Studies on the Regulation and Localization of 5-Lipoxygenase in Human B-Lymphocytes

Authors


Correspondence to H. E. Claesson, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden
Fax:+46 8 728 7627.

Abstract

Stimulated B-lymphocytes, isolated from patients with chronic lymphocytic leukemia of B-cell type (B-CLL cells) or from human tonsils, produced similar amounts of leukotriene (LT) B4 and 5–hydroxy-eicosatetraenoic acid (5-HETE) as polymorphonuclear granulocytes. Unlike intact granulocytes or monocytes, human B-lymphocytes require calcium ionophore, exogenous arachidonic acid and an oxidative environment in order to produce 5-lipoxygenase products. Several thiol-reactive compounds such as N-ethylmaleimide, methyl methanethiosulfonate, azodicarboxylic acid bisfdimethylamide] (diamide) as well as hydrogen peroxide were all found to stimulate cellular leukotriene biosynthesis. Reverse transcriptase (RT)-PCR analysis demonstrated the expression of 5-lipoxygenase, 5-lipoxygenase-activating protein (FLAP) and LTA4 hydrolase mRNA in B-CLL cells. Western blot analysis demonstrated a band corresponding to the molecular size of FLAP in the B-CLL cell membrane. Furthermore, MK886, the FLAP-binding cellular leukotriene biosynthesis inhibitor, reduced both LTB4 and 5-HETE formation. Immunocy-tochemistry showed that 5-lipoxygenase was mainly localized in the nuclei of non-activated B-CLL cells, tonsillar B-lymphocytes and monoclonal B-cells. In contrast, neither human peripheral T-lymphocytes nor Jurkat cells were stained. These results suggest that 5-lipoxygenase and its products function in the nucleus of B-lymphocytes.

Abbreviations
cPLA2

cytosolic phospholipase A2

FLAP

5-lipoxygenase-activating protein

B-CLL

chronic lymphocytic leukemia of B-cell type; reverse transcriptase

LT

leukotriene

HETE

hydroxyeicosatetraenoic acid

diamide

azodicarboxylic acid bis(dimethylamide)

Dnp-Cl

1-chloro-2,4–dinitrobenzene

MeSO2SMe

methyl methanethiosulfonate

MalNET

N-ethylmaleimide

SAC

Staphylococcus aureus Cowan 1

IL-1

interleukin-1

PMNL

polymorphonuclear leukocytes

HUVEC

human umbilical vein endothelial cells

HBSS

Hanks' balanced salt solutions, pH 7.4

FITC

fluorescein

 

isothiocyanate

MPETE

hydroperoxyeicosatetraenoic acid

Enzymes
 

Cytosolic phospholipase A2 (EC 3.1.1.4)

 

5-lipoxygenase (EC 1.13.11.12)

 

leukotriene-A4 hydrolase (EC 3.3.2.6)

Ancillary