Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Jun 2;133(6):1391–1402. doi: 10.1083/jcb.133.6.1391

Intracellular pH regulation during spreading of human neutrophils

PMCID: PMC2120889  PMID: 8682873

Abstract

The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.

Full Text

The Full Text of this article is available as a PDF (3.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Babior B. M. Oxygen-dependent microbial killing by phagocytes (first of two parts). N Engl J Med. 1978 Mar 23;298(12):659–668. doi: 10.1056/NEJM197803232981205. [DOI] [PubMed] [Google Scholar]
  2. Bengtsson T., Jaconi M. E., Gustafson M., Magnusson K. E., Theler J. M., Lew D. P., Stendahl O. Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium. Eur J Cell Biol. 1993 Oct;62(1):49–58. [PubMed] [Google Scholar]
  3. Berton G., Laudanna C., Sorio C., Rossi F. Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gp150/95, but not CR3, are able to stimulate the respiratory burst of human neutrophils. J Cell Biol. 1992 Feb;116(4):1007–1017. doi: 10.1083/jcb.116.4.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borregaard N., Schwartz J. H., Tauber A. I. Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst. J Clin Invest. 1984 Aug;74(2):455–459. doi: 10.1172/JCI111442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brundage R. A., Fogarty K. E., Tuft R. A., Fay F. S. Calcium gradients underlying polarization and chemotaxis of eosinophils. Science. 1991 Nov 1;254(5032):703–706. doi: 10.1126/science.1948048. [DOI] [PubMed] [Google Scholar]
  6. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  7. Chow C. W., Demaurex N., Grinstein S. Ion transport and the function of phagocytic cells. Curr Opin Hematol. 1995 Jan;2(1):89–95. doi: 10.1097/00062752-199502010-00012. [DOI] [PubMed] [Google Scholar]
  8. Clark R. A. The human neutrophil respiratory burst oxidase. J Infect Dis. 1990 Jun;161(6):1140–1147. doi: 10.1093/infdis/161.6.1140. [DOI] [PubMed] [Google Scholar]
  9. Counillon L., Scholz W., Lang H. J., Pouysségur J. Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties. Mol Pharmacol. 1993 Nov;44(5):1041–1045. [PubMed] [Google Scholar]
  10. DeCoursey T. E., Cherny V. V. Potential, pH, and arachidonate gate hydrogen ion currents in human neutrophils. Biophys J. 1993 Oct;65(4):1590–1598. doi: 10.1016/S0006-3495(93)81198-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Demaurex N., Grinstein S., Jaconi M., Schlegel W., Lew D. P., Krause K. H. Proton currents in human granulocytes: regulation by membrane potential and intracellular pH. J Physiol. 1993 Jul;466:329–344. [PMC free article] [PubMed] [Google Scholar]
  12. Demaurex N., Schrenzel J., Jaconi M. E., Lew D. P., Krause K. H. Proton channels, plasma membrane potential, and respiratory burst in human neutrophils. Eur J Haematol. 1993 Nov;51(5):309–312. doi: 10.1111/j.1600-0609.1993.tb01613.x. [DOI] [PubMed] [Google Scholar]
  13. Downey G. P., Chan C. K., Lea P., Takai A., Grinstein S. Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C. J Cell Biol. 1992 Feb;116(3):695–706. doi: 10.1083/jcb.116.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Downey G. P. Mechanisms of leukocyte motility and chemotaxis. Curr Opin Immunol. 1994 Feb;6(1):113–124. doi: 10.1016/0952-7915(94)90042-6. [DOI] [PubMed] [Google Scholar]
  15. Edmonds B. T., Murray J., Condeelis J. pH regulation of the F-actin binding properties of Dictyostelium elongation factor 1 alpha. J Biol Chem. 1995 Jun 23;270(25):15222–15230. doi: 10.1074/jbc.270.25.15222. [DOI] [PubMed] [Google Scholar]
  16. Foskett J. K., Spring K. R. Involvement of calcium and cytoskeleton in gallbladder epithelial cell volume regulation. Am J Physiol. 1985 Jan;248(1 Pt 1):C27–C36. doi: 10.1152/ajpcell.1985.248.1.C27. [DOI] [PubMed] [Google Scholar]
  17. Grinstein S., Furuya W., Biggar W. D. Cytoplasmic pH regulation in normal and abnormal neutrophils. Role of superoxide generation and Na+/H+ exchange. J Biol Chem. 1986 Jan 15;261(2):512–514. [PubMed] [Google Scholar]
  18. Grinstein S., Furuya W. Cytoplasmic pH regulation in phorbol ester-activated human neutrophils. Am J Physiol. 1986 Jul;251(1 Pt 1):C55–C65. doi: 10.1152/ajpcell.1986.251.1.C55. [DOI] [PubMed] [Google Scholar]
