Published online before print June 3, 2005

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(Journal of Leukocyte Biology. 2005;78:612-619.)
© 2005 by Society for Leukocyte Biology

Simultaneous measurements of cytoplasmic Ca²⁺ responses and intracellular pH in neutrophils of localized aggressive periodontitis (LAP) patients

Jens Martin Herrmann^*,, Alpdogan Kantarci^*, Heidi Long, John Bernardo, Hatice Hasturk^*, Lewis V. Wray, Jr., Elizabeth R. Simons and Thomas E. Van Dyke^*,1

^* Goldman School of Dental Medicine and
School of Medicine, Boston University, Massachusetts

¹Correspondence: Department of Periodontology and Oral Biology, Boston University, Goldman School of Dental Medicine, 100 East Newton St., Boston, MA 02118. E-mail: tvandyke{at}bu.edu

In view of the reports that polymorphonuclear leukocytes (PMN) of patients with localized aggressive periodontitis (LAP) exhibit hyper-responsiveness to stimulation, it has been suggested that such abnormalities could lead to PMN-mediated tissue damage during inflammation. To determine whether these abnormalities include signal transduction, we compared cytoplasmic calcium concentration ([Ca²⁺]_i) and cytoplasmic pH (pH_i) changes, early stimulus responses to chemotactic agents, of LAP versus control (C)-PMN and explored whether these could be modulated by sensitizing cytokines or calcium channel-blocking agents. PMN responses of LAP patients were compared with age- and gender-matched controls. [Ca²⁺]_i and pH_i were measured fluorimetrically using 1H-indole-6-carboxylic acid, 2-[4-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-3-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-1 and 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein as respective probes. Not only was the maximal calcium response to chemoattractants higher in LAP-PMN, but also their subsequent intracellular calcium redistribution was significantly slower. The slower calcium redistribution of LAP-PMN, but not their higher maximal calcium response, was successfully mimicked in C-PMN treated with Nifedipine^TM or 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole-HCl, both known to be inhibitors of membrane-associated calcium influx, but this redistribution was not affected when inhibitors of other calcium influx mechanisms, Diltiazem^TM or Verapamil^TM, were used. Taken together, our findings indicate that certain early stimulus responses are aberrant in LAP-PMN, that internal redistribution of cytoplasmic-free calcium is compromised, and, additionally, that a membrane-associated Ca²⁺ transport defect may be present.

Key Words: human polymorphonuclear neutrophils • stimulus responses • spectrofluorimetry

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