Endogenously Produced Interleukin 6 is an Accessory Cytokine for Dendritic Cell Hematopoiesis

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1 Endogenously Produced Interleukin 6 is an Accessory Cytokine for Dendritic Cell Hematopoiesis Frances Santiago-Schwarz, John Tucci, Steven E. Carsons Division of Rheumatology, Allergy & Immunology, Winthrop University Hospital, Mineola, New York, USA; Department of Medicine, State University of New York at Stony Brook, New York, USA Key Words. Dendritic cells Cord blood Interleukin 6 Accessory cells Hematopoiesis Abstract. The aim of this study was to investigate the role of interleukin 6 (IL-6) in normal dendritic cell (DC) hematopoiesis. We used an enzyme-linked immunosorbent assay to quantitate IL-6 levels in CD34 + progenitor cell cultures favoring monocyte (mono) development versus those supporting mono- DC growth, and studied the neutralizing effects of αil-6 antibody on DC hematopoiesis. IL-6 levels in mono cultures (GM-CSF alone) were detected by day 4 and remained constant (~100 pg/ml) for 18 days. In mono-dc cultures, higher IL-6 levels correlated with DC content and development. Shortterm mono-dc cultures initiated with GM-CSF + tumor necrosis factor (TNF) + stem cell factor (SCF) exhibited increases in IL-6 levels until day 11 (peak DC growth). By day 18, the levels had declined and cells expressing typical DC features were no longer present. Long-term mono-dc cultures sustained with GM-CSF + TNF + SCF contained the highest IL-6 levels (671 pg/ml) on day 11. In these cultures, DCs and higher IL-6 levels persisted beyond 18 days. Anti-IL-6 profoundly inhibited cell proliferation associated with DC hematopoiesis when added on days 0, 2 and 5 to GM-CSF + TNF + SCF cultures, indicating that various stages of mono-dc development rely on IL-6. There was no reduction in the T cell response when αil-6 was added to mixed leukocyte reaction cultures containing mature DCs as stimulators. Thus, αil-6 appears to downregulate developmental processes associated with optimal mono-dc growth, but not the effector functions of mature DCs. These studies substantiate the importance of IL-6 as a secondary cytokine during DC development and provide insight into another control point in the DC pathway. Stem Cells 1996;14: Correspondence: Dr. Frances Santiago-Schwarz, Division of Rheumatology, Winthrop University Hospital, 222 Station Plaza North, Suite 430, Mineola, NY 11501, USA. Received September 12, 1995; provisionally accepted September 28, 1995; accepted for publication November 15, AlphaMed Press /96/$5.00/0 Introduction Human dendritic cells (DCs) and monocytes (monos) develop simultaneously from a common progenitor when CD34 + cells from cord blood or bone marrow are treated with tumor necrosis factor (TNF) and GM-CSF [1-4]. TNF is the first signal required in this synergistic interaction and promotes high levels of GM-CSF receptors (Rs) and the inhibition of granulocyte differentiation [5]. Anti-GM-CSF severely inhibits development of all myeloid progeny in TNF + GM-CSF cultures, including monos, DCs and granulocytes. Anti-TNF selectively inhibits mono-dc development, while sparing granulocytic growth [5]. When combined with GM-CSF + TNF, stem cell factor (SCF) enhances the DC pathway without altering the developmental commitment instituted by GM-CSF and TNF [4-8]. It augments selfrenewal events of mono-dc progenitors, as assessed by increases in colony size and number, and replating capacity [4, 6]. SCF also facilitates DC survival in long-term mono-dc cultures by suppressing DC apoptosis [9]. Interleukin 6 (IL-6) is an important secondary cytokine in GM-CSF-induced monocytopoiesis and a survival factor for colony-forming unit-granulocyte-macrophage (CFU-GM) [10, 11]. It is also produced as a secondary cytokine in response to TNF, when acute myeloid leukemia cells are treated with IL-3 + TNF [12]. Moreover, the terminal development of DCs from a myelodendritic cell leukemia (ORL47) is not induced by GM- CSF + TNF or GM-CSF + TNF + SCF, but is strictly dependent on the exogenous addition of IL-6 [13]. We therefore speculated that endogenous IL-6 production might be important for the development of monos-dcs from normal CD34 + progenitors. In this study, we investigate the STEM CELLS 1996;14:

2 226 IL-6 Facilitates Dendritic Cell Hematopoiesis secondary production of IL-6 in mono-dc cultures and study the effects of αil-6 antibodies on the mono-dc pathway. Our results support that IL-6 is an accessory cytokine which promotes mono-dc hematopoiesis from normal CD34 + progenitors. Materials and Methods Enrichment of Neonatal Cord Blood-Derived Progenitor Cells Cord blood was collected from healthy fullterm infants into sterile heparinized containers during repeat caesarean sections, according to institutional guidelines. Mononuclear cells were prepared by density centrifugation on Lymphoprep gradients (endotoxin poor, Nyegaard, Norway) and placed on nylon wool columns for the isolation of nonadherent cells, as previously described [1, 4, 5]. Separation of CD34 + progenitor cells from the nonadherent population employed positive immunoselection using immunomagnetic beads (Dynabeads, Dynal Inc.; Oslo, Norway) as described elsewhere [1, 4, 5]. Liquid Cultures CD34 + cells were adjusted to cells/ml in RPMI 1640 medium (GIBCO; Grand Island, NY) containing 2 mm L-glutamine, 10 mm HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, 5% pooled normal human serum (NHS/RPMI) and incubated in Teflon vials (Scientific Specialties Service; Randalstown, MD) at 37 C in a 5% CO 2 humidified incubator. Monos and DCs were generated using previously established strategies. For induction of the mono-dc pathway, cultures were supplemented with GM-CSF + TNF (GT) and GM-CSF + TNF + SCF (GTS) at the onset of the culture period [1, 4, 5]. DCs generated under these conditions were: DR +, CD14, nonphagocytic, negative for nonspecific esterase (NSE) activity and potent stimulators of a mixed leukocyte reaction (MLR), as previously defined [14-17]. The cells exhibited classical DC features upon transmission electron microscopic examination. Birbeck granules were rare and 10% of the cells expressed CD1a antigens on day 10. Long-term mono-dc cultures were maintained by supplementing the GM-CSF + TNF + SCF cultures with the same cytokines every 48 h (GTS FED), as previously described [4, 6]. Cultures favoring mono development were instituted with GM-CSF alone [1]. Human recombinant (r)scf (Genzyme; Boston, MA) was employed at 50 ng/ml, human rtnf-α (Knoll Pharmaceuticals; Whippany, NJ) at 500 U/ml, human rgm-csf (Genzyme) at 100 U/ml. The optimal concentration for each factor was calculated after dose analysis, as previously described [1, 4, 5]. Rabbit (rb) polyclonal (IgG and IgM) α human IL-6 antibody (Ab) (Genzyme) was tested at 10 and 50 µg/ml where indicated. Normal rb serum (1:10 dilution) was used as a negative control for rb αil-6. For each experiment, cultures containing NHS/RPMI without cytokines were compared to cultures supplemented with the various cytokine combinations. For in situ analyses and examination under phase microscopy, cultures were also established in either 24-well plates or Lab-Tek chamber slides (Nunc; Naperville, IL). As previously demonstrated by us and others, these conditions do not support the development of lymphocytes [1, 3, 4]. Because endotoxin (LPS) can induce the secondary production of IL-6 [18, 19], all cultures were maintained under endotoxin poor conditions, as previously described [20]. IL-6 Production Cell-free supernatants were collected on days 4, 7, 11 and 18 from the various cultures and screened for IL-6 levels using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Genzyme, Predicta IL-6), exactly as recommended by the manufacturer. The sensitivity range of this assay is 18 pg/ml. Proliferation Proliferative events were measured on various days by the uptake of [ 3 H] thymidine and by manual hemacytometer-assisted cell counts (Improved Neubauer). For thymidine uptake, 0.5 µci of [ 3 H] thymidine (specific activity, 25 Ci/mmol, Amersham; Arlington, IL) was added to 100 µl aliquots taken from Teflon cultures and placed in 96-well microtiter plates. After 5 h of incubation, cells were harvested using an automated sample harvester and counted in a liquid scintillation counter. Results are expressed as the mean of triplicate samples and the SE was 20% in all experiments. Allogeneic MLR Cells grown under various culture conditions were removed from Teflon cultures after 11 days, centrifuged twice in RPMI, adjusted

