1391

Testing Neutrophil Function

THE LANCET

THE centenary of METSCHNIKOFF’S description’of the antibacterial role of phagocytes coincidedwith a burst of interest in their biological actions.The stimulus was a realisation that neutrophil dys-function might be important in the genesis of dis-ease, both infective and non-infective, coupled withan expansion and refinement of investigative tech-niques. Advances in cell-labelling2 have enormouspotential in studies on cell kinetics and traffic andfor localisation of disease.3 Granulocytes can nowbe concentrated to an extent that makes trans-

fusion therapeutically useful4 (though we do notyet know how best to separate cells for transfusionnor which patients are likely to benefit). And wecan examine separately some of the processeswhich, operating together, determine the activity ofneutrophils.The sequence of events in the life of a con-

scripted neutrophil includes egress from themarrow, circulation to the site of inflammation,recognition of, and attachment to the object of pha-gocytosis, ingestion, intravacuolar killing of

microbes, and digestion of phagocytosed materials.Studies of neutrophil kinetics and distribution havebeen hampered by inadequate methods of labellingcells. Until lately the tracers5 employed were

tritium, phosphorus-32, and chromium-51 which,although useful for measuring the rate of

disappearance of cells from the circulation, are

unsuitable for external detection and thus fordetermination of organ distribution and eventualfate. Experiments with these isotopes showed that,after release from the bone-marrow, neutrophilsremain in the bloodstream for about 6 hours,6either circulating or adhering to the endotheliallining of the vessels. Ultimately they migrate intothe tissues where their fate is unknown. Probablythey act as scavengers, percolating through the

1. Metschnikoff, E. Immunity in Infective Diseases (translated by F. G. Bin-nie). London, 1905.

2. Thakur, M. L., Coleman, R. E., Mayhall, C. G., Welch, M. J. Radiology,1976, 119, 731.

3. Segal, A. W., Thakur, M. L., Arnot, R. N., Lavender, J. P. Lancet, 1976,ii, 1056.

4. International Symposium on Leukocyte Separation and Transfusion (editedby J. M. Goldman, and R. M. Lowenthal). London, 1974.

5. Craddock, C. G. in Hematology (edited by W. J. Williams, E. Beutler, A. J.Erslev, and R. W. Rundles); p. 593. New York, 1972.

6. Cline, M J. The White Cell. Boston, 1975.

tissues and draining to regional lymph-nodeswhence they may, if they have not alreadyencountered a phagocytosable object, re-enter thecirculation. An important newcomer is the

technique employing the gamma-emitting isotopeindium-III.2 Already this isotope has been used tolabel autologous leucocytes for localisation ofabscesses in man,3 and it may enable study of thedistribution and extravascular movement of

neutrophils-though we shall need to be sure thatthe observed shifts of radioactivity accuratelyrepresent natural phenomena and that themovements of the cells are not modified by thelabelling procedure.

Motility can be examined in vivo or in vitro.Accumulation of pus at sites of infection is perhapsthe best evidence that chemotactic factors 7 are

being released from the tissues and that, throughsome as yet undefined sensory system, the muscu-loskeletal system of the neutrophil (composed ofthe microfilaments actin and myosin, and micro-tubules9) can respond normally. These functions canbe examined qualitatively by determining whetherneutrophils will pass through abraded skin onto anadherent glass slide,IO or semiquantitatively bymeasuring the rate of accumulation of cells in asuperimposed fluid-filled chamber12 to whichvarious mediators or drugs can be added. Tests ofin-vitro migration require the separation of leuco-cytes from whole blood. Cell movements can thenbe directly observed on a slide by light microscopy,and measured by progress along capillary tubes,through filters, 12 or under agarose13 in the absenceand presence of chemotactic mediators.

