Figure 4.
Hypoxia induces adenosine production and signaling in NLCs derived from CLL patients. (A) RT-PCR analysis of CD73 (NT5E, n = 9, left panel) and immunofluorescence confocal microscopy (right panels) of CD73 surface expression on NLCs generated from CLL patients under normoxic or hypoxic conditions. Cumulative data of CD73 mean pixel intensity confirmed a robust increase in expression under hypoxia. Original magnification ×63. (B) RT-PCR data on CD39 (ENTPD1) and CD26 (DPP4) expression indicated that NLCs are constitutively CD39+ and CD26+ but fail to significantly modulate the 2 molecules under hypoxia (n = 9). (C) HPLC analysis showing increased adenosine accumulation in NLCs exposed to 200 µM AMP (30 min, 37°C) under hypoxia (n = 11). Pretreatment with the adenosine deaminase inhibitor EHNA (10 µM, 30 min, 37°C) further increased adenosine production. (D) RT-PCR analysis of A2A (ADORA2A, left panel, n = 9) and immunofluorescence confocal microscopy (right panels) of the A2A receptor on NLC under normoxia or hypoxia. Cumulative data of A2A mean pixel intensity from 7 different experiments obtained by quantifying green fluorescence intensity in 3 randomly chosen fields using the ImageJ software. Original magnification ×63. (E) Immunohistochemical (left panel) and confocal microscopy analyses (right panels) of A2A and CD68 expression in LN sections showed enhanced staining in CLL proliferation centers, particularly by CD68+ myeloid cells. Triple staining with anti-CD23 (green), anti-A2A (red), and anti-CD68 (white) shows that CD68+ cells are strongly A2A+ (white arrows). Original magnification ×63. Scale bars, 25 µm. (F-G) Ligation of the A2A receptor with the pharmacological agonist CGS21680 (10 µM, 30 min, 37°C) in NLCs under normoxic conditions is followed by the phosphorylation of AKT, ERK1/2, STAT3 (F, top panels) and p65 (G, top panel). Original magnification ×63. The boxplots (F-G, bottom panels) show cumulative data of mean pixel intensity from NLC preparations obtained from 5 different patients. p65 activation was measured considering immunofluorescence within the nuclei, identified by DAPI staining. Statistical differences were analyzed using the Wilcoxon signed rank test and the Mann-Whitney U tests followed by the Tukey test. ADO, adenosine; CGS, CGS21680; H, hypoxia (gray boxes); N, normoxia (open boxes); SCH, SCH58261.

Hypoxia induces adenosine production and signaling in NLCs derived from CLL patients. (A) RT-PCR analysis of CD73 (NT5E, n = 9, left panel) and immunofluorescence confocal microscopy (right panels) of CD73 surface expression on NLCs generated from CLL patients under normoxic or hypoxic conditions. Cumulative data of CD73 mean pixel intensity confirmed a robust increase in expression under hypoxia. Original magnification ×63. (B) RT-PCR data on CD39 (ENTPD1) and CD26 (DPP4) expression indicated that NLCs are constitutively CD39+ and CD26+ but fail to significantly modulate the 2 molecules under hypoxia (n = 9). (C) HPLC analysis showing increased adenosine accumulation in NLCs exposed to 200 µM AMP (30 min, 37°C) under hypoxia (n = 11). Pretreatment with the adenosine deaminase inhibitor EHNA (10 µM, 30 min, 37°C) further increased adenosine production. (D) RT-PCR analysis of A2A (ADORA2A, left panel, n = 9) and immunofluorescence confocal microscopy (right panels) of the A2A receptor on NLC under normoxia or hypoxia. Cumulative data of A2A mean pixel intensity from 7 different experiments obtained by quantifying green fluorescence intensity in 3 randomly chosen fields using the ImageJ software. Original magnification ×63. (E) Immunohistochemical (left panel) and confocal microscopy analyses (right panels) of A2A and CD68 expression in LN sections showed enhanced staining in CLL proliferation centers, particularly by CD68+ myeloid cells. Triple staining with anti-CD23 (green), anti-A2A (red), and anti-CD68 (white) shows that CD68+ cells are strongly A2A+ (white arrows). Original magnification ×63. Scale bars, 25 µm. (F-G) Ligation of the A2A receptor with the pharmacological agonist CGS21680 (10 µM, 30 min, 37°C) in NLCs under normoxic conditions is followed by the phosphorylation of AKT, ERK1/2, STAT3 (F, top panels) and p65 (G, top panel). Original magnification ×63. The boxplots (F-G, bottom panels) show cumulative data of mean pixel intensity from NLC preparations obtained from 5 different patients. p65 activation was measured considering immunofluorescence within the nuclei, identified by DAPI staining. Statistical differences were analyzed using the Wilcoxon signed rank test and the Mann-Whitney U tests followed by the Tukey test. ADO, adenosine; CGS, CGS21680; H, hypoxia (gray boxes); N, normoxia (open boxes); SCH, SCH58261.

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