Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells

A panning method has been developed to enrich Langerhans cells (LC) from murine epidermis. In standard culture media, the enriched populations progressively lose viability over a 3-d interval. When the cultures are supplemented with keratinocyte-conditioned medium, LC viability is improved and the cells increase in size and number of dendritic processes. Accessory function, as monitored by stimulating activity in the mixed lymphocyte reaction (MLR), increases at least 10- 20-fold. The conditioned media of stimulated macrophages and T cells also support the viability and maturation of cultured LC. A panel of purified cytokines has been tested, and only granulocyte/macrophage colony-stimulating factor (GM-CSF) substitutes for bulk-conditioned medium. The recombinant molecule exhibits half-maximal activity at 5 pM. Without activity are: IL-1-4; IFN-alpha/beta/gamma; cachectin/TNF; M- and G-CSF. A rabbit anti-GM-CSF specifically neutralizes the capacity of keratinocyte-conditioned medium to generate active LC. However, GM-CSF is not required for LC function during the MLR itself. We conclude that the development of immunologically active LC in culture is mediated by GM-CSF. The observation that these dendritic cells do not respond to lineage-specific G- and M-CSFs suggests that LC represent a distinct myeloid differentiation pathway. Because GM-CSF can be made by nonimmune cells and can mediate the production of active dendritic cells, this cytokine provides a T-independent mechanism for enhancing the sensitization phase of cell-mediated immunity.

of the contaminating keratinocytes had adhered to the dishes. The nonadherent population with >95% LC was removed by pipetting and cultured an additional 0-3 d. Before testing accessory function, these cultured LC were washed free of most of the culture medium by centrifuging the cells in the microculture plates . We spun the plates twice, each time at 1,000 rpm for 2 min in a Sorvall RT6000 centrifuge followed by resuspension in fresh medium . The cells were finally resuspended to 0.2 ml for viable cell counts and for use as accessory cells in stimulating the primary mixed leukocyte reaction (MLR) .

Conditioned Media
Keratinocyte-conditioned Medium (KCM). Epidermal cell suspensions were plated in 100mm plastic dishes (3003 ; Falcon Labware) at 15-20 X 106 cells/plate in culture medium (see above) . After overnight culture, few if any LC but most of the keratinocytes had adhered forming a dense but subconfluent monolayer (1) . Nonadherent cells were removed by pipetting over the monolayer . The adherent cells were cultured for 2-3 d to provide KCM . Three different batches of KCM were prepared in this way and found to be active in supporting the maturation of enriched LC in culture (see Results) . The medium was centrifuged at 280 g, filtered through 0-45-jum Millipore filters, aliquoted, and stored frozen at -20'C .
Natural Sources of CSFs . The WEHI and L cell lines were used to provide conditioned media with mouse IL-3 (12) and M-CSF respectively (13). Both media actively supported the development of macrophages in liquid cultures of 0.5 X 106 mouse bone marrow cells in 16-mm plastic wells in DMEM with 10% FCS and antibiotics. Purified natural mouse M-CSF (13) was kindly provided by Dr. R. Stanley (Albert Einstein Medical College, Bronx, NY) .
MLR-conditioned Medium . Primary MLRs were generated between 3 X 104 dendritic cells and 3 X 106 allogeneic T lymphocytes in 1-ml macrocultures (14). The conditioned medium was collected at 3 d.

Anticytokine Antibodies
Three high-titer polyclonal antisera were tested for neutralization of keratinocyte conditioned media . Rabbit anti-mouse GM-CSF serum (16) was kindly provided by Dr. S. Gillis, Immunex Corp. A 1 % vol/vol dose fully neutralized 10 ng/ml rGM-CSF . Ig fractions of rabbit anti-mouse rTNF and sheep anti-mouse natural IFN-a/# were gifts of Dr. E. A. Havel], Trudeau Institute and were used at a dose of 1-5 X 10' neutralizing U/ml. The antibodies were mixed with conditioned media for 1 h before addition to the LC cultures .
Accessory Function Accessory function was tested in the primary MLR. It was shown previously that the capacity of primary populations of leukocytes to act as MLR stimulators required some accessory function m addition to the expression ofMHC-encoded transplantation antigens . For example, Ia' lymphoid dendritic cells and cultured epidermal LC were powerful MLR stimulators (1,4), while freshly isolated LC and IFN-.y-stimulated macrophages were weak in spite of the expression of large amounts of class 11 MHC products (1,4,8,14) . Therefore stimulatory activity in the primary MLR was used as a measure ofthe accessory function that is required for the activation of antigen-specific, resting T cells . We added graded doses of LC, or splenic dendritic cells (10) to a fixed dose of 3 x 105 allogeneic, la-, nylon wool-nonadherent, spleen or lymph node T cells in 0.2-ml culture medium in flat microtest wells. The LC were used with or without treatment with 900-1,500 rad of ionizing "'Cs irradiation, with similar results . On day 4 or 5 of the MLR, 1 J,Ci [5H]TdR was added, and ['H]TdR uptake was measured 8 h later. Standard deviations of triplicate cultures were <10%, and were not shown.

Results
Enrichment ofLC by Panning. la' LC compose -1 % of ear epidermal suspensions (1), so that an enrichment method had to be developed to study the properties of LC in the absence of keratinocytes . A panning technique provided LC in sufficient purity. The bulk of the epidermal keratinocytes were killed with anti-Thy-I and complement, and then the LC were positively selected by exposure to specific mAb and panning on anti-Ig-coated plates . In the first experiments we used anti-la, and in subsequent experiments, anti-leukocyte common antigen . Neither mAb bound significantly to keratinocytes (20).
By phase-contrast and immunofluorescence microscopy ( Fig. 1, A and B), it was evident that panning enriched the LC from a starting incidence of -I% to a level of 80-85% . A small subpopulation of LC stained more brightly with antila (arrows, Fig . 1, B and C). Quantitation by flow cytometry revealed that the mean level of LC la approached that of the la-rich dendritic cells from spleen ( Fig. 1 C). Staining with the mAb FITC-1121-2, which specifically stains I-Ad and not I-A k LC ( Fig. 1 C), was used to monitor LC content in subsequent experiments .
To test the effect of the LC enrichment procedure on accessory function, the MLR-stimulating activity of splenic dendritic cells and cultured LC was evaluated with or without treatment with anti-Thy-1 and complement followed by panning . In eight experiments, panning shifted the dose-response curve of MLR-stimulating function by a factor of two at most, and sometimes not at all (Fig. 2, A and B, respectively) .
When fresh LC were tested as MLR stimulators and compared with splenic dendritic cells (Fig. 2C) or to cultured LC (Fig. 2D), the day 0 LC were 10-20 times less active, which is in accordance with previous results in which LC were enriched by another method (4) . We conclude that LC can be significantly enriched by panning, and that the use of this method allows one to observe the marked increase in accessory function that occurs during bulk epidermal culture .
LC Viability and Function Is Mediated by KCM . LC were isolated from fresh epidermal suspensions and cultured with or without supplementation with KCM . During the first day of culture, many of the contaminating keratinocytes adhered to the surface of the culture dish, and then the nonadherent, highly enriched LC could be transferred to fresh wells . LC morphology, expression of la, and viability ( Fig. 3A) were not measurably changed by the addition of KCM during the first day of culture . The expression of la increased fivefold with or without KCM (not shown), as noted before in bulk epidermal cultures (3). However, the addition of KCM was critical for viability on the second and third days of culture . In the absence of KCM, viable cell yields fell markedly, while with KCM, viable FIGURE 1 . Features of LC after enrichment by panning. A. Phase-contrast microscopy following adherence to poly-L-lysine-coated multiwell slides . Most of the cells have the appearance of leukocytes. The small numbers of keratinocytes (black arrows) are larger, have a smooth surface, and often contain cytoplasmic granules. x 300. B. Immunofluorescence with anti-Ia. Same field as A, but stained with FITC-1321-2, anti-I-A b.d . Note that some of the LC (white arrows) stain more brightly . This fraction typically is <5% of the total Ia' cells. C. Cytofluorography . LC were panned from (C x D2)F, (I-A d ) and A (I-A'°) mice with antileukocyte mAb and compared with (C x D2)F, spleen dendritic cells (DC) (---). The staining reagents were FITC-1321-2, anti-I-A b,d (thick line) or the isotype-matched, IgG2b FITC-Thy-1 .2 as control (dotted line). Thy-1 is expressed on contaminating keratinocytes but not LC (20). Note that FITC-1321-2 stains I-Ad but not I-A' LC, and that the mean level of staining is comparable to splenic DC . The small subset of Ia-rich LC is evident (arrow). after culture in a dose of 10-30% vol/vol KCM. We conclude that the viability and function of cultured LC is markedly enhanced in KCM.
Effects of Other Conditioned Media on LC Viability and Function. The conditioned medium from LPS-stimulated macrophages was, like KCM, active in supporting the viability and function of cultured LC (Fig. 5A). Active medium was obtained from all LPS-treated macrophage populations that we tested, including the J774 and RAW cell lines, thioglycollate-elicited peritoneal macrophages, and Fc receptor-positive splenic adherent cells. LPS had no direct effect on cultured LC. Even when mixed with the medium from unstimulated macrophages, LPS did not enhance LC viability or function (not shown). rIFN-y and r1L-1 a were weak or inactive in stimulating the release of the required cytokines from macrophages (Fig. 5A) . In contrast to mouse macrophages, LPS-stimulated human monocytes did not produce a factor that could act on mouse LC (not shown) . The conditioned medium from the syngeneic and allogeneic MLR also mediated the maturation of LC in culture (data not shown) . However, the medium from the WEHI cell line and from L cells, which are used as standard sources of IL-3 and M-CSF, were repeatedly inactive in inducing LC maturation (Fig. 5B) . These media did stimulate the expected development of macrophages in marrow cultures (not shown) .
GM-CSF Is the Factor that Mediates the Maturation of LC in Culture . A panel ofpurified cytokines (Table I)  However, when rGM-CSF (Fig. 6) and purified natural GM-CSF (not shown) were evaluated, the LC behaved identically to cultures supplemented with KCM or with macrophage-conditioned medium . In the presence of low doses of GM-    CSF, the LC remained viable, developed many processes, stained brightly with anti-la mAb, and become very active MLR stimulators (Fig. 6, A-C) . Halfmaximal activity was observed at^-5 pM (0 .1 ng/ml; Fig. 6C). Three polyclonal antisera were then evaluated to test if the function of KCM could be inhibited. Rabbit anti-GM-CSF blocked the effect of KCM, and the block was reversed by adding back an excess (100 ng/ml) of rGM-CSF (Fig. 7A). Rabbit anti-TNF and sheep anti-IFN-a//3 did not block the effect of KCM (Fig.  7B). Taken together, the results indicate that GM-CSF is the principal if not exclusive mediator of LC maturation in culture.
Effects of GM-CSF on the MLR. To ensure that the observed effects of GM-CSF were on the maturation of LC before addition to the MLR, we added GM-CSF to MLRs that were induced by graded doses of cultured LC (Fig. 8) or splenic dendritic cells (not shown). Anti-GM-CSF did not block the MLRstimulating activity of LC that had matured in the presence of cytokine (Fig.  8A). Exogenous GM-CSF did not enhance the MLR, but it did induce myeloid colonies and some background proliferation in the unstimulated T cells (Fig.  8B). We conclude that GM-CSF mediates the immunologic maturation of LC in culture, but it does not influence subsequent dendritic or T cell function in the MLR.

