (radiolabel per weight of tissue). DC were unable to enter Peyer's patches, or mesenteric and other peripheral lymph nodes from the bloodstream. This was also true in splenectomized recipients, where the otherwise spleen-seeking DC were quantitatively diverted to the liver. In contrast, T cells homed readily to the spleen and lymph nodes of normal mice and increased numbers were present in these tissues in splenectomized mice. Thus, unlike T cells, DC cannot recirculate from blood to lymph via the nodes. We then show that migration of DC from the blood into the spleen is dependent on the presence of T cells: DC did not enter the spleens of nude mice, but when they were reconstituted with T cells the numbers entering the spleen resembled those in euthymic mice. In nude mice, as in splenectomized recipients, the DC that would normally enter the spleen were quantitatively diverted to the liver. These findings suggest that there is a spleen-liver equilibrium for DC, that may be akin to that existing between spleen and lymph node for T cells. Finally, we followed the traffic of radiolabeled DC via the afferent lymphatics after subcutaneous footpad inoculation. DC accumulated in the popliteal nodes but did not migrate further to the inguinal nodes. There was no difference between euthymic and nude mice, showing that unlike traffic to the spleen, this route probably does not require T cells. These migration patterns were not affected by major histocompatibility barriers, and were only seen with viable, but not glutaraldehyde-fixed, DC.(ABSTRACT TRUNCATED AT 400 WORDS)

Migration of dendritic leukocytes from cardiac allografts into host spleens. A novel pathway for initiation of rejection.

It has been a long-standing dogma that host sensitization against fully-vascularized organ allografts occurs peripherally within the graft itself. In this report we show that donor-derived MHC class II-positive (Ia+) DL migrate rapidly out of mouse cardiac allografts into the recipients' spleens where they home to the peripheral white pulp and associate predominantly with CD4+ T lymphocytes. This provides a novel route for central sensitization against fully vascularized allografts, and most likely represents a pathway by which immune responses are generated against antigens on blood-borne DL emigrating from peripheral tissues.

Phagocytosis of antigens by Langerhans cells in vitro.

Dendritic cells (DC) isolated from lymphoid tissues are generally thought to be nonphagocytic in culture. It has therefore been unclear how these cells could acquire particulate antigens such as microorganisms for initiation of primary immune responses. Lymphoid DC derive in part from cells that have migrated from nonlymphoid tissues, such as Langerhans cells (LC) of skin. The ability of LC to internalize a variety of particles was studied by electron, ultraviolet, phase, and differential interference contrast microscopy, and by two-color flow cytometry. Freshly isolated LC in epidermal cell suspensions phagocytosed the yeast cell wall derivative zymosan, intact Saccharomyces cerevisiae, representatives of two genera of Gram-positive bacteria, Corynebacterium parvum and Staphylococcus aureus, as well as 0.5-3.5-microns latex microspheres. During maturation in culture, the phagocytic activity of these cells was markedly reduced. Likewise, freshly isolated splenic DC were more phagocytic than cultured DC for two types of particle examined, zymosan and latex beads. Unlike macrophages, LC did not bind or internalize sheep erythrocytes before or after opsonization with immunoglobulin G or complement, and did not internalize colloidal carbon. The receptors mediating zymosan uptake by LC were examined. For this particle, C57BL/6 LC were considerably more phagocytic than BALB/c LC and exhibited a reproducible increase in phagocytic activity after 6 h of culture followed by a decline, whereas this initial rise did not occur for BALB/c LC. These differential kinetics of uptake were reflected in the pattern of zymosan binding at 4 degrees C, and endocytosis of the soluble tracer fluorescein isothiocyanate-mannose-bovine serum albumin at 37 degrees C. Zymosan uptake by LC from both strains of mice was inhibited in the presence of mannan or beta-glucan, although to different extents, but not by antibodies specific for CR3 (CD11b/CD18). These data indicate that zymosan uptake by LC can be mediated by a mannose/beta-glucan receptor(s) that is differentially expressed in the two strains of mice and that is downregulated during maturation of LC in culture.

Migration patterns of dendritic cells in the mouse. Homing to T cell-dependent areas of spleen, and binding within marginal zone.

