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Testing Neutrophil Function
1391
THE LANCET
Testing Neutrophil Function THE centenary of METSCHNIKOFF’S description’ of the antibacterial role of phagocytes coincided with a burst of interest in their biological actions.
The stimulus was a realisation that neutrophil dysfunction might be important in the genesis of disease, both infective and non-infective, coupled with an expansion and refinement of investigative techniques. Advances in cell-labelling2 have enormous potential in studies on cell kinetics and traffic and for localisation of disease.3Granulocytes can now be concentrated to an extent that makes transfusion therapeutically useful4 (though we do not yet know how best to separate cells for transfusion nor which patients are likely to benefit). And we can examine separately some of the processes which, operating together, determine the activity of
neutrophils. The sequence of
in the life of a conscripted neutrophil includes egress from the marrow, circulation to the site of inflammation, recognition of, and attachment to the object of phagocytosis, ingestion, intravacuolar killing of microbes, and digestion of phagocytosed materials. Studies of neutrophil kinetics and distribution have been hampered by inadequate methods of labelling cells. 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 for determination of organ distribution and eventual fate. Experiments with these isotopes showed that, after release from the bone-marrow, neutrophils remain in the bloodstream for about 6 hours,6 either circulating or adhering to the endothelial lining of the vessels. Ultimately they migrate into the tissues where their fate is unknown. Probably they act as scavengers, percolating through the events
tissues and draining to regional lymph-nodes whence they may, if they have not already encountered a phagocytosable object, re-enter the circulation. An important newcomer is the technique employing the gamma-emitting isotope indium-III.2 Already this isotope has been used to label autologous leucocytes for localisation of abscesses in man,3and it may enable study of the distribution and extravascular movement of neutrophils-though we shall need to be sure that the observed shifts of radioactivity accurately represent natural phenomena and that the movements of the cells are not modified by the
labelling procedure. Motility can be examined
in vivo or in vitro. Accumulation of pus at sites of infection is perhaps the best evidence that chemotactic factors 7 are being released from the tissues and that, through some as yet undefined sensory system, the musculoskeletal system of the neutrophil (composed of the microfilaments actin and myosin, and microtubules9) can respond normally. These functions can be examined qualitatively by determining whether neutrophils will pass through abraded skin onto an adherent glass slide,IO or semiquantitatively by measuring the rate of accumulation of cells in a superimposed fluid-filled chamber12 to which various mediators or drugs can be added. Tests of in-vitro migration require the separation of leucocytes from whole blood. Cell movements can then be directly observed on a slide by light microscopy, and measured by progress along capillary tubes, through filters, 12 or under agarose13 in the absence and presence of chemotactic mediators. Accurate measurement of phagocytosis9 is complicated by difficulty in distinguishing adherent from ingested particles. Attempts to surmount this obstacle include removal of unphagocytosed material exposed to the extracellular environment-for example, immune complexes may be re-dissolved by addition of an excess of antigen 14-or by measuring the ingestion of materials which are modified within the phagocytic vacuole (e.g., the dye nitroblue tetrazoliumI5). Among the objects of phagocytosis that have been employed are yeast and polystyrene particles and erythrocytes (counted microscopically), bacteria (cultured), paraffin-oil particles (measured spectrophotometrically), and radiolabelled bacteria or immune complexes. 6 9 16 Phasecontrast and electron microscopy may be useful for detecting qualitative abnormalities of phagocytosis.9
1. Metschnikoff, E.
Immunity in Infective Diseases (translated by F. G. Binnie). 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 (edited by 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.
