Formation of Leukotrienes From Calcium Ionophore-A23187 Stimulated Rabbit, Rat and Mice White Blood Cells

Leukotrienes (LTs) producing capacity was investigated in calcium ionophore A23 187-stimulated rabbit. rat and mice peripheral white blood cells suspension. A reverse phase high performance liquid chromatography technique and computerized UV spectroscopy were employed to isolate and quantitate the released LTs namely. LTC4 and LTB4. Preincubation of rabbit white blood cells at 37°C for 5 min followed by calcium ionophore-A23 187 (1 pM) stimulation for another 5 min produced an equal amounts of LTC4 as compared to LTC4 produced by human white blood cells (105i11 versus 9529.5 pmol/107 cells respectively; mean +SEM). In contrast rabbit white blood cells synthesized significantly lower LTB4 in comparison with LTB4 produced by peripheral white blood cells from healthy control (168i18 versus 228219 pmol/107 cells respectively: mean +SEM). When rat and mice white blood cells suspension were stimulated with calcium ionophore A23187 (1 pM) after preincubation at 37°C for 5 min, equivalent amounts of LTC4 and LTB4 were observed. However, LTB4 and LTC4 produced by rat and mice white blood cells were significantly lower in comparison with LTB4 and LTCj produced by human white blood cells stimulated with calcium ionophore-A23 187. These results demonstrate that rabbit. rat and mice white blood cells suspension possess the capacity to produce LTC4 and LTBj from endogenous substrate after calcium ionophore-A23 187 stimulation.


Introduction
Leukotrienes are a family of oxygenated metabolities of arachidonic acid, which synthesized by 5-lipoxygenase enzyme (EC 2.5.1.18)'. During cell activation, LTs biosynthesis is initiated by translocation of a specific high molecular weight cytosolic phospholipase A, (cPLA,) from cytoplasm to selectively hydrolyze nuclear envelope phospholipids releasing arachidonic acid2. The liberated arachidonic acid binds to arachidonate transfer protein, 5-lipoxygenaseactivating protein (FLAP) which makes the fatty acid available to 5-lipoxygenase In a calcium-and ATP-dependent reaction the fatty acid is transformed to unstable epoxide intermediate leukotriene Aq ( L T~) ' .~. In various human cell types and tissues, leukotriene Aq is metabolized via three main pathways; by cytosolic LTAq hydrolase (EC 3.3.2.6) to the potent leukocyte activator, LTB~' or by membrane bound LTC4 synthase (EC 2.5. 1 3 7 ) to the spasmogenic cysteinyl containing leukotriene vir LTC~' or spontaneously non-enzymaticaly hydrolysed to the biologically inactive stereoisomers namely; 6-trans LTB4 and 12-epi-6trans L T B~~. Endogenous and exogenous stimuli including platelet activating factor, chemotactic peptide C5a and calcium ionophore A23187 have been shown to trigger the release of LTS". LTC4 once formed is exported from the cells via an active transport to the extra-cellular space1'. The subsequent conversion of LTC4 into LTDd via removal of y -glutamyl moiety from glutathione is catalyzed by y-glutamyl-transpepetidase, an enzyme located at the external site of plasma membranet2. This reaction can not be considered as biological inactivation. since LTD4 generally possess equal or even higher potency than L T C~'~. The next metabolic step results in substantially loss of the biological activity through removal of glycine results in LTE4 formation. The cysteinyl-containing leukotrienes bind to specific receptors and mediate a wide variety of inflammatory response1". In the airway system. I,TC4 is one of the relevant mediators involved during bronchial asthma and is responsible for many Formation of Leukotrienes From Calcium lonophore-A23187 Stimulated Rabbit .. . 235 of the cardinal symptoms of the disease15. It increases mucus secretion, edema formation and being a potent spasmogenic16. The cysteinyl-containing leukotrienes receptors antagonists are now in clinical use for pharmacological treatment of these diseasesI7 A key pro-inflammatory role has been postulated for LTB4 in the light of its ability to recruit and activate inflammatory cells'! It stimulates the production of a number of proinflammatory mediators including cytokines7, release of lysosomal enzymesI9, the generation of superoxide radicals in neutr~~hils'~. as well as it activates NADPH oxidase (EC 1.6.99.1) of human eosinophil and H202 production by human monocytes2'. These effects reflect an ability to accentuate free radicals generation and indicating an ability to augment and prolong tissue inflammation.
The knowledge concerning leukotrienes biosynthesis by white blood cells of different species is limited. Previously it has been demonstrated that calcium ionophore-A23187 induced LTB4 generation from rabbit and rat white blood cells2'. However LTC4 formation has not been described.
The goal of the present study was to extend the previous observations to study the capacity of white blood cells from different species rabbit, rat and mice to produce LTB4 and LTCj. To achieve this goal. white blood cells from rabbit, rat and mice were isolated and stimulated with calcium ionophore-A23187 to produce LTB4 and LTC4 in comparison with LTBj and LTC4 produced by peripheral white blood cells from the healthy donors were investigated.

Animals Experiments:-
White rabbit, swiss albino rats and normal mice were obtained from Experimental Animal Care Center of King Saud University, Riyadh, Saudi Arabia. They were housed in a room maintained at a temperature of 24*1 "C and 55*5% relative humidity with a regular 12 h light: 12 h dark cycle and free access to standard laboratory food (Purina Chow) and water.

Preparation o f white blood cells suspension
Blood samples from the rabbit, rats and mice as well as from healthy controls were taken into heparinized tubes. The white blood cells were separated following standard laboratory technique23. Briefly, 10 ml of whole blood was mixed with an equal volume of dextran T500 (2% in saline), then, allowed to stand for 30 min at 4 "C to sediment erythrocytes. The leukocytes-rich upper plasma layer was then aspirated and centrifuged at 280 xg for 10 min.
The resultant leukocyte pellet was washed twice with PBS then, re-suspended in 20 ml of hypotonic ammonium chloride (0.74% in PBS) for another 30 min at 4°C to lyse any remaining erythrocytes. After centrifugation at 280 xg for 10 min, the cell pellets were washed twice and suspended in PBS. The cells were counted in a Neubauer counting chamber. The final white blood cell counted was adjusted at 10x10~ cells/ml PBS. The viability was about 95% as judged by trypan blue exclusion test.

Incubation procedure:
Human, rabbit, rat and mice white blood cells 10x10~ cells/ml PBS were incubated at 37°C for 5 min. After pre-incubation, the cells were immediately stimulated for the release of LTs with calcium ionophore-A23 187 (Final concentration 1 pM) 1 pl of calcium ionophore-A23187 (1mM) was added to each aliquote (1 ml) to give final concentration of 1 pM. The release reaction was terminated after 5 min by adding 5 volumes of 99% ethanol and placing the tubes in ice. The samples were stored at -20°C until required for further processing.

Purification, Identification and Ouantitation o f leukotrienes: -
The samples dissolved in ethanol were centrifuged and the clear supernatants were aspirated and then evaporated to dryness. The obtained residues were dissolved in mobile phase and then centrifuged. Identification and quantitation of leukotrienes were performed by RP-HPLC and UV spectroscopy, as previously reported24. Chromatographic separation was performed at room temperature using Shimadizu programmable module apparatus (Japan) and a mobile phase consisting of acetonitrile/methanol/water/acetic acid (30: 19:50:0.8, pH 5.6).
The absorbance of the effluent was continuously monitored at 280 nm using a variable wavelength UV detector. Authentic standard of LTs was co-chromatographed with the samples for the identification and quantitation of the eluted LTs in the samples. The areas under the curve of each type of LTs in the samples were correlated with that of the standard.
The results were expressed as pmol/l 0' cells.

Statistics
Data are expressed as (means+SEM). Statistical comparisons of leukotrienes production in suspension of rabbit, rat and mice white blood cells and human white blood cells were performed by using student's t-test. Significance was accepted at P< 0.05.

Leukotrienes -formation in suspension of human and rabbit white blood cells.
When human and rabbit white blood cells were stimulated with 1 pM calcium ionophore-A23 187 for 5 min. a release of 5-lipoxygenase derived products viz. LTC, and LTB, can be identified. LTB, was the major compound formed. Rabbit white blood cells produced an equivalent amount of LTC, as compared to that produced by peripheral white blood cell separated from the control healthy donor. Thus. 105*11 (n=8; mean * SEM) and 953t9.7 (n=4) pmol LTC,IIO' cells were produced by rabbit white blood cells and white blood cells separated from healthy controls respectively (Fig 1). However, the production of LTB, was significantly lower in rabbit white blood cells suspension as compared to the human white blood cells. Thus, 168t18 versus 228+19 pmol LTB ~1 0 ' cells were produced by rabbit and human white blood cell suspensions respectively (Fig 1).

Fig (1): Leukotrienes formation by rabbit and human peripheral white blood cells suspension stimulated with calcium ionophore-A23187.
Peripheral white blood cells separated from rabbit and control healthy donors (1 0 x 1 o6/ml PBS) were preincubated at 37°C for 5 min, then stimulated with A23187 (1 pM) for leukotriene release for another 5 min. The reaction was terminated by addition of 5 ml of ethanol 99% and placing the tube in ice. Leukotrienes were identified and quantified by RP-HPLC.

Leukotrienes formation in suspension of human, rat and mice white blood cells.
Rat and mice white blood cells preincubated at 37°C for 5 min and stimulated with calcium ionophore-A23187 for another 5 min, produced a similar amounts of LTC4 and LTB4.
However, stimulation of white blood cells separated from healthy donor produced LTC4 5 times more than LTC4 produced from rat and mice (Fig 2). In addition, human white blood cells produced 5 and 7 times LTB4 as compared to LTB4 produced from rat and mice, respectively (Fig 2).
Formation of Leukotrienes From Calcium lonophore-A23187 Stimulated Rabbit . . . 239 k §Z! Human white blood W Rat white blood Mice white blood

Fig (2): Leukotrienes formation by rat, mice and human peripheral white blood cells suspension stimulated with calcium ionophore-A23187.
Peripheral white blood cells separated from rat, mice and control healthy donors (10 x10~1ml PBS) were preincubated at 37'C for 5 min, then stimulated with A23 187 (1 pM) for leukotriene release for another 5 min. The reaction was terminated by addition of 5 ml of ethanol 99% and placing the tube in ice. Leukotrienes were identified and quantified by RP-HPLC

Discussion
Stimulation of human white blood cells with calcium ionophore-A23 187 (1 pM) led to the generation of leukotrienes, predominantly LTB, which is known as the major mediator released from human leukocytes2'. By virture of its calcium ionopheric property, A23187 produces an elevation of intracellular calcium concentration that is required for the binding of 5-lipoxygenase to an activating protein (FLAP)). This binding activates enzyme results in its association with the nuclear membrane and consequently increases the synthesis of LTs from arachidonic a~i d~~.~' .
The present study showed that, rabbit, rat and mice white blood cells suspension possess the capacity to produce LTC, and LTB, after calcium ionophore-A23187 stimulation. This finding indicates that calcium ionophore A23 187 activates 5-lipoxygenase enzyme in white blood cells isolated from rabbit, rat and mice and the stimulus is sufficient to make endogenous arachidonic acid available for leukotrienes synthesis in these cells.
The results of the present study demonstrate that rabbit white blood cells produced an equivalent amount of LTC, as compared to human peripheral white blood cells. In contrast, the synthesis of LTB, was significantly lowered in rabbit white blood cells suspension as compared with LTB, produced by human white blood cells (Fig. 1). Stimulation of rat and mice white blood cells suspensions produced similar amounts of LTC, and LTB,. However. LTC, produced from calcium ionophore-A23 187 stimulated human white blood cells was 5 times more than LTC, produced by rat and mice white blood cell suspension. In addition human white blood cells produced 5 and 7 times LTB, more than LTB, produced from white blood cells separated from rat and mice respectively.
The present results are consistent with the previous study demonstrated that rabbit and rat white blood cells produced LTB, from endogenous arachidonic acid after calcium ionophore-A23 187 (1 p~) 2 2

White blood cells suspension contains heterogeneous cells populations mainly neutrophils
which produce LTB?~, while esinophils and basophils produce LTC:~. In addition, it has been shown that B-lymphocytes produce leukotrienes only when stimulated with calcium ionophore-A23187 in the presence of exogenous arachidonic acid and reducing agents3'.
Thus. in the present study, LTB, and LTC, were produced by neutrophils and eosinophils respectively. However, when studying in vitro leukotrienes producing capacity, samples containing identical number of the cells and a similar percentage of different cell types should be compared. Therefore, it has been assumed that the decreased level of LTC, and LTB, produced by rat and mice white blood cells suspension in the present study may be due to the difference in the percentage of various cell types in comparing with human white blood cell suspensions. In the present study. differential cell identification count revealed that there was no significant difference between number of neutrophils, eosinophils and mononuclear cells in the different white blood cells suspensions (data not shown).
Formation of Leukotrienes From Calcium lonophore-A23187 Stimulated Rabbit .. . 241 The reason why the rat and mice cells produced significantly lowered LTC4 and LTB, as compared to human white blood cells may be due to decreased arachidonic acid content in phospholipid of nuclear envelope of white blood cells of rat and mice. Thus less of the substrate (arachidonic acid) will be available for 5-lipxygenase enzyme. In addition. the identity of the specific phospholipaseA2 involved in the release of arachidonic acid utilized for leukotrienes synthesis is still a matter of controversial between different species3'.
Moreover it has been demonstrated that leukotrienes synthesis in intact cells from various species depend on the activity of 5-lipoxygenase-activating protein (FLAP), which binds arachidonic acid, thereby facilitating the 5-lipoxygenase-catalyzed conversion of fatty acid into L T A~"~* .
These may indicate a difference in the level of arachidonic acid content or difference in 5-lipoxygenase activating protein level in rat and mice as compared to human white blood cells.
In conclusion: This investigation demonstrates that white blood cells of rabbit, rat and mice possess the capacity to produce different amounts of leukotrienes from endogenous substrate after short term incubations with calcium ionophore-A23 187.