1. Hyaluronate-Enhanced Hematopoiesis: Two Different Receptors Trigger the Release of Interleukin-1β and Interleukin-6 From Bone Marrow Macrophages
by Sophia Khaldoyanidi, Jürgen Moll, Svetlana Karakhanova, Peter Herrlich, and Helmut Ponta
Blood
Volume 94(3):
August 1, 1999
©1999 by American Society of Hematology

2. Influence of HA’ase on myelopoiesis in LTBMC
Influence of HA’ase on myelopoiesis in LTBMC. (A) Nonadherent cells from myeloid LTBMC in the absence of HA’ase (control) or treated with HA’ase (Streptomyces HA’ase, Calbiochem; 0.1 U/mL) were harvested once per week, and their number was counted.
Influence of HA’ase on myelopoiesis in LTBMC. (A) Nonadherent cells from myeloid LTBMC in the absence of HA’ase (control) or treated with HA’ase (Streptomyces HA’ase, Calbiochem; 0.1 U/mL) were harvested once per week, and their number was counted. Standard error (SE) was calculated from results obtained with 3 independent cultures. The insert shows the dose-dependent effect of HA’ase on the production of nonadherent cells in LTBMC after 4 weeks in culture. The bars indicate SE calculated from 3 independent wells each. (B) The production of clonogenic cells in either control LTBMC or in LTBMC treated with HA’ase (0.1 U/mL) was measured by the CFU assay (Materials and Methods). Nonadherent cells were harvested from LTBMC and plated in semisolid culture in the presence of IL-3 (Materials and Methods). SE was calculated from results obtained from 3 independent wells.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology

3. Influence of HA and of HA’ase on formation of the adherent layer.
Influence of HA and of HA’ase on formation of the adherent layer. Primary bone marrow cells were cultured in 6-well plates without any addition (A, D, and G) or with the addition of 100 μg/mL of HA (B, E, and H) or with 0.1 U/mL HA’ase (C, F, and I). Photographs were taken after 1 (top panel), 4 (middle panel), and 8 (bottom panel) weeks of culture. Only adherent cells are shown. In (D), (E), (G), and (H), typical cobblestone-like cells are visible, which are increased in number in (E) and (H), whereas in (F) and (I) such cells are hardly detectable. The magnification was 400-fold.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology

4. HA stimulates myelopoiesis in LTBMC
HA stimulates myelopoiesis in LTBMC. (A) Nonadherent cells from myeloid LTBMC in the absence of HA (control) or treated with HA (100 μg/mL; rooster comb HA from Sigma) were determined each week.
HA stimulates myelopoiesis in LTBMC. (A) Nonadherent cells from myeloid LTBMC in the absence of HA (control) or treated with HA (100 μg/mL; rooster comb HA from Sigma) were determined each week. SE was calculated from results obtained from 3 independent wells. The inset shows the dose-dependent effect of HA on the production of nonadherent cells in myeloid LTBMC after 4 weeks in culture. The asterisk indicates the use of heat-treated HA (1 hour at 95°C). The bars indicate the SE determined from results obtained from 3 independent wells. (B) Clonogenic cells in myeloid LTBMC grown in the absence of HA (control) or presence of HA (100 μg/mL) were determined at the time indicated by plating of nonadherent cells in semisolid methylcellulose culture medium in the presence of IL-3 (Materials and Methods). SE was calculated from results obtained from 3 different wells.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology

5. HA stimulates the production of a colony promoting activity in LTBMC
HA stimulates the production of a colony promoting activity in LTBMC. (A) LTBMC was treated with HA (100 μg/mL).
HA stimulates the production of a colony promoting activity in LTBMC. (A) LTBMC was treated with HA (100 μg/mL). CM was harvested at day 28 and tested for colony-promoting activity. Freshly isolated bone marrow cells were added to methylcellulose cultures and the colony formation by myelopoietic progenitors was determined in the presence of suboptimal doses of IL-3 (5% CM of Wehi-3B cells) and the addition of 20% of CM from either control LTBMC (lanes 1 and 3) or of CM from LTBMC treated with HA (lanes 2 and 4). After 7 days of culture, the numbers of colonies were counted. In lanes 3 and lane 4, hematopoietic progenitors were determined similarly to lanes 1 and 2, but antibodies directed against IL-6 (100 μg/mL) were added in addition. SE was calculated from 3 independent assays. (B) CM of control LTBMC or LTBMC cultured for 4 weeks in the presence of HA (100 μg/mL) or in the presence of HA’ase (0.1 U/mL) was assayed for IL-6 by ELISA.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology

6. Binding of HA to macrophage cells.
Binding of HA to macrophage cells. After 4 weeks, LTBMC nonadherent cells were washed out, adherent cells were treated for 30 minutes at 37°C with HA’ase (5 U/mL), and then fixed with 3% paraformaldehyde and incubated with FITC-labeled HA at 4°C for 30 minutes. Immunofluorescence-staining of a representative cell is shown. Magnification was 1,000-fold. Similar staining was observed without HA’ase treatment.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology

7. HA treatment of BMDM upregulates IL-6 production.
HA treatment of BMDM upregulates IL-6 production. BMDM were isolated as described in Materials and Methods and were then treated with HA (100 μg/mL). CM was collected at the indicated times and tested for the presence of IL-6 by ELISA. The error bars indicated SE calculated from results obtained from 3 independent wells.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology

8. HA-induced actin rearrangement in LTBMC adherent cells.
HA-induced actin rearrangement in LTBMC adherent cells. Adherent cells of LTBMCs after 4 weeks in culture were incubated with HA (100 mg/μL) for 10 minutes, 4 hours, and 16 hours, respectively. Staining of the actin cytoskeleton by phalloidine-rhodamine (Materials and Methods) is shown. Magnification was 1,000-fold.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology

9. Influence of KM81 MoAb on cytokine production by macrophages upon HA treatment.
Influence of KM81 MoAb on cytokine production by macrophages upon HA treatment. BMDM were treated with HA (100 μg/mL) for the times indicated either in the presence or absence of F(ab′)2 fragments of the pan CD44 antibody KM81 (20 μg/mL). The concentration of IL-6 (A) and IL-1β (B) was determined in the supernatant by ELISA. SE was calculated from results obtained from 3 independent wells each.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology

10. HA-induced activation of proline-directed protein kinases in BMDM
HA-induced activation of proline-directed protein kinases in BMDM. BMDM were stimulated with HA (100 μg/mL) for the indicated times, then lysed, and protein samples were analyzed by Western blotting for the presence of phosphorylated p38 kinase and phosphor...
HA-induced activation of proline-directed protein kinases in BMDM. BMDM were stimulated with HA (100 μg/mL) for the indicated times, then lysed, and protein samples were analyzed by Western blotting for the presence of phosphorylated p38 kinase and phosphorylated Erk. The same membranes were stripped and stained with anti p38 or Erk antibodies, respectively. Where indicated, cells were preincubated for 15 minutes at 4°C with KM81 F(ab′)2 fragments (20 μg/mL) before HA stimulation.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology

11. Influence of HA and HA’ase on myeloid LTBMC from CD44 knock-out mice.
Influence of HA and HA’ase on myeloid LTBMC from CD44 knock-out mice. Myeloid LTBMC from CD44-deficient mice were untreated or treated with HA or HA’ase as described in the legends to Figs 1 and3. The number of nonadherent cells (A) and hematopoietic progenitors (B) were measured once per week. SE was calculated from results obtained from 3 independent wells.
Sophia Khaldoyanidi et al. Blood 1999;94:
©1999 by American Society of Hematology