Björn Odlander

Leukotriene B4 in the immune system

Leukotriene (LT) B4 is a biologically active molecule derived from arachidonic acid via the 5-lipoxygenase pathway. It mediates certain inflammatory and immunological reactions. The role of LTB4 in the immune system has been questioned since lymphocytes have been regarded to lack the enzymes involved in LTB4 formation. This review focuses on the recently described biosynthesis of LTB4 in B-lymphocytes and the effects of this compound on lymphocyte functions.

Human B lymphocytes possess 5-lipoxygenase activity and convert arachidonic acid to leukotriene B4

Incubation of cell sonicates from monoclonal B cells with arachidonic acid led to the formation of leukotriene (LT) B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). In contrast, stimulation of intact B cells with the calcium ionophore A23187 +/- arachidonic acid did not, under similar conditions, lead to formation of LTB4. The identification of these products was based on reverse phase- and straight phase-HPLC analysis, UV-spectroscopy and gas chromatography-mass spectrometry. Cell sonicates of highly enriched human tonsillar B lymphocytes also converted arachidonic acid to LTB4 and 5-HETE. Activation of these cells with B cell mitogen and cytokines for three days led to an upregulation of 5-lipoxygenase activity. This study provides evidence for the biosynthesis of LTB4 from arachidonic acid in B cell lines and in normal human tonsillar B lymphocytes.

Human B and T lymphocytes convert leukotriene A4 into leukotriene B4

Incubation of human tonsillar B lymphocytes and peripheral blood T lymphocytes with leukotriene A4 led to the formation of leukotriene B4. The purity of these cell suspensions was more than 99%, containing less than 0.5% monocytes. Incubation of purified B or T lymphocytes with the calcium ionophore A23187 did not lead to the formation of any detectable amounts of leukotrienes. Several established cell lines of B and T lymphocytic origin were also found to convert leukotriene A4 into leukotriene B4, showing that monoclonal lymphocytic cells possess leukotriene A4 hydrolase activity.

Leukotriene A4 hydrolase in the human B-lymphocytic cell line Raji: Indications of catalytically divergent forms of the enzyme

Leukotriene A4 hydrolase was purified 1400-fold, with an approximate yield of 25%, to apparent homogeneity from the human B-lymphocytic cell line Raji. The purification included ammonium sulfate precipitations followed by anion exchange, hydrophobic interaction, and molecular exclusion fast protein liquid chromatography. Kinetic properties at 2 degrees C varied between different enzyme preparations. Two patterns were observed, one with a Km of about 12 microM and Vmax of about 1.1 mumol LTB4/mg protein/min which correlated well with the properties of the human leukocytic LTA4 hydrolase. In other enzyme preparations a higher catalytic activity was observed. These enzyme batches did not obey Michaelis-Menten kinetics but were compatible with a mixture of enzymatic species. Heat treatment (60 degrees C) led to a time-dependent decline in catalytic activity. However, certain enzyme preparations contained a subfraction of enzymatic activity which was more resistant to heat treatment, yielding a biphasic inactivation pattern. It is thus suggested, on the basis of the kinetic properties and the heat-inactivation pattern, that these enzyme preparations contained an addition form of LTA4 hydrolase.

B-lymphocytic cell line Raji expresses the leukotriene A4 hydrolase gene but not the 5-lipoxygenase gene

The expression of LTA hydrolase and 5-lipoxygenase genes was studied in Raji cells, a Burkitt lymphoma derived B-cell line. Northern and Western blot analyses clearly showed the expression, both at the transcriptional and translational level, of the LTA4 hydrolase gene in these cells. However, expression of the 5-lipoxygenase gene was undetectable. Thus, the genes coding for the two enzymes required for biosynthesis of leukotriene B4 from arachidonic acid, 5-lipoxygenase and LTA4 hydrolase, were differentially expressed in the Raji cells.

The Role of Leukotriene A4 Hydrolase in Cells and Tissues Lacking 5-Lipoxygenase

Leukotriene (LT) A4 hydrolase converts the unstable epoxide intermediate LTA4 into the potent proinflammatory compound LTB4. The formation of LTA4 is catalyzed by the enzyme 5-lipoxygenase and involves the dioxygenation of arachidonic acid with subsequent epoxide formation (1).

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes.

Incubation of human leukocytes with opsonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B4 and C4. After 3-4 min, the levels of LTB4 were 93 and 35 pmol/3*10(7) cells, respectively [corrected]. These amounts were 2-4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC4 were 8 and 20 times lower than those of LTB4 after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.

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

The expression of the 5-lipoxygenase and the 5-lipoxygenase activating protein (FLAP) genes in human tonsillar B cells and in a lymphoblastoid B cell line was demonstrated at the transcriptional level by the polymerase chain reaction (PCR) technique. Intact B cells produced very low amounts of leukotriene (LT) B4 and 5-hydroxyeicosatetraenoic acid upon stimulation with the calcium ionophore A23187 and arachidonic acid in comparison to those formed by sonicates of these cells. Incubation of intact lymphoblastoid B cells with the glutathione depleting agent, azodicarboxylic acid bis[dimethylamidel] (diamide), prior to the addition of the calcium ionophore A23187 and arachidonic acid, led to the formation of similar amounts of LTB4 as those produced by sonicated cells. These results indicate that the glutathione status is of importance for the activity of 5-lipoxygenase in B lymphocytes.