Heparanase promotes engraftment and prevents graft versus host disease in stem cell transplantation

Abstract

Background: Heparanase, endoglycosidase that cleaves heparan sulfate side chains of heparan sulfate proteoglycans, plays important roles in cancer metastasis, angiogenesis and inflammation.

Design and methods: Applying a mouse model of bone marrow transplantation and transgenic mice over-expressing heparanase, we evaluated the effect of heparanase on the engraftment process and the development of graft-versus-host disease.

Results: Analysis of F1 mice undergoing allogeneic bone marrow transplantation from C57BL/6 mice demonstrated a better and faster engraftment in mice receiving cells from donors that were pretreated with heparanase. Moreover, heparanase treated recipient F1 mice showed only a mild appearance of graft-versus-host disease and died 27 days post transplantation while control mice rapidly developed signs of graft-versus-host disease (i.e., weight loss, hair loss, diarrhea) and died after 12 days, indicating a protective effect of heparanase against graft-versus-host disease. Similarly, we applied transgenic mice over-expressing heparanase in most tissues as the recipients of BMT from C57BL/6 mice. Monitoring clinical parameters of graft-versus-host disease, the transgenic mice showed 100% survival on day 40 post transplantation, compared to only 50% survival on day 14, in the control group. In vitro and in vivo studies revealed that heparanase inhibited T cell function and activation through modulation of their cytokine repertoire, indicated by a marked increase in the levels of Interleukin-4, Interleukin-6 and Interleukin-10, and a parallel decrease in Interleukin-12, tumor necrosis factor-alfa and interferon-gamma. Using point mutated inactive enzyme, we found that the shift in cytokine profile was independent of heparanase enzymatic activity.

Conclusions: Our results indicate a significant role of heparanase in bone marrow transplantation biology, facilitating engraftment and suppressing graft-versus-host disease, apparently through an effect on T cell activation and cytokine production pattern.

Figure 1. Heparanase potentiates engraftment of WBC.

F1 mice were sublethally irradiated (750 cGy) and transplanted intravenously with 10×10⁶ spleen cells taken from heparanase (5 µg/mouse/day, i.p. for 5 days) or saline (control) treated C57BL/6 mice. The recipient mice were treated with heparanase (5 µg/mouse/day, i.p.) from the day of transplantation until day +7. Control recipient mice were injected with saline alone. Each group consisted of 8 mice. Heparanase treatment of both the donor and recipient mice caused a significant increase in the mean WBC count on day +14 post transplantation. 1.36×10⁹/L (range 1.2–1.68×10⁹/L) (□) vs. 0.48×10⁹/L (range 0.3–0.74×10⁹/L) in the control group (▪). Significantly higher WBC counts were maintained in the heparanase treated group 3 weeks post transplantation. Chimerism was assessed by the ameloginin gene expression method. Each bar represents mean ± SD (n = 8 mice) and the experiment was performed 3 times with similar results.

Figure 2. Effect of heparanase on GVHD.

A. Prolonged survival of mice treated with heparanase. Mice were sublethally irradiated (750 cGy) and transplanted i.v with 10×10⁶ spleen cells from heparanase treated or un-treated C57BL/6 mice (5 µg/mouse/day, i.p. for 3 days). The recipient mice were injected with heparanase (5 µg/mouse/day, i.p. daily) from the day of transplantation until day +7, or with saline for the same period of time. Altogether, 4 experimental groups (10 mice each) were tested: Donor and recipient mice treated with heparanase (•); Only donor mice treated with heparanase (□); Only recipient mice treated with heparanase (○); Donor and recipient mice treated with saline alone (Δ). A significant prolongation of survival was documented when both the donor and recipient mice were treated with heparanase (•), as compared to the control group where both the donor and recipient mice were treated with saline alone (Δ). In the two other groups, where heparanase was administered to either the donors (•) or recipients (□), a partial effect was achieved. B. Mice treated with different doses of heparanase. Mice were sublethally irradiated (750 cGy) and transplanted with 10×10⁶ spleen cells from heparanase treated C57BL/6 mice (5 µg/mouse/day, i.p. for 3 days). The recipient mice were sub-grouped with each arm (8 mice each) receiving a different dose of heparanase per day. Injections were given from the day of transplantation as follows: 1 µg/mouse/day, for 7 days until day +7 post transplantation (□); 5 µg/mouse/day, for 7 days until day +7 post transplantation (○); 35 µg/mouse twice weekly (Δ); Both the donor and recipient mice treated with saline alone, as control (•). All the control mice died of GVHD. In contrast, all the mice treated with 35 µg heparanase/mouse and all the mice (except one in each group) in the two other groups, remained alive until the end of the experiment (>45 days). Mice that received 1 µg heparanase/mouse/day exhibited clinical signs of mild GVHD. C. Transgenic mice over-expressing heparanase. Spleen-derived progenitor cells obtained from C57BL/6 mice were injected with 25×10⁶ cells/mouse (▵), or 50×10⁶ cells/mouse (▴) into heparanase transgenic (Hpa-tg) and control mice (n = 10). All the Hpa-tg mice survived until the end of the experiment. In contrast, more than 80% of the control mice, receiving either 25×10⁶ cells/mouse (□), or 50×10⁶ cells/mouse (○), died of GVHD. The experiment was performed twice with similar results.

Figure 3. Effect of heparanase on activation of T lymphocytes.

A. ConA activation. Mouse spleen cells were isolated and subjected to activation with ConA in the absence (control) (▪) and presence of 10 or 30 µg/ml recombinant latent (65 kDa) () or active (8+50 kDa) (□) heparanase, followed by measurements of ³H-thymidine incorporation, as described under ‘Materials & Methods’. Addition of heparanase to the culture medium resulted in a significant (p<0.01) dose dependent decrease in ConA activation and proliferation of the spleen cells. The asterisk (*) indicates statistically significant differences between the control and the different treatments. B. Heparanase-mediated inhibition of ConA stimulated T-cell proliferation is independent of its enzymatic activity. Mouse spleen cells were isolated and subjected to activation with ConA in the absence (control) and presence of active heparanase, active heparanase plus glycol split heparin (100 µg/ml, compound 1514), or inactive heparanase (point mutated in glutamic residues 225 and 343). ³H-thymidine incorporation was inhibited to a similar extent regardless of whether the heparanase was enzymatically active or inactive (p<0.001). C. Mixed lymphocyte culture (MLC). One way MLC reaction was performed in the absence (control) (▪) or presence () of 30 µg/ml recombinant latent (65 kDa) heparanase. A marked decrease in activation (³H-thymidine incorporation) of Balb/c-derived lymphocytes sensitized against C57BL/6-derived lymphocytes was noted in the heparanase treated culture (p<0.01). D. Killing capacity of activated T cells. ConA activated splenocytes were co-cultured with target Yac cells in the absence (▪) or presence () of 5 µg/ml active (8+50 kDa) or 30 µg/ml latent (65 kDa) heparanase in order to evaluate their killing capacity. Treatment with either the latent or active forms of heparanase markedly inhibited the ability of the activated lymphocytes to kill their target cells (p<0.01). Each bar represents mean ± SD from triplicate wells. All experiments were performed at least three times; variations between different experiments did not exceed ±20%.

Figure 4. Effect of heparanase on cytokine production.

A, B. In vivo. A. C57BL/6 mice were subjected to a daily injection of active (8+50 kDa) heparanase (3 days, 5 µg/mouse/day) or saline (control). Splenocytes were then harvested, activated with ConA (24 h, 37°C, RPMI + 10% FCS) and aliquots of the culture medium were subjected to ELISA analysis of IL-4, IL-6, IL-10 and IL-12. The amounts of secreted Th2-type cytokines such as IL-4, IL-6 and IL-10, were increased following in vivo administration of heparanase (□) vs. saline (▪). In contrast, under the same conditions, there was a marked decrease in the level of IL-12, representing a Th1-associated cytokine. B. TNF-α and IFN-γ. C57BL/6 mice were subjected to a daily injection of active (8+50 kDa) heparanase (3 days, 5 µg/mouse/day) or saline (control). Supernatants from ConA activated cells were subjected to ELISA analysis of TNF-α and IFN-γ. The amounts of secreted TNF-α and IFN-γ were decreased following administration of heparanase (□) as compared to saline (▪). C. In vitro. C57BL/6 derived spleen lymphocytes were harvested and co-activated with IL-2 (24 h, 37°C, RPMI + 10% FCS) in the presence of 65 kDa latent heparanase (30 µg/ml) (□) or saline (▪). Aliquots of the culture medium were subjected to ELISA analysis as above. Each bar represents mean±SD from triplicate wells. All experiments were performed at least three times; variations between different experiments did not exceed ±15%. The amounts of the secreted Th2-type cytokines IL-6 and IL-10 were increased following exposure to heparanase as compared to saline. In contrast, there was a marked decrease in the level of IL-12, representing a Th1 cytokine, in cells that were similarly treated with IL-2 and heparanase for 24 h.