Effect of heparanase on activation of T lymphocytes.

<p><b>A. ConA activation.</b> 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 ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010135#s2" target="_blank">Materials & Methods</a>’. 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>B. Heparanase-mediated inhibition of ConA stimulated T-cell proliferation is independent of its enzymatic activity.</b> 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). <b>C. Mixed lymphocyte culture (MLC).</b> 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). <b>D. Killing capacity of activated T cells.</b> 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%.</p

Effect of heparanase on cytokine production.

<p><b>A, B. In vivo. A.</b> 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 <i>in vivo</i> 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>B. TNF-α and IFN-γ.</b> 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 (▪). <b>C. In vitro.</b> 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.</p

Heparanase potentiates engraftment of WBC.

<p>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.</p

Effect of heparanase on GVHD.

<p><b>A. Prolonged survival of mice treated with heparanase.</b> 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>B. Mice treated with different doses of heparanase.</b> 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. <b>C. Transgenic mice over-expressing heparanase.</b> Spleen-derived progenitor cells obtained from C57BL/6 mice were injected with 25×10⁶ cells/mouse (▵), or 50×10⁶ cells/mouse (▴) into heparanase transgenic (<i>Hpa-tg</i>) and control mice (n = 10). All the <i>Hpa-tg</i> 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.</p

CD44-null NOD females display relative resistance to T1D: role of inflammatory cells.

<p>A) and B) Development of T1D in the spontaneous (A) and cell transfer (B) models of WT and CD44-deficient mice was monitored by measuring blood glucose. In the transfer model, irradiated CD44-deficient and WT young (6–8 weeks-old) male recipients were respectively transplanted with splenocytes from CD44-deficient and WT diabetic females. Percentage of diabetes-free mice (showing <250 mg/dL blood glucose) was recorded versus time. Statistical analysis by Breslow; <i>A</i>, <i>P</i> < 0.05; <i>B</i>, <i>P</i> < 0.005. C) The invasion capacity of infiltrating cells derived from WT (n = 6) and CD44-deficient (n = 6) NOD females (spontaneous model) was measured as indicated in <i>Materials and Methods</i>. A total of 754 pancreatic islets in each mouse group were scored by an uninformed observer. The percentage of islets showing each one of the infiltrating scores was calculated for each mouse, and the average values are presented. Islets from CD44-deficient mice display higher percentage of infiltrating scores (2, 3 and 4) than islets from wild type mice. <i>P</i> < 0.0001 by Pearson's χ2 test comparing the distribution of scores in the two mouse groups. D) Wild type or CD44-deficient pre-diabetic spleen cells were added to the top compartment of transwell migration chamber, separated by HA-coated filter (10 μg/filter) from the bottom compartment, and the % of cells migrating through the filter toward the chemoatractant in the bottom compartment was calculated by flow cytometry. Statistical analysis by Student’s-<i>t-</i> test. Accumulated data of 4 experiments (n = 8; error bars, SEM).</p

CD44 involvement in T1D.

<p>Three CD44-dependent pathways affect the fate of T1D in NOD mice. I. CD44 involvement in inflammatory cell migration (blue). Inflammation upregulates CD44 on the islet-infiltrating cells. The firm adhesion of the CD44 receptor to the HA substrate slows the motility of the inflammatory cells on the endothelium. II. CD44 involvement in peripheral insulin sensitivity and glucose uptake (green). CD44 expression on peripheral tissue, increases insulin sensitivity and glucose uptake by, for example, muscle and liver cells, resulting in reduced hyperglycemia. Compensation for insulin-deficiency (see pathway III) may explain this event. III. CD44 involvement in β cell apoptotic death (red). Inflammation-induced cytokines up-regulate CD44 expression on β cells. CD44 expression on β cells is associated with increased β cell dysfunction and susceptibility to apoptosis, which could be triggered by the binding of HA fragments to CD44 receptor. However, large HA fragments could interfere with the detrimental effect of LMW-HA on β cells. The β cell dysfunction and susceptibility to apoptosis is indicated by increased iNOS induction and subsequent NO production, increased caspase-3 activity (Western blot, not shown), reduced glucose-stimulated insulin secretion, and reduced insulin content. As an outcome of β cell demise, hyperglycemia is detected, implying the development of T1D. Broken arrows and question marks represent pathways and factors that are yet to be established.</p

CD44-deficient pancreatic islet cells retain their functionality under pro-inflammatory conditions.