Using quantitative techniques we have shown elsewhere that dendritic cells (DC) migrate from blood into the spleen, under the control of T cells. Here we traced the localization of DC within the spleen and sought to explain the means by which they entered. DC were labeled with a fluorochrome, Hoescht 33342, and injected intravenously. Spleens were removed 3 or 24 h later and DC were visualized within particular areas that were defined by mAbs and FITC anti-Igs. At 3 h most DC were in the red pulp, whereas by 24 h the majority had homed to T-dependent areas of the white pulp and may have become interdigitating cells. Lymphoid DC, isolated from spleen and perhaps normally present in blood, may thus be a migratory stage distinct from the relatively fixed interdigitating cells. We also developed a frozen section assay to investigate the interaction of DC with various lymphoid elements. When DC were incubated on sections of spleen, at 37 degrees C but not at 4 degrees C they attached specifically within the marginal zone and did not bind to T areas; in contrast, macrophages attached only to red pulp and T cells did not bind specifically. However, DC did not bind to sections of mesenteric lymph node, whereas T cells localized in particular regions at 4 degrees C but not at 37 degrees C, probably the high endothelial venules. DC may thus express "homing receptors," similar to those of T cells, for certain endothelia. We propose that T cells can modify the vascular endothelium in certain areas to allow egress of DC from the bloodstream.

Dendritic cells initiate a two-stage mechanism for T lymphocyte proliferation.

T cells oxidized with sodium periodate proliferate polyclonally in response to accessory cells. We confirmed previous work showing that DC are potent stimulators of this response. In addition, the accessory function of unfractionated mouse spleen and spleen adherent cells was markedly reduced after elimination of DC with a specific monoclonal antibody and complement. Therefore oxidative mitogenesis was used as a model to study the mechanism by which DC stimulate T cell proliferative responses. A two-stage mechanism was identified. The first stage occurred during the first 20 h of culture, required live DC, and involved the progressive release of interleukin 2 (IL-2) into the medium and acquisition of responsiveness to this growth factor. The second stage occurred between 20 and 40 h, did not require live DC, and involved DNA synthesis in response to IL-2. Similar events occurred during culture of DC with unmodified T cells (syngeneic MLR) but were quantitatively reduced. The experimental approach was to co-culture DC and T cells for up to 20 h and then kill the DC with specific antibody, or anti-Ia antibody, and complement. Subsequent proliferation was inhibited if the T cells were cultured in fresh medium. However, proliferation was restored when the lymphocytes were cultured in the original DC-T cell medium, or with a crude or a purified preparation of IL-2. IL-2 did not induce the proliferation of T cells that had been cultured in the absence of DC, and did not synergize with viable DC. We conclude that DC induce proliferation by tightly coordinating the release of, and responsiveness to, T cell growth factor or IL-2.

Migration and maturation of Langerhans cells in skin transplants and explants.

The behavior of Langerhans cells (LC) has been examined after skin transplantation and in an organ culture system. Within 24 h (and even within 4 h of culture), LC in epidermal sheets from allografts, isografts, and explants dramatically increased in size and expression of major histocompatibility complex class II molecules, and their numbers were markedly decreased. Using a new procedure, dermal sheets were then examined. By 24 h, cells resembling LC were found close to the epidermal-dermal junction, and by 3 d, they formed cords in dermal lymphatics before leaving the skin. In organ culture, the cells continued to migrate spontaneously into the medium. These observations establish a direct route for migration of LC from the epidermis into the dermis and then out of the skin. These processes are apparently induced by a local inflammatory response, and are independent of host-derived mediators. The phenotype of migratory cells was then examined by two-color immunocytochemistry and FACS analysis. The majority of migratory leukocytes were Ia+ LC, the remainder comprised Thy-1+, CD3+, CD4-, CD8- presumptive T cell receptor gamma/delta+ dendritic epidermal cells, which clustered with the LC, and a small population of adherent Ia-, FcRII+, CD11a/18+ macrophages. In contrast to the cells remaining within the epidermis of grafted skin at 1 d, the migratory cells were heterogeneous in phenotype, particularly with respect to F4/80, FcRII, and interleukin 2 receptor alpha expression, which are useful markers to follow phenotypic maturation of LC. Moreover, cells isolated from the epidermis of grafts at 1 d were more immunostimulatory in the allogeneic mixed leukocyte reaction and oxidative mitogenesis than LC isolated from normal skin, though less potent than spleen cells. The day 1 migratory cells were considerably more immunostimulatory than spleen cells, and day 3-5 migratory cells even more so, suggesting that functional maturation continues in culture. Thus, maturation of LC commences in the epidermis and continues during migration, but the cells do not need to be fully mature in phenotype or function before they leave the skin. In vivo, the migration of epidermal LC via the dermis into lymphatics and then to the draining nodes, where they have been shown previously to home to T areas, would provide a powerful stimulus for graft rejection.

Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide, tumor necrosis factor, and interleukin 1.

Dendritic cells (DC) in nonlymphoid organs can internalize and process foreign antigens before migrating to secondary lymphoid tissues to initiate primary immune responses. However, there is little information on which stimuli promote migration of DC from the tissues. Systemic administration of lipopolysaccharide (LPS), which induces in vivo production of cytokines, led to a reduction in the numbers of major histocompatibility complex class II-positive (Ia+) leukocytes in mouse hearts and kidneys: > 95% of DC were depleted 1-3 d after injection of 50 micrograms LPS. Several lines of evidence indicated that this response was due to migration of DC rather than loss of Ia expression or cytotoxic effects. In skin of treated mice, the number of Ia+ epidermal Langerhans' cells (LC) was reduced, and "cords" of Ia+ leukocytes became evident in the dermis. The latter cells expressed little NLDC145 and may have originated from recruited or resident DC progenitors. Systemic administration of recombinant tumor necrosis factor (rhTNF)-alpha resulted in a decrease in numbers of Ia+ cells in heart and kidney and of epidermal LC, and it also induced dermal cords. Administration of a rh-interleukin (IL)-1 resulted in a decrease in Ia+ cells only in renal medulla, appeared to activate a subset of epidermal LC, and induced dermal cords. Similar microgram doses of rhIL-2 had no obvious effect. Treatment with a neutralizing anti-TNF antiserum before LPS administration inhibited the depletion of LC from skin but not from heart or kidney. Therefore, TNF-alpha and IL-1 alpha may promote DC migration from nonlymphoid tissues and may have differential effects on different DC populations, but it is unclear whether they act on DC directly or indirectly (e.g., via other cytokines).

Macrophage-tropic HIV induces and exploits dendritic cell chemotaxis.

Immature dendritic cells (iDCs) express the CC chemokine receptor (CCR)5, which promotes chemotaxis toward the CC chemokines regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta. By contrast, mature DCs downregulate CCR5 but upregulate CXC chemokine receptor (CXCR)4, and as a result exhibit enhanced chemotaxis toward stromal cell-derived factor (SDF)-1alpha. CCR5 and CXCR4 also function as coreceptors for macrophage-tropic (M-tropic) and T cell-tropic (T-tropic) human immunodeficiency virus (HIV)-1, respectively. Here, we demonstrate chemotaxis of iDCs toward M-tropic (R5) but not T-tropic (X4) HIV-1. Furthermore, preexposure to M-tropic HIV-1 or its recombinant envelope protein prevents migration toward CCR5 ligands. The migration of iDCs toward M-tropic HIV-1 may enhance formation of DC-T cell syncytia, thus promoting viral production and destruction of both DC and T helper lymphocytes. Therefore, disturbance of DC chemotaxis by HIV-1 is likely to contribute to immunosuppression in primary infection and AIDS. In addition, migration of iDCs toward HIV-1 may aid the capture of R5 HIV-1 virions by the abundant DC cell surface protein DC-specific intercellular adhesion molecule (ICAM)3-grabbing nonintegrin (DC-SIGN). HIV-1 bound to DC cell-specific DC-SIGN retains the ability to infect replication-permissive T cells in trans for several days. Consequently, recruitment of DC by HIV-1 could combine with the ability of DC-SIGN to capture and transmit the virus to T cells, and so facilitate dissemination of virus within an infected individual.

Formation and kinetics of MHC class I-ovalbumin peptide complexes on immature and mature murine dendritic cells.

Dendritic cells (DC) are professional antigen-presenting cells that are able to induce primary T cell responses. Therefore, several strategies employ peptide-pulsed DC in tumor immunotherapy. For efficient antigen presentation and induction of an immune response by DC the number and stability of MHC I-peptide complexes is crucial. We studied this issue by using the antibody 25-D1.16 that specifically detects OVA peptide SIINFEKL in conjunction with H-2 Kb molecules, and determined its kinetics on mature and immature bone marrow-derived murine DC. Optimal peptide loading was reached after 8-16 h at 50 microM peptide pulse, and was comparable in serum-free versus serum-containing medium. Stimulation of DC with LPS or Poly I:C, and to a lesser extent TNF-alpha, upregulated the total number of surface MHC I molecules and thus improved peptide loading. Pulse-chase experiments revealed a constant half-life of peptide/Kb complexes independent of preceding DC stimulation or their maturation stage. The duration of peptide/Kb complex expression on mature DC, however, could be extended from 24 h to 72 h when the cultures were pretreated with LPS or Poly I:C, but not TNF-alpha. These data might have important implications for the clinical application of peptide-pulsed DC in tumor immunotherapy.