Accurate measurement of phagocytosis9 is com-plicated by difficulty in distinguishing adherentfrom ingested particles. Attempts to surmount thisobstacle include removal of unphagocytosed mater-ial exposed to the extracellular environment-forexample, immune complexes may be re-dissolved byaddition of an excess of antigen 14-or by measur-ing the ingestion of materials which are modifiedwithin the phagocytic vacuole (e.g., the dye nitro-blue tetrazoliumI5). Among the objects of phagocy-tosis that have been employed are yeast and poly-styrene particles and erythrocytes (counted micro-scopically), bacteria (cultured), paraffin-oil particles(measured spectrophotometrically), and radio-labelled bacteria or immune complexes. 6 9 16 Phase-contrast and electron microscopy may be useful fordetecting qualitative abnormalities of phagocy-tosis.9

7 Sorkin, E , Stecher, V. J., Borel, J. F. Ser. Hœmat. 1970, 3, 131.8. Wilkinson, P. L. Chemotaxis and Inflammation. Edinburgh, 1974.9 Stossel, T P. New Engl J. Med. 1974, 290, 717

10. Rebuck, J. W., Crowley, J. H. Ann N.Y. Acad. Sci. 1955, 59, 757.11 Senn, H. Fektalswehr bei Hömoblastosen, p. 36. Berlin, 1972.12. Boyden, S. J. exp. Med. 1962, 115, 453.13. Nelson, R. D., Quie, P. G., Simmons, R. L. J. Immun. 1975, 115, 1650.14. Ward, P A , Zvaifler, N. J. ibid. 1973, 111, 1771.15. Segal, A. W Lancet, 1974, ii, 1248.16. Hallgren, R., Stalenheim, G. Immunology, 1976, 30, 755.

1392

The metabolic changes that are normally associ-ated with phagocytosisl’ seem to be predominantlyconcerned with the generation of microbicidal freeradicals,18 and with free-radical scavenging sys-tems19 which protect the cell from autolysis.Although not conclusively proven, the prime micro-bicidal compounds18 seem to be superoxide, singletoxygen or hydrogen peroxide which form spon-taneously from superoxide, or hydroxyl radicalswhich are generated by the combination of super-oxide and hydrogen peroxide. The toxic effect ofhydrogen peroxide may be amplified by or

mediated through myeloperoxidase.18 Superoxide,the initial compound in this sequence is probablygenerated from reduced nicotinamide adeninedinucleotide (N.A.D.H) through the action of anoxidase situated in the plasma membrane of thecell.20 21 The observed burst of hexose-monophos-phate shunt activity is likely to be a secondaryeffect20 concerned with the regeneration of reducingequivalents consumed by free-radical scavengers,including glutathione and ascorbic acid, in thedetoxification of oxygen radicals and peroxidewhich diffuse out of the phagocytic vacuole into thecytoplasm.The burst of metabolic activity can be measured

by oxygen consumption,1’ by chemiluminescencefrom singlet oxygen, 22 by superoxide-mediatedreduction of N.B.T.23 or cytochrome C,24 or in-

directly by 14CO2 generated from glucose-1-14C bythe hexose-monophosphate shunt.25 The action ofmyeloperoxidase on endogenously generatedhydrogen peroxide can be examined by measuringthe iodination of phagocytosed bacteria after theaddition of radioactive iodine to the medium. 26

Killing of bacteria or fungi can be determined di-rectly by incubating the microbes and cells togetherand then assessing the viability of the organisms.Bacterial survival can be assessed by colony countson culture plates27 or by measuring bacterial in-corporation of tritiated thymidine,6 and dead fungiare identified by their failure to exclude the dyemethylene-blue. 211The discharge of granule contents into the

vacuole can be observed by light29 and electronmicroscopy. It can be quantitated indirectly by themethod of "frustrated phagocytosis"3O in which the

17. Sbarra, A. J., Karnovsky, M. L. J. biol. Chem. 1959, 234, 1355.18. Klebanoff, S. J. Semin. Hemat. 1975, 12, 117.19. Reed, P. W. J. biol. Chem. 1969, 244, 2459.20. Segal, A. W., Peters, T. J. Lancet, 1976, i, 1363.21. Briggs, R. T., Drath, D. B., Karnovsky, M. L., Karnovsky, M. J. J. Cell

