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Paracrinally stimulated expansion of early human hemopoietic progenitors by stroma-generated cyclic ADP-ribose¹

ELENA ZOCCHI^*2,3, MARINA PODESTÀ^,3, ANNA PITTO, CESARE USAI, SANTINA BRUZZONE^*, LUISA FRANCO, LUCREZIA GUIDA^*, ANDREA BACIGALUPO and ANTONIO DE FLORA^*

^* Department of Experimental Medicine, Section of Biochemistry, University of Genova, Italy;
Department of Hematology, S. Martino Hospital, Genova, Italy;
Institute of Cybernetics and Biophysics, National Research Council, Genova, Italy; and
G. Gaslini Institute, Genova, Italy

²Correspondence: Department of Experimental Medicine, Section of Biochemistry, Viale Benedetto XV/1, 16132 Genova, Italy. E-mail: toninodf{at}unige.it

SPECIFIC AIMS

Coexpression of transmembrane NAD⁺-exporting activity (via connexin 43 hemichannels) and of ectocellular ADP-ribosyl cyclase activity (via CD38 and BST-1) on stromal cells in the bone marrow microenvironment potentially enables the extracellular production of cyclic ADP-ribose (cADPR) in the hemopoietic tissue. We previously demonstrated that cADPR stimulates the proliferation of human committed hemopoietic progenitors (HP), the colony forming cells (CFC); the major aim of the present work was to study the effect of cADPR on the most immature HP, i.e., the long-term culture initiating cells (LTC-IC), which include the HP capable of repopulating the irradiated host.

PRINCIPAL FINDINGS

1. Exogenously added cADPR induces an in vitro expansion of LTC-IC
Pretreatment of cord blood-derived mononuclear cells (CB MNC) with cADPR (100 µM for 24 h) induced a significant increase (P<0.04) of the LTC-IC frequency over ADP-ribose (ADPR) -treated controls; median values were 69 vs. 39 LTC-IC/10⁶ MNC, respectively (n=14). In limiting dilution experiments, cADPR-primed and control CB MNC generated a similar number of colonies per LTC-IC: 6.0 vs. 7.5, respectively (P=0.6). Thus, cADPR priming (100 µM for 24 h) did not increase the number of colonies generated by one LTC-IC, but rather determined a true expansion of the number of LTC-IC during the subsequent 5 wk culture of the HP on the irradiated stroma.

2. CD38-transfected murine stromal cell lines produce extracellular cADPR
To test whether functionally significant cADPR concentrations could be produced by an ADP-ribosyl cyclase-positive stroma, two murine stromal cell lines were transduced with human CD38. The fibroblast cell lines M210B4, which is routinely used for long-term culture of HP, and NIH-3T3 were transfected with the sense (CD38⁺) or antisense (CD38^-) cDNA encoding for human CD38. The ecto-ADP-ribosyl cyclase activity expressed by either CD38⁺ cell line was 20-fold higher than that expressed by human stroma, obtained from normal bone marrow (BM) or CB through in vitro expansion of the few mesenchymal cell precursors present in BM and CB MNC (<10 and <3 cells/10⁶ MNC, respectively): ADP-ribosyl cyclase activities were 0.6 and 0.03 nmol cADPR/min/mg for transfected murine cell lines and native human stroma, respectively. GDP-ribosyl cyclase activities were 10-fold higher than the corresponding value of ADP-ribosyl cyclase for each cell type. However, occurrence of ectocyclase activity per se would not be sufficient to generate cADPR in the medium unless in the presence of its substrate, i.e., extracellular NAD⁺. This can be accounted for by release of intracellular NAD⁺ into the medium, mediated by NAD⁺ transport across the plasma membrane. M210B4 and 3T3 are among a number of cell lines we had previously shown to express Connexin 43 (Cx43) -mediated, NAD⁺-transporting activity on the plasma membrane. This feature was conserved in the transduced cell lines: NAD⁺ efflux occurred across the membrane of intact CD38^- cell lines, as well as of human BM- and CB-derived stroma (Fig. 1 ). Indeed, extracellular cADPR was detectable in the supernatant from confluent CD38⁺ feeder layers: its concentration was similar for 3T3 and M210B4 cells and was estimated to be 0.4 ± 0.03 nM by a sensitive, cADPR-specific RIA.

View larger version (22K): [in this window] [in a new window]   Figure 1. Release of NAD+ from intact CD38- murine cell lines and from human hemopoietic stroma. Efflux of NAD+ from intact CD38- murine M210B4 () and 3T3 () cell lines and from confluent layers of human BM-derived () or CB-derived () hemopoietic stroma. Each point is the mean of duplicate assays, performed on 3 different samples, giving comparable results.

3. Stroma-produced cADPR increases the LTC-IC frequency of MNC cocultured over CD38⁺ feeders
The LTC-IC frequency was significantly higher (P<0.04) for MNC cocultured for 24 h on the CD38⁺, cADPR-producing feeders (then transferred on CD38^- layers for 5 wk) than for control MNC, cultured continuously over CD38^- feeders: median values were 80 vs. 29 LTC-IC/10⁶ MNC, respectively (n=9). Similar results were obtained with the stromal cell lines 3T3 and M210B4. Addition of NAD⁺-glycohydrolase (2 U/ml) to the cocultures during the first 24 h prevented the stimulatory effect of the CD38⁺ feeder on LTC-IC frequency by removing the substrate for ectocellular cADPR production. LTC-derived colonies grown in semisolid medium were harvested and cells were reseeded in growth factors-supplemented methylcellulose to evaluate growth of second generation colonies. Although control cells cultured on CD38^- feeders did not produce colonies beyond the second generation, cells cultured for 24 h on CD38⁺ feeders produced colonies up to the fifth generation, with a median expansion of the number of LTC-derived colonies of 250-fold that of controls (range 77–420, n=5). A comparable increase in LTC-IC frequency after 5 wk LTC was observed when CB MNC were cultured for 24 h over CD38^- feeders supplemented with the purified ADP-ribosyl cyclase from the invertebrate Aplysia californica (0.3 nmol cADPR/min), then transferred for LTC over CD38^- feeders.

4. Coculture of MNC over CD38⁺ feeders increases the [Ca²⁺]_i of MNC
Since expansion of human HP by extracellularly added cADPR was causally related to the increased [Ca²⁺]_i it induced on target cells, we investigated the effect of a 24 h coculture of CB MNC over either CD38⁺ or CD38^- feeders on the [Ca²⁺]_i of MNC. During the first 24 h culture over CD38⁺ 3T3 feeders, the [Ca²⁺]_i of MNC increased from a basal value of 20 ± 1 nM to a maximal value of 43 ± 2 nM. Prolonging the coculture did not result in any further calcium increase. Preincubation of MNC for 1 h with 20 µM 8-Br-cADPR, a cADPR antagonist, prior to the coculture prevented the subsequent calcium increase. When MNC were cultured over CD38^- feeders, the [Ca²⁺]_i kept constant at 20 ± 1 nM, which is the same value recorded in freshly isolated MNC. Incubation of MNC with exogenously added cADPR in the absence of stroma induced a higher [Ca²⁺]_i increase at 24 h, i.e., 92 ± 5 nM. Upon removal of cADPR by either washing the cells or a transfer from the CD38⁺ onto the CD38^- feeders, the [Ca²⁺]_i of MNC decreased slowly to basal values within 48 h. Comparable results were obtained with the M210B4 CD38^± feeders.

5. Long-term culture of MNC over CD38⁺ layers inhibits colony growth via an increased interferon (IFN-) production by the engineered stroma
When MNC were maintained on CD38⁺ feeders for the entire duration of the liquid culture (5 wk), however, a decrease in the total cell number and colony output was observed throughout LTC. In these conditions, the LTC-IC frequency dropped dramatically to 10% of control values recorded on MNC cultured over CD38^- feeders (Fig. 2 ). Similar results were observed with both stromal cell lines. The same effect (10-fold reduction in LTC-IC frequency) was observed when cADPR (100 µM) was added twice weekly to MNC maintained on the CD38^- feeder for 5 wk, but not when added to cells cultured in the medium conditioned by CD38^- feeders without the stromal layer. Accordingly, the decrease of LTC-IC output observed in the cocultures was not elicited by cADPR itself, but by its interaction with the stromal layer: the presence of extracellular cADPR, either endogenously produced by the transduced stroma or exogenously added to the CD38^- feeder, might induce (over)expression of factor(s) inhibiting hemopoiesis, possibly via the increased [Ca²⁺]_i induced by the cyclic nucleotide in stromal cells as in MNC. Indeed, both CD38⁺ cell lines released into the medium fivefold more IFN-, one of the most potent hemopoiesis-inhibiting cytokines, than the corresponding CD38^- controls: 6.5 ± 1.2 vs. 1.3 ± 0.2 pg/ml, respectively, for M210B4 (Fig. 2) and 3.8 ± 0.8 vs. 0.7 ± 0.2 pg/ml, respectively, for 3T3. Addition of the cADPR antagonist 8-Br-cADPR (20 µM) to the medium inhibited IFN- production by the CD38⁺ stroma, demonstrating a causal correlation between the increased [Ca²⁺]_i induced by autocrine cADPR production in the stromal cells and cytokine generation. Moreover, after incubating the CD38^- feeder with 100 µM cADPR for 1 wk, production of IFN- was increased to values similar to those observed for CD38⁺ cells (Fig. 2) . When CB MNC were cultured in transwells over a mixed feeder, obtained by diluting the CD38⁺ with the CD38^- cells in a proportion of 1:10, respectively, a fourfold increase of LTC-IC output over controls cultured over CD38^- cells was observed. Dilution of the CD38⁺ feeder to yield an ectocyclase activity as low as that of native hemopoietic stroma decreased IFN- induced growth inhibition and allowed the stimulatory effect of cADPR to become apparent (Fig. 2) .