Bacterial lipopolysaccharide contamination of commercial collagen preparations may mediate dendritic cell maturation in culture.

Dendritic cells (DC) are potent antigen presenting cells, which are responsible for the initiation of naive T and T-dependent immune responses. The present studies were based upon recent reports that commercial collagen I preparations induce the maturation of human DC in vitro. We show that human blood monocyte-derived (GM-CSF and IL-4 cultured) DC pulsed on collagen I-coated plates undergo a dose-dependent increase in stimulatory capacity in oxidative mitogenesis assays. This is accompanied by the upregulation of costimulatory molecules (CD40, CD80, CD86), CD25, ICAM-1 and the DC-specific marker CD83. The maturation effect is more potent than TNF-alpha, which is a known mediator of DC function. However, bacterial lipopolysaccharide (LPS), a powerful inducer of DC maturation, was found to be present at very high levels in one commercial collagen solution that was tested. The effect of LPS upon DC maturation was similar to culture with collagen. Furthermore, a different collagen I preparation with low levels of LPS contamination was less effective at inducing DC maturation, while spiking the collagen solution with LPS prior to plastic coating equalised these effects. Finally, human monocyte-derived DC were found not to express typical collagen receptors VLA-1, 2 and 3. We therefore propose that LPS contamination may at least partially explain reported collagen I induced DC maturation.

Failure of mature dendritic cells of the host to migrate from the blood into cardiac or skin allografts.

Precisely where sensitization occurs after transplantation is uncertain, but it has been immunological dogma that sensitization to skin grafts occurs "centrally" in the draining lymph nodes. On the other hand, sensitization to fully-vascularized organs (kidney, heart, etc.) has been thought to occur "peripherally" within the graft itself. We have previously shown that mature dendritic cells migrate from the blood into the spleens of normal mice in a T cell-dependent manner, raising the possibility that circulating host dendritic leukocytes might be recruited from the blood into allografts where T cells had accumulated. This would provide a precedent for peripheral sensitization after transplantation. We examined whether 111indium-labeled mature DC host strain could migrate from the blood into cardiac or skin grafts. We were unable to detect migration into either allografts or isografts of these tissues, and found instead that the cells migrated to the spleen as in unmanipulated animals. This was despite the fact that accumulation of resting T cells was readily demonstrable in cardiac or skin allografts. In addition, we found that T cells sensitized against donor or third-party alloantigens had equal access to cardiac allografts, indicating that their migration into transplants is independent of their antigen specificity. The data of this study are discussed in the light of our other recent findings that donor DL migrate from fully-vascularized allografts into the recipients' spleens. Our current hypothesis is that allograft rejection is predominantly initiated centrally in host lymphoid tissues.

Inability of dendritic cells to prevent the blood transfusion effect in a mouse cardiac allograft model.

The beneficial effect of blood transfusions before clinical renal transplantation is well established, but this can result in sensitization of some potential first graft recipients. Dendritic cells (DC), present in human blood, are potent stimulators of immune responses in vitro and in vivo, and in different systems they can overcome immune unresponsiveness. We therefore investigated whether DC could prevent the transfusion effect in a murine cardiac allograft model. C57BL/10 (C57) hearts were normally rejected by untreated DBA/2 recipients with a median survival time (MST) of 17 days. Long-term survival (MST greater than 100 days) was induced when the recipients were transfused with C57 blood 14 days before transplantation (d-14). Similar survival times were obtained when up to 1.5 x 10(5) splenic DC were added to the transfused blood. This number of DC or as few as 10(3), in "unsorted" preparations that were 70-85% pure, similarly enhanced when transfused alone at d-14. This enhancement was probably due to the contaminating cells rather than the DC since 10(3) lymphocytes prolonged survival, but an equivalent dose of "sorted" DC (ca. 93% pure, most contaminating cells being removed by sorting on an Ortho Cytofluorograf) did not. Transfusion of unsorted preparations containing 1.5 x 10(5) DC at d-3 led to accelerated graft rejection (MST 4 days). This sensitization was most likely due to the DC because equivalent numbers of lymphocytes were ineffective. Nevertheless, if a blood transfusion was given at d-14 followed by a normally sensitizing dose of DC at d-3, graft survival was still prolonged. Thus in no case were DC able to prevent the blood transfusion effect in this strain combination. Furthermore, DC of donor origin given to DBA/2 recipients with long-surviving C57 hearts, produced by prior blood transfusion, did not trigger rejection of the hearts.