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 associated with phagocytosisl’ seem to be predominantly concerned with the generation of microbicidal free radicals,18 and with free-radical scavenging systems19 which protect the cell from autolysis. Although not conclusively proven, the prime microbicidal compounds18 seem to be superoxide, singlet oxygen or hydrogen peroxide which form spontaneously from superoxide, or hydroxyl radicals which are generated by the combination of superoxide and hydrogen peroxide. The toxic effect of hydrogen peroxide may be amplified by or mediated through myeloperoxidase.18 Superoxide, the initial compound in this sequence is probably generated from reduced nicotinamide adenine dinucleotide (N.A.D.H) through the action of an oxidase situated in the plasma membrane of the cell.20 21 The observed burst of hexose-monophosphate shunt activity is likely to be a secondary effect20 concerned with the regeneration of reducing equivalents consumed by free-radical scavengers, including glutathione and ascorbic acid, in the detoxification of oxygen radicals and peroxide which diffuse out of the phagocytic vacuole into the
cytoplasm. The burst of metabolic activity can be measured by oxygen consumption,1’ by chemiluminescence from singlet oxygen, 22 by superoxide-mediated reduction of N.B.T.23 or cytochrome C,24 or in-
directly by 14CO2 generated from glucose-1-14C by the hexose-monophosphate shunt.25 The action of myeloperoxidase on endogenously generated hydrogen peroxide can be examined by measuring the iodination of phagocytosed bacteria after the addition of radioactive iodine to the medium. 26 Killing of bacteria or fungi can be determined directly by incubating the microbes and cells together and then assessing the viability of the organisms. Bacterial survival can be assessed by colony counts on culture plates27 or by measuring bacterial incorporation of tritiated thymidine,6and dead fungi are identified by their failure to exclude the dye
methylene-blue. 211 The discharge
22. 23. 24. 25.
Sbarra, A. J., Karnovsky, M. L. J. biol. Chem. 1959, 234, 1355. Klebanoff, S. J. Semin. Hemat. 1975, 12, 117. Reed, P. W. J. biol. Chem. 1969, 244, 2459. Segal, A. W., Peters, T. J. Lancet, 1976, i, 1363. Briggs, R. T., Drath, D. B., Karnovsky, M. L., Karnovsky, M. J. J. Cell Biol. 1975, 67, 566. Allen, R. L., Stjernholm, R. L., Steele, R. H. Biochem. biophys. Res. Commun. 1972, 47, 679. Baehner, R. L., Nathan, D. G. New Engl. J. Med. 1968, 278, 971. Curnutte, J. T., Whitten, D. M., Babior, B. M. ibid. 1974, 290, 593. 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. 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.
the vacuole and release of its
contents
into the
sur.
rounding medium, or directly by causing the cell to ingest material, such as oil droplets,3’ which facilitate separation of the vacuole and its contents from other organelles of the disrupted cell. The extent to which microbes are degraded can be assessed morphologically by electron microscopy," by changes in permeability or protein synthesis,33 and by the release of products of catabolism from the organism.34 The importance of preliminary degradation in microbial killing and in subsequent generation of antibodies is unknown. Incomplete modification of microbial macromolecules may be important in the aetiology of chronic inflammatory disease.32 At the clinical level, which patients should be investigated for defective neutrophil function and with what tests? Impaired neutrophil function35 should be suspected when an infectious disease has an unduly severe or protracted course or when it becomes recurrent or chronic in the absence of other predisposing conditions such as hypogammaglobulinaemia. A history of similar troubles in other members of the family is an important pointer to a hereditary defect. One sign which can alert the clinician to the possibility of defective chemotaxis or motility is an unusually small amount of pus at infection sites in a patient with adequate numbers of circulating granulocytes. Morphological aberrations may be detected by light microscopy. The nylon-column dye test36 is a simple rapid screening test which examines the properties of adherence, stimulation, phagocytosis, and superoxide production. This is a useful qualitative test to exclude chronic granulomatous disease, and it can also be employed as a quantitative test of neutrophil function. Routine hospital laboratories often do not have the facilities to test chemotaxis and cell motility (whether in vivo by the skin-window techniquelU 11 or in vitro 12 13) or to measure bacteriap7
fungal2g killing. Myeloperoxidase deficiency can, however, be readily excluded biochemicallyY Patients with such disorders are best served by or
of granule contents into the vacuole can be observed by light29 and electron microscopy. It can be quantitated indirectly by the method of "frustrated phagocytosis"3O in which the 17. 18. 19. 20. 21.
exposed to a large object which cannot bi completely engulfed, leading to failure of closure oi cell is
1967, 46,
with a research interest in these disorders, where the morphology and cell biology of adherence, motility, phagocytosis, killing mechanisms, degranulation, and digestion can be individually examined and detailed. Such centres are best placed to administer neutrophil transfusions-—the logical accompaniment to specific antimicrobial therapy. referral
to a centre
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.