3 Santiago-Schwarz, Tucci, Carsons 227 to equal concentrations in 5% NHS/RPMI and irradiated with a 60 Co source (for a total of 2000 rads). Varying numbers of these stimulator cells were then added to 96-well microtiter plates containing responder cells/well (nylon wool enriched T cell populations obtained from normal peripheral blood). A proliferative response was measured after seven days of incubation by adding 0.5 µci of [ 3 H] thymidine to each well and harvesting the cells as described above. Thymidine uptake in irradiated stimulator cells and responder cells cultured alone as controls was <200 cpm. Cytochemistry Cells cultured in suspension (Teflon) were prepared for Wright stain (Hemacolor) and NSE analysis by depositing them onto slides by means of cytocentrifugation (Shandon; Pittsburgh, PA). Cells in chamber slides were stained in situ. No fewer than 500 cells were analyzed. Statistics Where indicated, Student s t-test was used to analyze data using a Crunch Interactive Statistical Package. to GTS, GTS FED cultures contained three times as much IL-6 (p = 0.05). Day 11 supernatants from cells cultured in NHS/RPMI alone contained 32 ± 5 pg/ml of IL-6. GM-CSF + SCF treatment did not produce increases in IL-6 compared to GM-CSF treatment (data not shown). Only GTS FED cultures, which exhibited prolonged DC growth, contained high levels of IL-6 on day 18. The differences in IL-6 levels between GTS and GTS FED cultures on day 18 were significant (55 ± 12 versus 380 ± 175, pg/ml, respectively, p = 0.046). IL-6 Levels are Positively Associated with DC Content In Figure 2, we depict IL-6 levels in relation to the absolute number of DCs in the various cultures during peak DC growth (day 11). In cultures containing combinations of GM-CSF, TNF and SCF, DCs comprise 40%-60% of the progeny [1, 4]. Most of the remaining cells are mono-mφs which have developed simultaneously with the DCs [1-6]. With GM-CSF treatment, 5%-10% of the progeny are DCs, while the remaining cells are monos and granulocytes [1, 4]. GM-CSF cultures containing ~ DCs/ml contained the lowest levels (~83 pg/ml) of IL-6. Increasing Results and Discussion IL-6 Levels During Development Temporal analysis revealed the pattern of endogenous IL-6 production during mono/dc hematopoiesis. Figure 1 illustrates IL-6 measurements in cultures supporting mono-dc hematopoiesis (GT, GTS, GTS FED) and those favoring mono (GM-CSF) development during 18 days of culture. With GM-CSF treatment, IL-6 was detected by day 4. The levels remained constant ( 100 pg/ml) throughout the culture period. In comparison, in cultures promoting DC growth, the pattern of IL-6 production was linked with the development of DCs, as previously detailed [1, 4, 5]. In GTS cultures, IL-6 levels increased until ~ day 11 (peak DC development) and declined by day 18, when typical DC features were no longer present. On day 11, IL-6 levels in GTS cultures had more than doubled compared to GM-CSF cultures (213 ± 15 versus 83 ± 8, pg/ml, respectively, p < 0.02). For GTS FED versus GM-CSF on day 11, IL-6 levels increased eight-fold (671 ± 157 versus 83 ± 8, pg/ml, respectively, p < 0.03). Compared Fig. 1. Pattern of endogenous IL-6 production during mono/dc development. Cell-free supernatants were harvested and subject to an ELISA measuring IL-6, as outlined in Materials and Methods. Mono development: = GM-CSF. Mono-DC development: = GTS FED, = GTS, = GT. For each time point, results represent the mean of two to five experiments.