Discussion
LC Enrichment by Panning. To study the mechanism whereby epidermal LC become active accessory cells during culture (1,2,4), it was necessary to separate this trace cell type from most of the keratinocytes in the starting epidermal suspension . We considered several enrichment methods at the outset . We were unable to remove all keratinocytes by cytotoxicity approaches (anti-Thy-1 or antidesmosomal antibodies and complement). Cytotoxicity was useful as a first step, however, and provided a preliminary enrichment from an initial level of^-1 LC to a level of 7-15% LC. Cell sorting of these partially enriched populations on the FACS could provide LC in >95% purity (4), but we encountered substantial losses in yield and viability during the sorting procedure. Therefore we coated the epidermal suspensions with either antileukocyte or anti-la mAb, washed, and panned on dishes coated with anti-Ig. Panning depleted >90% of the LC from the applied suspension and did not markedly reduce the accessory function of the attached cells (Fig. 2). The panned LC were sufficiently pure to establish a dependence on exogenous cytokines for viability and function (Figs.

3-8) .
The Effects of Keratinocyte-conditioned Medium and GM-CSF on Cultured LC. The addition of appropriate cytokines to enriched populations of fresh LC had three effects over a 2-3-d period of culture: cell viability was maintained at "50% of the starting inoculum; the LC became larger and exhibited many processes and veils; and stimulating capacity in the primary MLR increased 10-20-fold. As a result of the combined effects on viability and function, the stimulating activity of 3-d cultured LC populations was typically enhanced 100fold by exogenous cytokines.
The actual increase in the stimulating activity of individual LC might have been even greater than is suggested by our data (Fig. 4). This is because some of the activity that was seen with fresh populations could be due to a fraction of larich LC (Fig. 3, B and C) that either were in the process of maturing in situ or had been exposed to small amounts of GM-CSF during the preparation of the epidermal suspension . It is possible that the majority of LC in the starting population have insignificant accessory function.
The increased accessory activity that was induced by KCM or GM-CSF might be due to three effects. One relates to improved viability (Fig. 3), which is a characteristic effect of CSFs on their target cells (21,22). We suspect that enhanced viability is not the only effect of GM-CSF since, in isolated instances (perhaps related to higher levels of contaminating keratinocytes), LC viability was maintained close to the level of that seen with GM-CSF, but little or no increase in accessory function was observed . A second factor is the increase in the level of la or class II products that occurs in culture (3). Freshly isolated LC have similar amounts of la to splenic dendritic cells (Fig. 1), but are much less active as MLR stimulators (Fig. 2C). We have also found that la levels increase in cells that are cultured in the absence of KCM for a day, yet there is little increase in accessory function (M. Witmer-Pack, unpublished observations). Therefore the levels of la on LC do not seem to be the sole determinant of the level of accessory function . We propose that GM-CSF exerts a third differentiation effect that allows the LC to become an active accessory cell comparable to the dendritic cells isolated from lymphoid organs and lymph. This differentiation results in an accessory cell that can cluster and activate resting, antigen-specific T cells in a manner analogous to that seen with lymphoid dendritic cells (4).
Implications of Our Data in Pinpointing the Lineage of Langerhans Cells. Both LC and lymphoid dendritic cells are bone marrow derived (23,24), but their precise lineage is not established. It is of interest that GM-CSF mediates differentiation of both major types of myeloid cells, granulocytes and macrophages, whereas G-and M-CSF are more lineage specific (21,22). Because LC are influenced by GM-CSF but not by G-or M-CSF, we suggest that LC represent a type of myeloid lineage that is distinct from typical phagocytes. This would be consistent with prior data demonstrating stable differences in phenotype and function between dendritic cells and mononuclear phagocytes (25).
