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Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils
International Immunopharmacology 6 (2006) 509 www.elsevier.com/locate/intimp
Letter to the Editor
Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils I wish to draw the following concerns regarding the report by Tuluc et al. [1]. It has been demonstrated that neutrophils disappear from the circulation with a half time (T 1/2) of 6.7 h [2], [3] and [4]. A minimum of 65 min is generally required to isolate neutrophils and an additional hour of incubation at room temperature is needed prior to evaluating results within studies of this nature. Therefore, we assume that nearly 2 h after isolation, the neutrophils would demonstrate a considerable loss in functional response within the studies of Tuluc et al. Consequently, these dead cells might lyse and inadvertently increase nucleotide levels in the storage medium. This increase in extracellular nucleotide could serve as substrate for ectonucleotidases [5]. It is known that ATP has a biphasic effect on neutrophils and the hydrolysis product, adenosine, has an inhibitory effect on neutrophil activation. It is also known that AMP can inactivate human neutrophil functions [6]. Induction of adhesion of neutrophils by CD39 could mask receptormediated functions in the vicinity of adhesive contact. This in turn could have both specific and non-specific effects on the overall signaling mechanism in the isolated neutrophil population. Taken together, these caveats regarding cell viability could compromise or limit the use of isolated neutrophils as a valid in vitro model of intracellular signaling. Further, the formation of neutrophil aggregates in the storage medium may be another significant factor affecting these results. There were no precautionary measures undertaken to avoid the effect(s) of extracellular nucleotides on the neutrophil membrane receptors (P2X/P2Y) or measurements of extracelullar nulcleotidases (ecto-ATPases, ecto-ATPDases, extracellular nucleotide diphosphokinases, ecto-adenylate kinases, and extracellular lysophosphate phosphatases) that could be 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.08.021
present to catalyze the degradation and formation of extracellular nucleotides (ATP and ADP). These factors are known to play a significant role in cell adhesion. Addressing these issues could clarify the extent to which these influences might confound intracellular signaling studies utilizing isolated neutrophils. In summary, it is critical for investigators to understand that neutrophil viability may decrease over a period of 60 to 120 min, which may lead to results that are difficult to interpret or properly control. Consequently, the reader should use caution in their interpretation of the results presented in the article by Tuluc et al. [1]. References [1] Tuluc F, Meshki J, Kunapuli SP. Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils. Int Immunopharmacol 2003;3:1775 – 90. [2] Athens JW, Haab OP, Raab SO, Mauer AM, Ashenbruker H, Cartwright GE, et al. Leukokinetic studies: IV. The total blood, circulating and marginal granulocyte pools and the granulocyte turnover rate in normal subjects. J Clin Invest 1961;40:989 – 95. [3] Bishop CR, Rothstein G, Ashenbrucker HE, Athens JW. Leukokinetic studies: XIV. Blood neutrophil kinetics in chronic, steady-state neutropenia. J Clin Invest 1971;50:1678 – 89. [4] Dresch C, Najean Y, Bauchet J. Kinetic studies of 51Cr and DF32P labeled granulocytes. Br J Haematol 1975;29:67 – 80. [5] Lazarowski ER, Boucher RC, Harden TK. Mechanisms of release of nucleotides and integration of their action as P2X-and P2Y receptor activating molecules. Mol Pharmacol 2003;64:785. [6] Fredholm BB. Purines and neutrophil leukocytes. Gen Pharmacol 1997;28:345.
Subburaj Kannan University of Texas, Medical Branch Pathology, 928-Postoffice Street Apt1, Galveston, TX 77550, United States E-mail address: [email protected]. Tel.: +1 409 750 9060.
Letter to the Editor
Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils I wish to draw the following concerns regarding the report by Tuluc et al. [1]. It has been demonstrated that neutrophils disappear from the circulation with a half time (T 1/2) of 6.7 h [2], [3] and [4]. A minimum of 65 min is generally required to isolate neutrophils and an additional hour of incubation at room temperature is needed prior to evaluating results within studies of this nature. Therefore, we assume that nearly 2 h after isolation, the neutrophils would demonstrate a considerable loss in functional response within the studies of Tuluc et al. Consequently, these dead cells might lyse and inadvertently increase nucleotide levels in the storage medium. This increase in extracellular nucleotide could serve as substrate for ectonucleotidases [5]. It is known that ATP has a biphasic effect on neutrophils and the hydrolysis product, adenosine, has an inhibitory effect on neutrophil activation. It is also known that AMP can inactivate human neutrophil functions [6]. Induction of adhesion of neutrophils by CD39 could mask receptormediated functions in the vicinity of adhesive contact. This in turn could have both specific and non-specific effects on the overall signaling mechanism in the isolated neutrophil population. Taken together, these caveats regarding cell viability could compromise or limit the use of isolated neutrophils as a valid in vitro model of intracellular signaling. Further, the formation of neutrophil aggregates in the storage medium may be another significant factor affecting these results. There were no precautionary measures undertaken to avoid the effect(s) of extracellular nucleotides on the neutrophil membrane receptors (P2X/P2Y) or measurements of extracelullar nulcleotidases (ecto-ATPases, ecto-ATPDases, extracellular nucleotide diphosphokinases, ecto-adenylate kinases, and extracellular lysophosphate phosphatases) that could be 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.08.021
present to catalyze the degradation and formation of extracellular nucleotides (ATP and ADP). These factors are known to play a significant role in cell adhesion. Addressing these issues could clarify the extent to which these influences might confound intracellular signaling studies utilizing isolated neutrophils. In summary, it is critical for investigators to understand that neutrophil viability may decrease over a period of 60 to 120 min, which may lead to results that are difficult to interpret or properly control. Consequently, the reader should use caution in their interpretation of the results presented in the article by Tuluc et al. [1]. References [1] Tuluc F, Meshki J, Kunapuli SP. Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils. Int Immunopharmacol 2003;3:1775 – 90. [2] Athens JW, Haab OP, Raab SO, Mauer AM, Ashenbruker H, Cartwright GE, et al. Leukokinetic studies: IV. The total blood, circulating and marginal granulocyte pools and the granulocyte turnover rate in normal subjects. J Clin Invest 1961;40:989 – 95. [3] Bishop CR, Rothstein G, Ashenbrucker HE, Athens JW. Leukokinetic studies: XIV. Blood neutrophil kinetics in chronic, steady-state neutropenia. J Clin Invest 1971;50:1678 – 89. [4] Dresch C, Najean Y, Bauchet J. Kinetic studies of 51Cr and DF32P labeled granulocytes. Br J Haematol 1975;29:67 – 80. [5] Lazarowski ER, Boucher RC, Harden TK. Mechanisms of release of nucleotides and integration of their action as P2X-and P2Y receptor activating molecules. Mol Pharmacol 2003;64:785. [6] Fredholm BB. Purines and neutrophil leukocytes. Gen Pharmacol 1997;28:345.
Subburaj Kannan University of Texas, Medical Branch Pathology, 928-Postoffice Street Apt1, Galveston, TX 77550, United States E-mail address: [email protected]. Tel.: +1 409 750 9060.