4 228 IL-6 Facilitates Dendritic Cell Hematopoiesis cultures remained unaffected (Fig. 4). Anti-IL-6 treatment did not alter the distribution of granulocytes ( 10%) or the NSE or phagocytic activity of the surviving progeny (data not shown). Cells obtained from GTS cultures treated with αil-6 Ab on day 0 exhibited similar MLR Fig. 2. Correlation of IL-6 levels with DC content on day 11. The absolute number of DCs per ml represents: Total number of cells per ml % DCs 100. The % of DCs in G (GM-CSF) = 10%, in GT, GTS and GTS FED the % = 60%. For all conditions, mono-macrophages comprised the bulk of the remaining cell population. For DC content, n = 5 for G, 6 for GT, 14 for GTS and 3 for GTS FED. The differences in DC content between groups were significant (p 0.035). levels of IL-6 (150, 213 and 671 pg/ml) were noted in GT, GTS and GTS FED cultures, which contained an average of , and DCs/ml, respectively. Effects of αil-6 Polyclonal Ab Figure 3A illustrates the effects of αil-6 Ab treatment on days 0, 2 and 5 in GTS cultures, as measured by thymidine uptake on day 8. Addition of αil-6 on days 0 and 2 yielded 50%-60% decreases in proliferation versus untreated GTS cultures (p = ). When added on day 5, αil-6 decreased proliferation by ~36% (p < 0.04). Nonimmune rb serum did not inhibit the GTS response on any day. Determination of hemacytometer-assisted cell counts revealed similar neutralizing effects of αil-6, as shown in Figure 3B. On all days, the addition of αil-6 to GTS cultures induced significant decreases (p 0.02) in the total number of cells/ml compared to the untreated GTS cultures. While αil-6 treatment reduced cell proliferation and number in GTS cultures, the proportion of surviving monos to DCs in these Fig. 3. The effect of rb polyclonal αil-6 Ab (10 µg/ml) added to GTS cultures on either day 0, 2 or 5 on proliferation. A) Peak proliferative events, as measured by thymidine uptake on day 8. For days 0 and 2 versus untreated GTS cultures, p = , n = 3-7. For day 5 versus untreated GTS cultures, p < 0.04, n = 4-7. Results represent the mean ± the SE. Nonimmune rb serum did not inhibit the GTS response. B) The total number of cells in mature mono-dc cultures, as determined by hemacytometer-assisted cell counts on day 11. Open bars = αil-6, black bars = parallel GTS cultures. For day 0, p = (n = 7), for day 2, p = 0.02 (n = 3), for day 5, p = (n = 3).

5 Santiago-Schwarz, Tucci, Carsons 229 Fig. 4. Phase photomicrographs of GTS cultures treated with αil-6 on day 0, taken on day 7. Although there were large decreases in cell density with αil-6, the morphological features of the surviving cells appeared unaffected. Cells exhibiting monocyte and DC features were present under both conditions. Original magnification = 40. stimulatory capacity to that of cells obtained from untreated GTS cultures (Fig. 5). Therefore, αil-6 treatment during mono-dc hematopoiesis did not alter the MLR capacity of DCs. The addition of αil-6 directly to MLR assays containing untreated GTS stimulators did not decrease the proliferative response (data not shown), indicating that the functional capacity of mature mono/dcs is also not affected by αil-6. The latter results are consistent with those of Leenaerts et al., who demonstrated that αil-6 did not inhibit the MLR stimulatory capacity of peripheral blood mononuclear cells [18]. Our results suggest a complex role for IL-6 in the development of DCs. Anti-IL-6 neutralized developmental processes associated with optimal mono-dc growth, but not the effector functions of mature DCs. Since αil-6 did not alter the proportion of surviving monos to DCs, it appears to affect a common mono-dc progenitor. In support of this observation, Kerst et al. described that IL-6 is a survival factor for the colony-forming unit-granulocyte-macrophage (CFU-GM), which precedes the mono-dc CFU in development [11]. Since SCF induces progenitor cell renewal, it is possible that common mono-dc progenitors are still present on days 2 and 5 in our GTS cultures. If so, they would be subject to the negative effects of αil-6. We further observed that ril-6 added to GTS cultures at the onset did not produce a significant increase in the number of mono-dcs generated (n = 3, p > 0.05), suggesting that some differentiation events must occur within the CD34 + progenitor cell compartment before the cells become responsive to IL-6 (data not shown). Recent studies also show that committed mono- DC precursors are present by day 5 in GTS cultures derived from CD34 + progenitors [21] and that DC maturation from peripheral blood precursors (contained in fractions enriched for monos) is dependent on the endogenous production of IL-6 [22]. Thus, the possibility that IL-6 also facilitates later phases of mono-dc development cannot be discounted. Jansen et al. established an important role for IL-6 in monocytopoiesis by demonstrating that the addition of αil-6 to human bone marrow progenitor cultures on day 0 inhibits the development of GM-CSF-induced monocytic colonies in semisolid cultures by about 50% [10]. The similar requirement of DC development on IL-6 provides another example of the close ontogenic relationship between monos and DCs. As suggested by Jansen et al., for GM-CSF-driven monocytopoiesis [10], IL-6 may be produced by progenitor cells and/or their offspring during mono-dc development with GM-CSF, TNF and SCF. Since monos clearly produce IL-6 during monocytopoiesis [10, 11], Fig. 5. The MLR potential of cells developing in the presence of αil-6. The slight decrease in MLR activity in day 0-treated GTS cultures was not significant (p > 0.05). Results represent the mean ±SE of triplicate samples, at stimulator:responder ratios of.025:1. One of two representative experiments is shown.