  19. Grinstein S., Woodside M., Waddell T. K., Downey G. P., Orlowski J., Pouyssegur J., Wong D. C., Foskett J. K. Focal localization of the NHE-1 isoform of the Na+/H+ antiport: assessment of effects on intracellular pH. EMBO J. 1993 Dec 15;12(13):5209–5218. doi: 10.1002/j.1460-2075.1993.tb06216.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Henderson L. M., Chappell J. B., Jones O. T. Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel. Biochem J. 1988 Apr 15;251(2):563–567. doi: 10.1042/bj2510563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hendey B., Maxfield F. R. Regulation of neutrophil motility and adhesion by intracellular calcium transients. Blood Cells. 1993;19(1):143–164. [PubMed] [Google Scholar]
  22. Jaconi M. E., Theler J. M., Schlegel W., Appel R. D., Wright S. D., Lew P. D. Multiple elevations of cytosolic-free Ca2+ in human neutrophils: initiation by adherence receptors of the integrin family. J Cell Biol. 1991 Mar;112(6):1249–1257. doi: 10.1083/jcb.112.6.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. MacFarlane G. D., Herzberg M. C., Nelson R. D. Analysis of polarization and orientation of human polymorphonuclear leukocytes by computer-interfaced video microscopy. J Leukoc Biol. 1987 Apr;41(4):307–317. doi: 10.1002/jlb.41.4.307. [DOI] [PubMed] [Google Scholar]
  24. Mandeville J. T., Ghosh R. N., Maxfield F. R. Intracellular calcium levels correlate with speed and persistent forward motion in migrating neutrophils. Biophys J. 1995 Apr;68(4):1207–1217. doi: 10.1016/S0006-3495(95)80336-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marks P. W., Maxfield F. R. Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils. J Cell Biol. 1990 Jan;110(1):43–52. doi: 10.1083/jcb.110.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nathan C., Srimal S., Farber C., Sanchez E., Kabbash L., Asch A., Gailit J., Wright S. D. Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins. J Cell Biol. 1989 Sep;109(3):1341–1349. doi: 10.1083/jcb.109.3.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pozzan T., Lew D. P., Wollheim C. B., Tsien R. Y. Is cytosolic ionized calcium regulating neutrophil activation? Science. 1983 Sep 30;221(4618):1413–1415. doi: 10.1126/science.6310757. [DOI] [PubMed] [Google Scholar]
  28. Roos A., Boron W. F. Intracellular pH. Physiol Rev. 1981 Apr;61(2):296–434. doi: 10.1152/physrev.1981.61.2.296. [DOI] [PubMed] [Google Scholar]
  29. Rothe G., Oser A., Valet G. Dihydrorhodamine 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes. Naturwissenschaften. 1988 Jul;75(7):354–355. doi: 10.1007/BF00368326. [DOI] [PubMed] [Google Scholar]
  30. Simchowitz L. Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes. J Biol Chem. 1985 Oct 25;260(24):13248–13255. [PubMed] [Google Scholar]
  31. Simchowitz L., Cragoe E. J., Jr Regulation of human neutrophil chemotaxis by intracellular pH. J Biol Chem. 1986 May 15;261(14):6492–6500. [PubMed] [Google Scholar]
  32. Smith R. M., Curnutte J. T. Molecular basis of chronic granulomatous disease. Blood. 1991 Feb 15;77(4):673–686. [PubMed] [Google Scholar]
  33. Springer T. A. Adhesion receptors of the immune system. Nature. 1990 Aug 2;346(6283):425–434. doi: 10.1038/346425a0. [DOI] [PubMed] [Google Scholar]
  34. Stossel T. P. On the crawling of animal cells. Science. 1993 May 21;260(5111):1086–1094. doi: 10.1126/science.8493552. [DOI] [PubMed] [Google Scholar]
  35. Theler J. M., Lew D. P., Jaconi M. E., Krause K. H., Wollheim C. B., Schlegel W. Intracellular pattern of cytosolic Ca2+ changes during adhesion and multiple phagocytosis in human neutrophils. Dynamics of intracellular Ca2+ stores. Blood. 1995 Apr 15;85(8):2194–2201. [PubMed] [Google Scholar]
  36. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
  37. Tsien R. Y. Fluorescent indicators of ion concentrations. Methods Cell Biol. 1989;30:127–156. doi: 10.1016/s0091-679x(08)60978-4. [DOI] [PubMed] [Google Scholar]
  38. el Benna J., Ruedi J. M., Babior B. M. Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation. J Biol Chem. 1994 Mar 4;269(9):6729–6734. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES