Quantitative determination of the chemotactic function of neutrophilic granulocytes
 | SUMMARY and EXPLANATION |
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Also, the decrease of expresssion of the cell adhesion molecule LECAM-1 can be analysed together with the shape-change of the cells by analysing the changes of the forward scatter signals.
APPLICATIONS
By MIGRATEST® it is intended to investigate the altered chemotactic function of neutrophils found in various disorders and to evaluate the effects of drugs on this process.
Abnormal chemotaxis was observed and described with a variety of clinical disorders (3, 4). These defects are usually accompanied by recurrent infections of the skin or respiratory tract. Clinical syndromes with frequent infections are already observed in the infant. A functional chemotaxis defect diminishes the number of granulocytes which migrate from the circulation into inflamed tissues and therefore the amount of active cells at the sites of inflammation.
Known inborn defects are Leukocyte Adhesion Deficiency (5), Hyperimmunoglobulin E or Job?s syndrome and Chédiak-Higashi syndrome (CHS). These disorders are associated with a reduced chemotactic activity of neutrophilic granulocytes (6, 7).
Acquired defects can be observed in renal failure, diabetes, hepatic cirrhosis (8) and Hodgkin?s disease. Circulating immune complexes in rheumatoid arthritis also inhibit chemotaxis (10).
Various drugs and immunomodulators can enhance or decrease the chemotactic activity of granulocytes. These effects can be investigated in vitro or ex vivo by application of MIGRATEST®.
TEST PRINCIPLES
The phagocytic process can be separated into several major stages: Chemotaxis as the initiating step of phagocytosis (migration of phagocytes to inflammatory sites towards a gradient of chemotactic factors), attachment of particles to the cell surface of phagocytes, ingestion (phagocytosis) and intracellular killing by oxygen-dependent and oxygen-independent mechanisms (1, 2). Phagocytosis by polymorphonuclear neutrophils and monocytes therefore constitutes an essential arm of host defense against bacterial or fungal infections.
MIGRATEST® allows the quantitative determination of neutrophil chemotaxis. The number of cells can be determined which have migrated through cell culture inserts with a pore size of 3.0 µm towards a concentration gradient of the chemoattractant fMLP. In parallel, the decrease of LECAM-1 (11) expression can be measured. Downregulation of this cell adhesion molecule correlates directly with the activation of neutrophils by exposing to chemotactic factors. The shape-change of the cells precedes the cell migration and can be measured by analysing the changes of the forward scatter signals using flow cytometry. The test kit contains the chemoattractant fMLP, cell culture inserts and necessary reagents. Leukocytes are isolated from heparinzed whole blood by spontaneous sedimenation over leukocyte SEPARATION MEDIUM and placed into cell culture inserts. Chemotaxis is conducted at 37°C towards a gradient of fMLP in comparison to a control of INCUBATION BUFFER. The cells are then stained with an antibody reagent (STAINING/COUNTING REAGENT) which also contains counting beads. Just prior flow cytometric analysis a special vital DNA dye is added.
In summary, chemotaxis is the initiating step of the multistep and multifactorial process of phagocytosis (1, 2). The whole cascade of events can be investigated individually under controlled conditions by separate assays: MIGRATEST® to measure chemotaxis, PHAGOTEST® to measure ingestion of bacteria, BURSTTEST (PHAGOBURST®) to measure oxidative burst.
REFERENCES
(1) Roitt IM, Brostoff J, Male DK. 1996. Immunology, 4th ed. Gower Medical Publishing Ltd., London
(2) Sawyer DW, Donowitz GR, Mandell, GL. 1989. Polymorphonuclear neutrophils: An effective antimicrobial force. Rev. Infect. Dis. 11: S1532-S1544.
(3) Donadebian HD, 1989. Congenital and acquired neutrophil abnormalities. In: Klempner, M.S. et al. (eds) Phagocytes and Disease. Kluwer, Dordrecht Boston New York, pp 103-118
(4) Robinson JP, Carter WO. 1993. Flow cytometric analysis of granulocytes. In: Bauer, K.D. et al. (eds) Clinical Flow Cytometry, Principles and Applications. Williams & Wilkins, Baltimore, pp 405-433.
(5) Anderson DC, Springer TA. 1987. Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1 and p150, 95 glycoproteins. Annu Rev Med 38: 175-194.
(6) Gallin JI, Wright DG, Malech HL, Davis JM, Klempner MS, Kirkpatrick CH. 1980. Disorders of phagocyte chemotaxis. Ann Intern Med 92: 520-538
(7) Ottonello L, Dapino P, Pastorino G, Dallegri F, Sacchetti C. 1995. Neutrophil dysfunction and increased susceptibility to infection. Eur J Clin Invest 25:687-92.
(8) Ward PA, Berenberg JL. 1974. Defective regulation of inflammatory mediators in Hodgkin's disease. Supernormal levels of chemotactic-factor inactivator. N Engl J Med 290: 76-80.
(9) DeMeo AN, Anderson BR. 1972. Defective chemotaxis associated with a serum inhibitor in cirrhotic patients. N Engl J Med 286: 735-740.
(10) Attia WM, Clark HW, Brown TM. 1982. Inhibition of polymorphonuclear leukocyte migration by sera of patients with rheumatoid arthritis. Ann Allergy 48: 21-24.
(11) Kishimoto TK, Jutila MA, Butcher EC. 1990. Identification of a human peripheral lymph node homing receptor: a rapidly down-regulated adhesion molecule. Proc Natl Acad Sci, USA 87: 2244-2248