6 230 IL-6 Facilitates Dendritic Cell Hematopoiesis it is likely that monos are primary producers of IL-6 during mono-dc hematopoiesis. Compared to monos, the cytokine profile of DCs is not well-defined [15]. It has been reported that murine cultures enriched in Langerhan s cells (an immature DC element of the skin) produce IL-6, but the possible contribution of contaminating keratinocytes in these cultures was not established [23]. Our preliminary in situ analysis with polyclonal αil-6 yielded cytoplasmic staining in adherent monos, but not in adherent DCs, supporting the notion that monos are the primary producers of IL-6 in our system. IL-6 has been shown to induce the expression of adhesion molecules on a variety of cell types [24, 25]. In the myeloid lineage, upregulation of adhesion molecules by IL-6 is most effective when combined with CSF factors inducing myelopoiesis, such as GM-CSF [24]. Since several classes of adhesion molecules are elevated on DCs and these are critical for DC functions [15, 16], we speculate that some IL-6 functions on the DC pathway reflect induction of adhesion molecules on the DC surface, including those involved in DC migration and homing. The effects of αil-6 are in marked contrast to the inhibitory effects of αtnf Ab, which selectively downregulates mono-dc hematopoiesis while sparing the development of granulocytes [5]. Because of the important role of granulocytes in innate immunity, αtnf may be more useful than αil-6 in developing therapeutic strategies designed to downregulate the DC pathway in autoimmune diseases characterized by an excess of DCs, such as rheumatoid arthritis [26, 27]. In other pathological situations characterized by deficiencies in DC development such as the ORL47 myelodendritic leukemia [13], it may be useful to promote the terminal maturation of DCs with exogenous IL-6 + GM-CSF + TNF + SCF. This type of strategy would not only serve to control leukemic cell growth, but also to increase the level of immunocompetence in these patients. Acknowledgments The authors gratefully acknowledge the cooperation of the nursing staff in Labor and Delivery, Department of Obstetrics and Gynecology at Winthrop University Hospital, in the collection of cord blood. We wish to thank in particular Ms. Joanne Pastore and Ms. Rhona Schlau. We are also grateful to Mr. Bob Eggers and Mr. Jay Podbielak for assistance with the irradiation protocol and to Dr. Donald Coppock for helpful discussions. References 1 Santiago-Schwarz F, Belilos E, Diamond B et al. TNF in combination with GM-CSF enhances the differentiation of neonatal cord blood stem cells into dendritic cells and macrophages. J Leukoc Biol 1992;52: Reid CDL, Stackpoole A, Meager A et al. Interactions of tumor necrosis factor with granuloctye-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34 + progenitors in human bone marrow. J Immunol 1992;149: Caux C, Dezutter-Dambuyant C, Schmitt D et al. GM-CSF and TNF-α cooperate in the generation of dendritic Langerhans cells. Nature 1992;360: Santiago-Schwarz F, Rappa D, Laky K et al. Stem cell factor augments tumor necrosis factor-granulocyte-macrophage colony-stimulating factor-mediated dendritic cell hematopoiesis. STEM CELLS 1995;13: Santiago-Schwarz F, Divaris N, Kay C et al. Mechanisms of TNF/GM-CSF induced dendritic cell development. Blood 1993;82: Santiago-Schwarz F, Laky K, Carsons SE. Stem cell factor enhances dendritic cell development. In: Banchereau J, Schmitt D, eds. Dendritic Cells in Fundamental and Clinical Immunology. New York: Plenum Press, 1995: Saraya K, Reid CDL. Synergistic interaction between c-kit ligand (SCF), GM-CSF and TNF promotes optimal dendritic Langerhans cell proliferation from primitive progenitors in human bone marrow. In: Banchereau J, Schmitt D, eds. Dendritic Cells in Fundamental and Clinical Immunology. New York: Plenum Press, 1995: Young J, Szabolcs P, Feller E et al. Large numbers of immunostimulatory DC can be generated from human CD34 + bone marrow progenitors under the aegis of c-kit ligand, GM-CSF, and TNFα. In: Banchereau J, Schmitt D, eds. Dendritic Cells in Fundamental and Clinical Immunology. New York: Plenum Press, 1995: Santiago-Schwarz F, Borrera M, Tucci J et al. The role of apoptosis in regulating dendritic cell development and survival. Arthritis Rheum 1995;38:R35.

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