Limits...
CXCL12-induced VLA-4 activation is impaired in trisomy 12 chronic lymphocytic leukemia cells: a role for CCL21.

Ganghammer S, Hutterer E, Hinterseer E, Brachtl G, Asslaber D, Krenn PW, Girbl T, Berghammer P, Geisberger R, Egle A, Zucchetto A, Kruschinski A, Gattei V, Chigaev A, Greil R, Hartmann TN - Oncotarget (2015)

Bottom Line: Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression.Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup.Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Immunological and Molecular Cancer Research, 3rd Medical Department with Hematology, Medical Oncology, Hemostaseology, Infectious Diseases and Rheumatology, Oncologic Center, Paracelsus Medical University Salzburg, Austria.

ABSTRACT
Homing to distinct lymphoid organs enables chronic lymphocytic leukemia (CLL) cells to receive pro-survival and proliferative signals. Cytogenetic aberrations can significantly affect CLL cell compartmentalization. Trisomy 12 (tri12) defines a CLL subgroup with specific clinical features and increased levels of the negative prognostic marker CD49d, the α4-subunit of the integrin VLA-4, which is a key regulator of CLL cell homing to bone marrow (BM). Chemokine-induced inside-out VLA-4 activation, particularly via the CXCL12-CXCR4 axis, increases the arrest of various cell types on VCAM-1 presenting endothelium. Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression. Dissecting functional consequences of these alterations, we observed that tri12 CLL cell homing to murine BM is not affected by CXCR4-CXCL12 blockage using AMD3100 or olaptesed pegol/NOX-A12. In line, CCL21-CCR7 rather than CXCL12-CXCR4 interactions triggered VLA-4-mediated arrests of tri12 CLL cells to VCAM-1 under blood flow conditions. Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup. Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.

No MeSH data available.


Related in: MedlinePlus

CCL21, but not CXCL12, induces an activated conformation on tri12 CLL cellsIn flow-cytometric real-time kinetic analysis, (A) no tri12 and (B) tri12 CLL cells were incubated with LDV-FITC and stimulated with (i) CXCL12 or (ii) CCL2, as well as Mn2+ or DMSO, resulting in a different equilibrium binding of LDV-FITC to VLA-4, depending on the respective integrin affinity state. Dissociation kinetics of the fluorescent LDV peptide from the cells is induced by 500-fold excess of unlabeled peptide. To obtain the dissociation rate constants (Koff), the data were fitted to a one-phase exponential decay equation. The calculated Koff values indicate the VLA-4 affinity state after stimulating the cells with the three different substances; Koff < 0.02 s−1 high affinity, Koff > 0.06 s−1 low affinity. The data represent one out of 2-3 independent experiments with no tri12 (n = 2) and tri12 (n = 3) CLL cells; each experiment has been performed in duplicates. (A/B(iii)) Summary of the Koff values after VLA-4 activation by DMSO, CXCL12 or CCL21 in (A(iii)) no tri12 and (B(iii)) tri12 CLL cells. The data represent the mean ± SD of 2-3 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494922&req=5

Figure 7: CCL21, but not CXCL12, induces an activated conformation on tri12 CLL cellsIn flow-cytometric real-time kinetic analysis, (A) no tri12 and (B) tri12 CLL cells were incubated with LDV-FITC and stimulated with (i) CXCL12 or (ii) CCL2, as well as Mn2+ or DMSO, resulting in a different equilibrium binding of LDV-FITC to VLA-4, depending on the respective integrin affinity state. Dissociation kinetics of the fluorescent LDV peptide from the cells is induced by 500-fold excess of unlabeled peptide. To obtain the dissociation rate constants (Koff), the data were fitted to a one-phase exponential decay equation. The calculated Koff values indicate the VLA-4 affinity state after stimulating the cells with the three different substances; Koff < 0.02 s−1 high affinity, Koff > 0.06 s−1 low affinity. The data represent one out of 2-3 independent experiments with no tri12 (n = 2) and tri12 (n = 3) CLL cells; each experiment has been performed in duplicates. (A/B(iii)) Summary of the Koff values after VLA-4 activation by DMSO, CXCL12 or CCL21 in (A(iii)) no tri12 and (B(iii)) tri12 CLL cells. The data represent the mean ± SD of 2-3 experiments.

Mentions: Upon stimulation with CXCL12 or CCL21, a rapidly enhanced binding of no tri12 CLL cells to the ligand was observed (Figure 7A(i, ii)). The Koff rates for the activation with CXCL12 and CCL21 were 0.03-0.04 s−1 and 0.035-0.045 s−1, respectively (Figure 7A(iii)), and thus comparable to that of inside-out VLA-4 activation of other cell types [21]. However, in contrast to no tri12 CLL cells, tri12 CLL cells largely failed to undergo the rapid affinity up-regulation triggered by CXCL12 stimulation, in keeping with tethering experiments (Figure 7B(i)). Dissociation rate constants were at about 0.06-0.075 s−1 and thus comparable to solvent controls (Figure 7B(iii)). On the other hand, CCL21 did not fail to induce VLA-4 activation in tri12 CLL cells (Figure 7B(ii)), yielding similar Koff values as in the no tri12 CLL subgroup (Figure 7A(ii)).


CXCL12-induced VLA-4 activation is impaired in trisomy 12 chronic lymphocytic leukemia cells: a role for CCL21.

Ganghammer S, Hutterer E, Hinterseer E, Brachtl G, Asslaber D, Krenn PW, Girbl T, Berghammer P, Geisberger R, Egle A, Zucchetto A, Kruschinski A, Gattei V, Chigaev A, Greil R, Hartmann TN - Oncotarget (2015)

CCL21, but not CXCL12, induces an activated conformation on tri12 CLL cellsIn flow-cytometric real-time kinetic analysis, (A) no tri12 and (B) tri12 CLL cells were incubated with LDV-FITC and stimulated with (i) CXCL12 or (ii) CCL2, as well as Mn2+ or DMSO, resulting in a different equilibrium binding of LDV-FITC to VLA-4, depending on the respective integrin affinity state. Dissociation kinetics of the fluorescent LDV peptide from the cells is induced by 500-fold excess of unlabeled peptide. To obtain the dissociation rate constants (Koff), the data were fitted to a one-phase exponential decay equation. The calculated Koff values indicate the VLA-4 affinity state after stimulating the cells with the three different substances; Koff < 0.02 s−1 high affinity, Koff > 0.06 s−1 low affinity. The data represent one out of 2-3 independent experiments with no tri12 (n = 2) and tri12 (n = 3) CLL cells; each experiment has been performed in duplicates. (A/B(iii)) Summary of the Koff values after VLA-4 activation by DMSO, CXCL12 or CCL21 in (A(iii)) no tri12 and (B(iii)) tri12 CLL cells. The data represent the mean ± SD of 2-3 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494922&req=5

Figure 7: CCL21, but not CXCL12, induces an activated conformation on tri12 CLL cellsIn flow-cytometric real-time kinetic analysis, (A) no tri12 and (B) tri12 CLL cells were incubated with LDV-FITC and stimulated with (i) CXCL12 or (ii) CCL2, as well as Mn2+ or DMSO, resulting in a different equilibrium binding of LDV-FITC to VLA-4, depending on the respective integrin affinity state. Dissociation kinetics of the fluorescent LDV peptide from the cells is induced by 500-fold excess of unlabeled peptide. To obtain the dissociation rate constants (Koff), the data were fitted to a one-phase exponential decay equation. The calculated Koff values indicate the VLA-4 affinity state after stimulating the cells with the three different substances; Koff < 0.02 s−1 high affinity, Koff > 0.06 s−1 low affinity. The data represent one out of 2-3 independent experiments with no tri12 (n = 2) and tri12 (n = 3) CLL cells; each experiment has been performed in duplicates. (A/B(iii)) Summary of the Koff values after VLA-4 activation by DMSO, CXCL12 or CCL21 in (A(iii)) no tri12 and (B(iii)) tri12 CLL cells. The data represent the mean ± SD of 2-3 experiments.
Mentions: Upon stimulation with CXCL12 or CCL21, a rapidly enhanced binding of no tri12 CLL cells to the ligand was observed (Figure 7A(i, ii)). The Koff rates for the activation with CXCL12 and CCL21 were 0.03-0.04 s−1 and 0.035-0.045 s−1, respectively (Figure 7A(iii)), and thus comparable to that of inside-out VLA-4 activation of other cell types [21]. However, in contrast to no tri12 CLL cells, tri12 CLL cells largely failed to undergo the rapid affinity up-regulation triggered by CXCL12 stimulation, in keeping with tethering experiments (Figure 7B(i)). Dissociation rate constants were at about 0.06-0.075 s−1 and thus comparable to solvent controls (Figure 7B(iii)). On the other hand, CCL21 did not fail to induce VLA-4 activation in tri12 CLL cells (Figure 7B(ii)), yielding similar Koff values as in the no tri12 CLL subgroup (Figure 7A(ii)).

Bottom Line: Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression.Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup.Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Immunological and Molecular Cancer Research, 3rd Medical Department with Hematology, Medical Oncology, Hemostaseology, Infectious Diseases and Rheumatology, Oncologic Center, Paracelsus Medical University Salzburg, Austria.

ABSTRACT
Homing to distinct lymphoid organs enables chronic lymphocytic leukemia (CLL) cells to receive pro-survival and proliferative signals. Cytogenetic aberrations can significantly affect CLL cell compartmentalization. Trisomy 12 (tri12) defines a CLL subgroup with specific clinical features and increased levels of the negative prognostic marker CD49d, the α4-subunit of the integrin VLA-4, which is a key regulator of CLL cell homing to bone marrow (BM). Chemokine-induced inside-out VLA-4 activation, particularly via the CXCL12-CXCR4 axis, increases the arrest of various cell types on VCAM-1 presenting endothelium. Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression. Dissecting functional consequences of these alterations, we observed that tri12 CLL cell homing to murine BM is not affected by CXCR4-CXCL12 blockage using AMD3100 or olaptesed pegol/NOX-A12. In line, CCL21-CCR7 rather than CXCL12-CXCR4 interactions triggered VLA-4-mediated arrests of tri12 CLL cells to VCAM-1 under blood flow conditions. Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup. Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.

No MeSH data available.


Related in: MedlinePlus