Systemic lipopolysaccharide recruits dendritic cell progenitors to nonlymphoid tissues.

Dendritic cells (DC) are thought to be the "passenger leukocytes" that sensitize the recipients of organ transplants against graft antigens and trigger allograft rejection. DC originate from MHC class II-negative (Ia-) progenitors in the bone marrow, which enter the tissues and develop into migratory cells with the specialized capacity to initiate primary immune responses. There is little information on which stimuli recruit DC progenitors to the tissues. Systemic administration of LPS to mice depletes Ia+ leukocytes from heart and kidney but recruits Ia- leukocytes (Roake JA, et al., see footnote 6). When these leukocytes were isolated and cultured overnight, Ia+ low density leukocytes developed that could stimulate primary T cell responses in vitro. Hearts from LPS-treated mice were transplanted to allogeneic recipients. One to 4 days after grafting, Ia+ donor cells were present in recipient spleens, localized to peripheral white pulp, and associated with CD4+, but not CD8+, T cells. Cells with the migratory characteristics of DC, therefore, originated from Ia- progenitors in the transplanted hearts. We conclude that LPS recruits Ia- DC precursors to the heart and kidneys. Hearts from LPS-treated donors were rejected by allogeneic recipients at the same tempo as normal hearts, implying that Ia- DC progenitors might ultimately contribute to heart graft rejection (direct sensitization). However, since hearts from cyclophosphamide-treated donors, which do not give rise to Ia+ cells in recipient spleens, were also rejected at a similar tempo, indirect sensitization could also play a role in heart graft rejection in this model.

Thymic dendritic cells: phenotype and function.

Interdigitating (IDC) cells of the thymus have been characterized in situ by their ultrastructure and phenotype. Thymic dendritic cells (DC), thought to represent their in vitro correlate, resemble splenic DC in their ability to initiate peripheral T cell responses. In vivo, however, DC of the thymus have been implicated in tolerance induction, although at one time they were thought to impart MHC-restriction on developing T cells. Our present understanding of these areas is reviewed here. An in vitro model has been developed to address directly the function of DC in the thymus. Mature DC and immature thymocytes migrate into deoxyguanosine-treated thymus lobes where they adopt a reciprocal distribution, DC homing primarily to the medulla while the thymocytes remain in the cortex. These observations support the close relationship between thymic DC and IDC and provide a powerful tool to examine the role of DC in thymocyte ontogeny.

The dendritic cell system and anti-tumour immunity.

Dendritic cells (DC) are essential for the initiation of T- and T-dependent immune responses by virtue of their capacities to acquire, process and present antigens and to deliver activation signals to mature resting T cells. In contrast, antigen presentation by other types of antigen-presenting cells (APC) may lead to peripheral T cell tolerance. This paper reviews the immunobiology of DC, and considers the possibility that immune unresponsiveness of tumour-bearing hosts may be due to clonal anergy of T cells. An important issue is whether it is possible to use DC to vaccinate tumour-free individuals, to overcome the immunological unresponsiveness of tumour-bearing hosts, and ultimately to cause rejection of primary and metastatic cancers.

Identification and isolation of CD1a positive putative tumour infiltrating dendritic cells in human breast cancer.

Identification of dendritic cells (DC) in human tissues has been technically problematic due to the lack of truly specific immunohistochemical markers for DC's. Human dendritic cells express CD1a glycoprotein at certain points in their life cycle. CD1a positive cells are present in many human tumours and have been associated with improved survival. However, little information exists concerning the separation of DC from human tumours. The current study reports that human breast carcinomas have low densities of CD1a positive cells with dendritic morphology, and details are shown of a technique for successful separation of these cells from tumour tissues.

Isolation of dendritic leukocytes from non-lymphoid organs.

These observation suggest that dendritic leukocytes from several different non-lymphoid organs in situ are functionally immature and that in this respect they more closely resemble epidermal LC than mature lymphoid DC. The exception appears to be the interstitial dendritic leukocytes from small and large intestinal lamina propria and Peyer's patches, where functional maturation could be attributed to constitutively secreted GM-CSF by lamina propria cell in situ, or alternatively to the isolation procedure which might lead to functional maturation of gut DC. After overnight culture, and possibly following organ transplantation, interstitial dendritic leukocytes may mature into potent activators of antigen-specific T-cell proliferation (immunostimulation). Further studies are needed to characterize dendritic leukocytes in solid non-lymphoid organs, and these may lead to new strategies for overcoming graft rejection by inhibiting the maturation of dendritic leukocytes after transplantation.