Comparison of the Effects of GM-CSF on LC with the Effects of Other Cytokines on Accessory Cells. It is becoming evident that the accessory function of dendritic cells for primary T cell responses like the MLR can be increased by cytokines. IL-I amplifies the function of lymphoid dendritic cells about threefold, but the cells are already very active before exposure to cytokine (26) . GM-CSF does not increase the function of mature LC (Fig. 8) or splenic dendritic cells (data not shown) . It instead acts on freshly isolated LC, which quantitatively are weak stimulators of the MLR and polyclonal T cell mitogenesis (1,4). GM-CSF therefore may be specialized to mobilize active dendritic cells from a weakly active, nonlymphoid tissue source .
Two other T cell-derived products enhance the expression of class II MHC products but do not appear to induce accessory cells for primary responses. IFN-.y increases the expression of la on mononuclear phagocytes (27) and many nonleukocytes. The lymphokine-treated macrophages do not act as stimulators of the primary MLR (8,26), but are better presenting cells for sensitized CD4+

4 6 GM-CSF MEDIATES LANGERHANS CELL MATURATION IN VITRO
T lymphocytes (14). IL-4 or BSF-1 is a cytokine that enhances la on small mouse B lymphocytes (28). Here too the upregulated B cells are weak in stimulating a primary MLR but are active in presenting alloantigens to freshly sensitized T lymphoblasts U. Metlay, M. Birkeland, R. Steinman, and E. Pure; unpublished results) .
It is noteworthy that both cytokines that modulate the function of dendritic cells for primary responses can be released in the absence of sensitized T cells. IL-1 and GM-CSF are produced by such non-T cells as macrophages and keratinocytes. Therefore these cytokines could be released during the afferent limb of the immune response, perhaps in nonlymphoid organs where many antigens are first deposited, where they may be critical for the production of active dendritic cells. In contrast, IFN-y and IL-4 are the products of T cells that have already begun to respond to antigen . IFN-y and IL-4 might be expected to enhance T cell-macrophage or T-B cell interactions in the efferent limb of immunity .
We are extending our studies to test if GM-CSF alters dendritic cell function in situ. Given the role of GM-CSF as a mediator of dendritic cell maturation in culture, we are interested in testing if this cytokine can mobilize dendritic cells from tissues and/or act as an adjuvant during primary immune responses.

Summary
A panning method has been developed to enrich Langerhans cells (LC) from murine epidermis. In standard culture media, the enriched populations progressively lose viability over a 3-d interval . When the cultures are supplemented with keratinocyte-conditioned medium, LC viability is improved and the cells increase in size and number of dendritic processes. Accessory function, as monitored by stimulating activity in the mixed lymphocyte reaction (MLR), increases at least 10-20-fold . The conditioned media of stimulated macrophages and T cells also support the viability and maturation of cultured LC. A panel of purified cytokines has been tested, and only granulocyte/macrophage colony-stimulating factor (GM-CSF) substitutes for bulk-conditioned medium. The recombinant molecule exhibits half-maximal activity at 5 pM. Without activity are: IL-1-4 ; IFN-a/,Q/'y; cachectin/TNF; M-and G-CSF. A rabbit anti-GM-CSF specifically neutralizes the capacity of keratinocyte-conditioned medium to generate active LC. However, GM-CSF is not required for LC function during the MLR itself.
We conclude that the development of immunologically active LC in culture is mediated by GM-CSF. The observation that these dendritic cells do not respond to lineage-specific Gand M-CSFs suggests that LC represent a distinct myeloid differentiation pathway. Because GM-CSF can be made by nonimmune cells and can mediate the production of active dendritic cells, this cytokine provides a Tindependent mechanism for enhancing the sensitization phase of cell-mediated immunity .