THE LANCET
Testing Neutrophil Function THE centenary of METSCHNIKOFF’S description’ of the antibacterial role of phagocytes coincided with a burst of interest in their biological actions.
The stimulus was a realisation that neutrophil dysfunction might be important in the genesis of disease, both infective and non-infective, coupled with an expansion and refinement of investigative techniques. Advances in cell-labelling2 have enormous potential in studies on cell kinetics and traffic and for localisation of disease.3Granulocytes can now be concentrated to an extent that makes transfusion therapeutically useful4 (though we do not yet know how best to separate cells for transfusion nor which patients are likely to benefit). And we can examine separately some of the processes which, operating together, determine the activity of
neutrophils. The sequence of
in the life of a conscripted neutrophil includes egress from the marrow, circulation to the site of inflammation, recognition of, and attachment to the object of phagocytosis, ingestion, intravacuolar killing of microbes, and digestion of phagocytosed materials. Studies of neutrophil kinetics and distribution have been hampered by inadequate methods of labelling cells. 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 for determination of organ distribution and eventual fate. Experiments with these isotopes showed that, after release from the bone-marrow, neutrophils remain in the bloodstream for about 6 hours,6 either circulating or adhering to the endothelial lining of the vessels. Ultimately they migrate into the tissues where their fate is unknown. Probably they act as scavengers, percolating through the events
tissues and draining to regional lymph-nodes whence they may, if they have not already encountered a phagocytosable object, re-enter the circulation. An important newcomer is the technique employing the gamma-emitting isotope indium-III.2 Already this isotope has been used to label autologous leucocytes for localisation of abscesses in man,3and it may enable study of the distribution and extravascular movement of neutrophils-though we shall need to be sure that the observed shifts of radioactivity accurately represent natural phenomena and that the movements of the cells are not modified by the
labelling procedure. Motility can be examined
in vivo or in vitro. Accumulation of pus at sites of infection is perhaps the best evidence that chemotactic factors 7 are being released from the tissues and that, through some as yet undefined sensory system, the musculoskeletal system of the neutrophil (composed of the microfilaments actin and myosin, and microtubules9) can respond normally. These functions can be examined qualitatively by determining whether neutrophils will pass through abraded skin onto an adherent glass slide,IO or semiquantitatively by measuring the rate of accumulation of cells in a superimposed fluid-filled chamber12 to which various mediators or drugs can be added. Tests of in-vitro migration require the separation of leucocytes from whole blood. Cell movements can then be directly observed on a slide by light microscopy, and measured by progress along capillary tubes, through filters, 12 or under agarose13 in the absence and presence of chemotactic mediators. Accurate measurement of phagocytosis9 is complicated by difficulty in distinguishing adherent from ingested particles. Attempts to surmount this obstacle include removal of unphagocytosed material exposed to the extracellular environment-for example, immune complexes may be re-dissolved by addition of an excess of antigen 14-or by measuring the ingestion of materials which are modified within the phagocytic vacuole (e.g., the dye nitroblue tetrazoliumI5). Among the objects of phagocytosis that have been employed are yeast and polystyrene particles and erythrocytes (counted microscopically), bacteria (cultured), paraffin-oil particles (measured spectrophotometrically), and radiolabelled bacteria or immune complexes. 6 9 16 Phasecontrast and electron microscopy may be useful for detecting qualitative abnormalities of phagocytosis.9
1. Metschnikoff, E.
Immunity in Infective Diseases (translated by F. G. Binnie). 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 (edited by 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.
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 associated with phagocytosisl’ seem to be predominantly concerned with the generation of microbicidal free radicals,18 and with free-radical scavenging systems19 which protect the cell from autolysis. Although not conclusively proven, the prime microbicidal compounds18 seem to be superoxide, singlet oxygen or hydrogen peroxide which form spontaneously from superoxide, or hydroxyl radicals which are generated by the combination of superoxide and hydrogen peroxide. The toxic effect of hydrogen peroxide may be amplified by or mediated through myeloperoxidase.18 Superoxide, the initial compound in this sequence is probably generated from reduced nicotinamide adenine dinucleotide (N.A.D.H) through the action of an oxidase situated in the plasma membrane of the cell.20 21 The observed burst of hexose-monophosphate shunt activity is likely to be a secondary effect20 concerned with the regeneration of reducing equivalents consumed by free-radical scavengers, including glutathione and ascorbic acid, in the detoxification of oxygen radicals and peroxide which diffuse out of the phagocytic vacuole into the
cytoplasm. The burst of metabolic activity can be measured by oxygen consumption,1’ by chemiluminescence from singlet oxygen, 22 by superoxide-mediated reduction of N.B.T.23 or cytochrome C,24 or in-
directly by 14CO2 generated from glucose-1-14C by the hexose-monophosphate shunt.25 The action of myeloperoxidase on endogenously generated hydrogen peroxide can be examined by measuring the iodination of phagocytosed bacteria after the addition of radioactive iodine to the medium. 26 Killing of bacteria or fungi can be determined directly by incubating the microbes and cells together and then assessing the viability of the organisms. Bacterial survival can be assessed by colony counts on culture plates27 or by measuring bacterial incorporation of tritiated thymidine,6and dead fungi are identified by their failure to exclude the dye
methylene-blue. 211 The discharge
22. 23. 24. 25.
Sbarra, A. J., Karnovsky, M. L. J. biol. Chem. 1959, 234, 1355. Klebanoff, S. J. Semin. Hemat. 1975, 12, 117. Reed, P. W. J. biol. Chem. 1969, 244, 2459. Segal, A. W., Peters, T. J. Lancet, 1976, i, 1363. Briggs, R. T., Drath, D. B., Karnovsky, M. L., Karnovsky, M. J. J. Cell Biol. 1975, 67, 566. Allen, R. L., Stjernholm, R. L., Steele, R. H. Biochem. biophys. Res. Commun. 1972, 47, 679. Baehner, R. L., Nathan, D. G. New Engl. J. Med. 1968, 278, 971. Curnutte, J. T., Whitten, D. M., Babior, B. M. ibid. 1974, 290, 593. 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. 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.
the vacuole and release of its
contents
into the
sur.
rounding medium, or directly by causing the cell to ingest material, such as oil droplets,3’ which facilitate separation of the vacuole and its contents from other organelles of the disrupted cell. The extent to which microbes are degraded can be assessed morphologically by electron microscopy," by changes in permeability or protein synthesis,33 and by the release of products of catabolism from the organism.34 The importance of preliminary degradation in microbial killing and in subsequent generation of antibodies is unknown. Incomplete modification of microbial macromolecules may be important in the aetiology of chronic inflammatory disease.32 At the clinical level, which patients should be investigated for defective neutrophil function and with what tests? Impaired neutrophil function35 should be suspected when an infectious disease has an unduly severe or protracted course or when it becomes recurrent or chronic in the absence of other predisposing conditions such as hypogammaglobulinaemia. A history of similar troubles in other members of the family is an important pointer to a hereditary defect. One sign which can alert the clinician to the possibility of defective chemotaxis or motility is an unusually small amount of pus at infection sites in a patient with adequate numbers of circulating granulocytes. Morphological aberrations may be detected by light microscopy. The nylon-column dye test36 is a simple rapid screening test which examines the properties of adherence, stimulation, phagocytosis, and superoxide production. This is a useful qualitative test to exclude chronic granulomatous disease, and it can also be employed as a quantitative test of neutrophil function. Routine hospital laboratories often do not have the facilities to test chemotaxis and cell motility (whether in vivo by the skin-window techniquelU 11 or in vitro 12 13) or to measure bacteriap7
fungal2g killing. Myeloperoxidase deficiency can, however, be readily excluded biochemicallyY Patients with such disorders are best served by or
of granule contents into the vacuole can be observed by light29 and electron microscopy. It can be quantitated indirectly by the method of "frustrated phagocytosis"3O in which the 17. 18. 19. 20. 21.
exposed to a large object which cannot bi completely engulfed, leading to failure of closure oi cell is
1967, 46,
with a research interest in these disorders, where the morphology and cell biology of adherence, motility, phagocytosis, killing mechanisms, degranulation, and digestion can be individually examined and detailed. Such centres are best placed to administer neutrophil transfusions-—the logical accompaniment to specific antimicrobial therapy. referral
to a centre
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.