View larger version (27K): [in this window] [in a new window]   Figure 2. Effects of long-term coculture of human CB MNC over CD38+ murine stromal cells. LTC-IC frequency of MNC cultured for 5 wk over CD38- (control), CD38+, or mixed CD38± M210B4 feeders (white bars). IFN- concentration in the medium conditioned by the feeders (black bars). *In this experiment, CB MNC were cultured for 24 h over the CD38+ feeder and then for 5 wk over the CD38- feeder.

CONCLUSIONS AND SIGNIFICANCE

The present results complete our previous observations on the effects of extracellular cADPR on the proliferation of human committed HP. Here we demonstrate that the same protocol of cADPR treatment also increases the proliferation and self-renewal of the LTC-IC, which represent the most immature HP, i.e., those responsible for repopulation of the irradiated host. It is noteworthy that a 24 h incubation of the cells with cADPR is sufficient to induce a priming effect whose consequences become apparent after 5 wk culture.

The second new finding provided by the present work is the demonstration that a similar (threefold) expansion of human LTC-IC is also produced by a 24 h coculture of MNC over murine stromal cell lines transfected with CD38: expression of ectocellular ADP-ribosyl cyclase activity, combined with the presence of an NAD⁺-releasing activity (recently demonstrated in our laboratory to be mediated by Connexin 43 hemichannels), enables the generation of cADPR at the outer surface of these engineered cell lines, as well as of native, weakly CD38⁺ human hemopoietic stroma. This coculture setup was intended to mimic the physiology of the BM microenvironment: here, HP grow in close contact with the stroma, a mixed cell population including fibroblasts, osteoclasts, and stromal cells, the latter expressing the ectocyclase activity of BST-1. The present data seem to shed light on the molecular mechanisms underlying several recent observations demonstrating an important role of Cx43 expression in the physiology of hemopoiesis. A paracrine interaction between a cADPR-producing stroma and cADPR-responsive parenchymal cells (Fig. 3 ) is not unprecedented; we recently described a similar interplay between epithelial mucosa and the underlying myocytes in bovine tracheal smooth muscle. In that tissue, cADPR produced by cyclase-positive epithelial cells increases both the [Ca²⁺]_i and the contractile response of myocytes to acetylcholine. Thus, the paracrine production of cADPR by a cyclase-expressing stroma seems to be a recurring motif in the physiology of mammalian tissue microenvironments where the targeted delivery of this cyclic nucleotide to cADPR-sensitive parenchymal cells is ensured by proximity between the two cell types.

View larger version (15K): [in this window] [in a new window]   Figure 3. Bone marrow microenvironment: NAD+/cADPR/IFN--mediated paracrine effects of stromal cells on the proliferation of hemopoietic progenitors (HP). Release of NAD+ from stromal cells across connexin 43 hemichannels is followed by CD38-catalyzed generation of cADPR. A fraction of the cyclic nucleotide is transported by CD38 itself into the stromal cell (autocrine loop), where it elicits a Ca2+-mediated overproduction of IFN- that can inhibit proliferation of HP. Extracellular cADPR permeates across the plasma membrane of HP (paracrine loop) and elicits a calcium-mediated expansion of the cells.

Finally, the third new finding emerging from these results is that the increased [Ca²⁺]_i induced in the CD38⁺-transfected stroma by autocrine cADPR generation determines an increased production of IFN-, a potent hemopoiesis-inhibiting cytokine. The clinical implications of this finding are potentially far-reaching: an extensive invasion of the hemopoietic bone marrow by activated lymphocytes, expressing high levels of CD38, is a common feature of aplastic syndromes and poorly engrafted BM transplants, where immature HP, though present in the BM, do not proliferate despite high blood levels of endogenous hemopoiesis-stimulating cytokines. The local generation of extracellular cADPR by these immune cells could induce production of IFN- by the stroma, similar to what was observed in this study for the cADPR-hyperproducing, transfected stromal cell lines. The low ADP-ribosyl cyclase activity expressed by normal human stroma (20-fold less than the transfected feeder cell lines used in this study) thus appears to be the best strategy to provide HP with growth-promoting, extracellular cADPR, while avoiding the risk of a cADPR-induced overproduction of growth-inhibiting IFN- by the same stroma (Fig. 3) .

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0803fje ; to cite this article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0803fje

³ These authors contributed equally to the work.

³ These authors contributed equally to the work.

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