The mechanism of essential thrombocythemia (ET) or polycythemia vera (PV) progresses to myelofibrosis (MF) is unknown. MF is mainly characterized by an increase in fibronectin deposition. VLA-4 and VLA-5 are important integrins that attach cells to fibronectin. Lysyl oxidase (LOX), a matrix cross-linking protein secreted by megakaryocytes (MK) , has been reported to be increased in MF. Abnormalities in MK numbers and morphology are also considered common features of Myeloproliferative Neoplasms (MPNs). VLA-4 and VLA-5 are also important factors for MK poiesis and adhesions. Therefore, VLA-4, VLA-5, and LOX are important in the MK and fibronectin attachments , were studied in the progression from ET and PV into MF.
Flow Cytometry, Isolated mononuclear cells (MNC) were incubated with fluorophore conjugated antibody for 60 min at room temperature and analyzed by BD Accuri C6 system . Data was collected and analyzed using BD Accuri C6 software. Fluorophore conjugated primary antibodies against human integrin α4 (CD49d), α5(CD49e) were purchased from Miltenyi Biotec . CD14 and CD34 isolation kits were also purchased from Miltenyi Biotec Adhesion Assay Isolated cells were labeled with calcein (Sigma-Aldrich) and then seeded on fibronectin (Sigma-Aldrich) coated 96-well plates. Cells were incubated at 37°C for one hour. Unattached cells were washed with PBS. Attached cells were detected and analyzed by Spark plate reader (Tecan, Switzerland).. ELISA . LOXL2 levels in the plasma were assayed using Human LOXL2 ELISA kit from Abcam (Waltham, MA). Cell Culture Human cell lines Hel and U937 were obtained from ATCC . Cells were maintained at 37°C in RPMI medium and 10% fetal bovine serum. To determine the effects of cytokines, cells were seeded on 6-well plates and incubated TNFα (Sigma-Aldrich,) at indicated concentrations overnight. Untreated and treated cells were collected for flow cytometry
Results.36 patients were studied ( 16 ET, 12PV, 4 PMF and 2 each of ET-MF and PV-MF)
1) Integrins (MFI) : No difference between ET & PV , therefore they were grouped together. a) α4 Values (MNC cells ) , ET & PV (mean+ SE ) were (180.3 + 28.91) , (n=28) , MF were (571.0+ 122.1) (n=8) and controls (593.7 + 77.05) (n=18) . ET & PV were significantly lower than Controls (P<0.0001) and MF ( p<0.01) while there was no difference between MF and controls. b) α5 values (MNC cell ) were as follows: ET & PV( 528.8 + 57.8) (n=28), MF (872.4 + 105.5) (n=8), controls ( 808.5 + 51.26) (n=18) . ET & PV were significantly lowered than controls ( P<0.01) or MF ( P<0.01) while no difference between MF and controls . C)β1 integrin .ET & PV ( 8041+ 484.9) (n=28) MF ( 8485 +393.9) (n=8) and controls ( 10924+ 421.6) (n=14) , ET & PV were significantly decreased compared with controls ( P<0.001) and not significantly different from MF , MF were not significantly different from controls .Then CD14+, CD 34 + cells were isolated and assayed for α4, α5. Also decreased expression in both α 4 and α 5 expression ( P<0.05) in ET & PV (n=6) than controls or MF . while MF were not different from controls . 2) Adhesion : CD14+ cells showed decreased adhesion ( %) in ET & PV ( 5.8 +0.6) than controls (16.7 + 1.6) or MF ( 16.4 + 1.5 ) ( P< 0.05) while MF were not different from controls . 3) LOXL2 Elisa Assay : The results showed PV ( 110+ 19.9 ) ( n=10), ET (97.9+16.4) (n=9) , MF (265.5+ 32.1) (n=11) ( p<0.0001 between MF Vs ET, or CTR ; P<0.001 between MF Vs PV ; P=NS between CTR Vs ET, or PV , or ET Vs PV ) . MF has a significantly increased LOXL2 levels than PV , ET or controls . 4)TNF- α stimulate α4, α5 integrins formation . TNF- α induced a 2.56 and 1.5 fold changes on α4 , α5 in Hel cells ( JAK2 mutation positive ) respectively , while it has no effects on the U937 cell ( JAK2 mutation negative )with 0.81 and 0.9 fold changes on α4 and α5 respectively
Conclusion .
Patients with ET and PV showed significantly lower levels of α4, α5, and LOXL2 compared to patients with MF. Inflammatory cytokines, specifically TNFα, promote the production of α4 and α5 in JAK-positive cell lines, but not in JAK2- negative cell lines. This suggests that inflammatory cytokines cause changes in VLA-4 and VLA-5 in MPN cells, leading to increased adhesion of megakaryocytes and monocytes, ultimately resulting in the higher levels of fibronectin seen in patients with MF after many years of having ET or PV.
No relevant conflicts of interest to declare.
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