Biol. 1975, 67, 566.22. Allen, R. L., Stjernholm, R. L., Steele, R. H. Biochem. biophys. Res. Com-

mun. 1972, 47, 679.23. Baehner, R. L., Nathan, D. G. New Engl. J. Med. 1968, 278, 971.24. Curnutte, J. T., Whitten, D. M., Babior, B. M. ibid. 1974, 290, 593.25. Baehner, R. L., Nathan, D. G., Karnovsky, M. L. J. clin. Invest. 1970, 49,

865.26. Pincus, S. H., Klebanoff, S. J. New Engl. J. Med. 1971, 284, 744.27. Quie, P. G., White, J. G., Holmes, B., Good, R. A. J. clin Invest. 1967, 46,

668.28. Lehrer, R. I., Cline, M. J. J. Bact. 1969, 98, 996.29. Hirsch, J. G. J. exp. Med. 1962, 116, 827.30. Henson, P M. ibid. 134, suppl. 114.

cell is exposed to a large object which cannot bicompletely engulfed, leading to failure of closure oithe vacuole and release of its contents into the sur.

rounding medium, or directly by causing the cellto ingest material, such as oil droplets,3’ whichfacilitate separation of the vacuole and its contentsfrom other organelles of the disrupted cell. Theextent to which microbes are degraded can beassessed morphologically by electron microscopy,"by changes in permeability or protein synthesis,33and by the release of products of catabolism fromthe organism.34 The importance of preliminarydegradation in microbial killing and in subsequentgeneration of antibodies is unknown. Incompletemodification of microbial macromolecules may be

important in the aetiology of chronic inflammatorydisease.32

At the clinical level, which patients should be in-vestigated for defective neutrophil function andwith what tests? Impaired neutrophil function35should be suspected when an infectious disease hasan unduly severe or protracted course or when itbecomes recurrent or chronic in the absence ofother predisposing conditions such as hypogamma-globulinaemia. A history of similar troubles in othermembers of the family is an important pointer toa hereditary defect. One sign which can alert theclinician to the possibility of defective chemotaxisor motility is an unusually small amount of pus atinfection sites in a patient with adequate numbersof circulating granulocytes. Morphological aber-rations may be detected by light microscopy. Thenylon-column dye test36 is a simple rapid screeningtest which examines the properties of adherence,stimulation, phagocytosis, and superoxide produc-tion. This is a useful qualitative test to excludechronic granulomatous disease, and it can also beemployed as a quantitative test of neutrophil func-tion. Routine hospital laboratories often do nothave the facilities to test chemotaxis and cell moti-

lity (whether in vivo by the skin-window tech-

niquelU 11 or in vitro 12 13) or to measure bacteriap7or fungal2g killing. Myeloperoxidase deficiency can,however, be readily excluded biochemicallyYPatients with such disorders are best served byreferral to a centre with a research interest in these

disorders, where the morphology and cell biologyof adherence, motility, phagocytosis, killingmechanisms, degranulation, and digestion can beindividually examined and detailed. Such centresare best placed to administer neutrophil transfu-sions-—the logical accompaniment to specific anti-microbial therapy.

31. Stossel, T. P., Pollard, T. D., Mason, R. J., Vaughan, M. J. clin Invest

1971, 50, 1745.32. Ginsberg, I., Sela, M. N. Crit. Rev. Microbiol. 1976, 4, 249.33. Elsbach, P. New Engl. J. Med. 1973, 289, 846.34. Cohn, Z. A. J. exp. Med. 1963, 117, 27.35. Quie, P. G. Semin. Hematol. 1975, 12, 143.36. Segal, A. W., Peters, T. J. Clin Sci. mol. Med. 1975, 49, 591.37. Klebanoff. S. J. Endocrinology, 1965, 76, 301.