<p>(A and B) <i>In vitro</i> pro-inflammatory conditions. A) Griess assay. Wild type and CD44-deficient pancreatic islet cells were incubated with medium (-) or Cytomix for 24 and 48 h. Nitrite release was assessed by Griess assay (n = 3). Inset: Cell extracts from pancreatic islets described in (<i>A</i>) were subjected to Western blot analysis, using anti-iNOS antibody. A representative Fig of three experiments. B) Glucose-stimulated insulin secretion (GSIS). Pancreatic islet cells from WT and CD44-deficient NOD mice were incubated with medium (-) or Cytomix for 48 h and then stimulated with 3.3 or 16.7 mM glucose for additional one hour and their ability to secrete insulin was measured by ELISA. Inset: Insulin content in each one of the samples (n = 4–5) (error bars, SEM). Statistical analysis for A and B by standard two-tailed Student’s <i>t</i>-test. <i>A</i>, one representative experiment of five. <i>B</i>, one representative experiment of two. (C and D) <i>Ex vivo</i> pro-inflammatory conditions. C) Freshly isolated pancreatic islet cells from WT and CD44-deficient pre-diabetic NOD females were processed as described in (<i>B</i>) (n = 6–8) (error bars, SEM). D) Content of insulin in pancreatic islets of WT and CD44-deficient pre-diabetic NOD females was analyzed by ELISA. Statistical analysis in <i>C</i> and <i>D</i> by standard two-tailed Student’s <i>t</i>-test. The statistical analysis in <i>C</i> shows that the <i>ex vivo</i> insulin release of CD44-deficient pancreatic islets at 12 and 14 weeks is significantly higher (p<0.05) than the insulin release of the corresponding WT cells.The statistical analysis in <i>D</i> shows that the insulin content in pancreatic islets of 14 wks-old pre-diabetic WT mice is significantly different (<i>P</i> < 0.05) from all other samples. (n = 6–8). In <i>C—D</i>, at least one representative experiment of two.</p

Insulin-producing β cells from WT NOD females display enhanced susceptibility to apoptosis.

<p>A) Enhanced caspase-3 activity is detected in cell extracts of WT than in those of CD44-deficient pancreatic islet cells. Pancreatic islet cells were freshly removed from WT and CD44-deficient pre-diabetic NOD females. Cell extracts from islets were subjected to Western blot analysis, using anti-cleaved caspase-3 antibody. A representative Fig of three experiments. (B and C) Pancreata were harvested from 10-weeks-old normoglycemic WT and CD44-deficient NOD females (n = 5 in each group). β-cell apoptosis was assessed using TUNEL assay (green) and co-staining for insulin (red) and DNA (blue). B) Apoptosis was calculated as percent of TUNEL positive nuclei (+/- SEM) in insulin-positive cells out of the total number of insulin-positive cells per mouse. WT (white bar, ~17,560 total β-cells); CD44-deficient (black bar, ~14,800 total β-cells). C) Pictures are representative images of islets from WT (left panel) and CD44-deficient NOD females (right panel). Original images were taken at a magnification of x40. Inset images were digitally increased x5. White arrowheads point to apoptotic β-cells.</p

CD44 expressed in the recipient’s tissue rather than on the infiltrating-cells enhances the development of T1D in the cell transfer model.

<p>A) CD44-deficient non-diabetic NOD male recipients were reconstituted with inflammatory spleen cells derived from WT (CD44^+/+→CD44^-/-; marked #1) or CD44-deficient (CD44^-/-→CD44^-/-; marked #2) diabetic NOD female donors. In addition, WT non-diabetic NOD male recipients were reconstituted with inflammatory spleen cells derived from WT (CD44^+/+→CD44^+/+; marked #3) or CD44-deficient (CD44^-/-→CD44^+/+; marked #4) diabetic NOD female donors. Inflammatory cells are marked by arrows and local islet cells by arrowhead. The pancreases of all 4 combinations were removed after detection of diabetes or 200 days after cell transfer. Doing so, pancreases that were harvested approximately at the same time, were stained with anti-CD44 mAb. B) The % of diabetes-free mice (exhibiting less than 250 mg/dL urine glucose) was determined in corresponding four mouse groups, described in <i>A</i>. Statistical analysis by Breslow: #2 versus #3, <i>P</i> < 0.01; #1 versus #2 (follow up 75 to 200 days), <i>P</i> < 0.03; #3 versus #4 (follow up 20 to 75 days), <i>P</i> < 0.03.</p

Levels of BMS originating from nSSL-BMS or free BMS in tissues of naive healthy mice and mice with ECM.

<p>Mice at initial clinical stages of ECM were injected (on day 6 p.i.) with either free or nSSL-BMS. Healthy mice were injected with nSSL-BMS. *n.d, not done.</p