patch amplifier. ... CA) patch-clamp amplifier with a 10-gigaohm feedback resistor and ..... Baiidii, S. A,, Mc Call, C. E., Cousart, S. L., and Bass, D. A. (1991) J. 9.
THEJOURNAL OF BIOLOGICAL CHEMISTRY B 1993 by The American Society for Biochemistry and Molecular BioloW, Inc.
Vol. 268, No. 3, Isaue of January 25, pp. 2134-2140,1993 Printed in U.S.A.
Recombinant Human TumorNecrosis Factor a! Induces Calcium Oscillation and Calcium-activated Chloride Current in Human Neutrophils THE ROLEOF
CALCIUM/CALMODULIN-DEPENDENTPROTEIN KINASE* (Received for publication, July 27, 1992)
Muhammad A. SchumannS, Phyllis Gardnerg, and Thomas A. RaffinS From the SDiuision of Pulmonary and Critical Care Medicine and the §Cardiology Division, Department of Medicine, Stanford University School of Medicine, Stanford, California94305-5236
The role of calcium in the action of tumor necrosis factor (TNF) on human neutrophils is not clear. With fluorescent cytometry, using the visible wavelength calcium probe, fluo-3, and patch clamping, we investigatedwhether TNF induces cytosolic free Ca2+ changes and Ca2+-activatedC1- current, respectively. Bath application of 1000 units/ml recombinant human TNFa (rhTNFa)induced a rise incytosolic free Ca” in 75% of fluo-3-loaded cells, 25% of which displayed irregular patterns of oscillation. Addition of rhTNFo activated C1- current in 80% of tested cells; the activated current was blocked by 10 p~ 5-nitro-2-3-phenylpropy1amino)benzoic acid, a C1- channel blocker. The current was similarly activated by 1 MM ionomycin, a Ca” ionophore. To studythe mechanismby which rhTNFa induced Ca2+-activatedC1- current, we examined the involvement of calcium/calmodulin-dependent protein kinase (CaM kinase). With intracellular application of the Ca2+chelator l,a-bis(a-aminophenoxy)ethane-N,N,N’,N’-tetraacetate(5 mM), the , kinase 11-(290calmodulin antagonist (2 p ~ ) CaM 309), or the inhibitorypeptide (10 PM), CaM kinase 11(273-302), the current was no longer activated by rhTNFa. The intracellular application of the control , kinase 11-(284-302), or the propeptide (10 p ~ ) CaM tein kinase C (PKC) inhibitory, PKC-(19-36), or control, [Glu2’]PKC-(19-36), peptide (5 PM) did not block the rhTNFa-induced C1- current. These results show that Ca” changes’are associated with the effects of rhTNFa and thatCaM kinase plays a role in themechanism underlying rhTNFa-induced activation of Ca2+activated C1- current inhuman neutrophils.
produced cytokines that has multiple, stimulatory effects on neutrophil functions such as phagocytosis, adherence, degranulation, and respiratory burst activity (3, 4). Since Ca2+also is vital to these functions (5),it was reasonable to consider that Ca2+or a Ca2+-dependent component may be involvedin the signal transduction pathway launched in these cells by TNF. Identifying the biochemical link between Ca2+and TNF action is therefore of great importance in understandingneutrophil activation by the cytokine. To explore this link, we studied the effect of recombinant human tumornecrosis factor a (rhTNFa) on the cytosolic free Ca2+ level and on Ca2+activated C1- current in human neutrophils. The nexus between TNF action and cytosolic free Ca2+ changes has been debatable. Although, many authors agree on the importance of a Ca2+role in TNF action (Refs. 6 and 7, for example), they disagree on whether such a role is initiated by TNF action (7, 8). Some authors have obtained either direct (9, 10) or indirect (11)evidence for cellular Ca2+ changes caused by TNF. Others have not obtained or could not substantiate such evidence (12, 13), and thusthey believe that prior Ca2+changes are necessary for TNF action which they relegate to “cell priming” (8,13-17). One of the problems leading to this disparity is the presence of spontaneous Ca2+ oscillations (7, 18, 19), in resting neutrophils, which masks the detection of Ca2+changes attributed to TNF action on these cells. Neutrophils plated on coverslips already coated with albumin, fibronectin, or heat-inactivated human serum, or on plastic or glass surfaces coated with poly@-lysine) exhibit spontaneous oscillations. Their counterparts plated on uncoated surfaces or put in suspensions, however, lack spontaneous oscillations (20-24). Another problem that may have hindered the observation of Ca2+changes triggered by Neutrophils exert a pivotal role in host defense against TNF is the utilization of fluorescent dyes that do not enable acute bacterial infection (1).Perhaps dependent on the extent detection of peak cytosolic free Ca2+in stimulatedneutrophils of their activation, they may also contribute to tissue injury due to theuse of high dye concentrations which are known to (2). Cytokines that may modify neutrophil functions are pro- buffer Ca2+changes or that generate in resting neutrophils duced by activated monocytes and macrophages at thefoci of undesirable cellular autofluorescences due to theexcitation of inflammation. Tumor necrosis factor (TNF)’ is one of the the dye at UV wavelengths. In thepresent work, we used the new fluorescent probe 2-(3,6-bis(acetyloxy)-2,7dichloro-9H* This work was supported by National Institutes of Health Grant xanthen-9-y1)benzoicacid, known as fluo-3, which, dueto its HL455330. The costs of publication of this article were defrayed in high Kd, allows more sensitive measurements at stimulated part by the payment of page charges. This article must therefore be hereby marked“aduertisement” in accordance with18U.S.C. Section cytosolic free Ca2+ levels, and, due to its visible excitation wavelength, circumvents inducing irrelevant cellular autoflu1734 solely to indicate this fact. The abbreviationsused are:TNF, tumornecrosis factor; rhTNFa, orescence (20). With this fluorescent probe, we examined the recombinant human tumor necrosis factor a;PKC, protein kinase C; effect of rhTNFa on the cytosolic free Ca2+level in human BAPTA AM, l,Z-bis(Z-aminophenoxy)ethane-N,~,N’,N’-tetraace- neutrophils that had been placed in uncoated plastic dishes. tate; CaM, calmodulin; CaM kinase, calcium/calmodulin-dependent protein kinase; fluo-3 AM, 2-(3,6-bis(acetoxy)-2,7-dichloro-9If-xan- To evaluate the role of Ca2+in the action of rhTNFa, we considered activation of ion channels in neutrophils by Ca2+ then-9-y1)benzoicacidacetoxymethyl ester;NMDG,N-methyl-Dto serve as a possible target for rhTNFa intervention. By glucamine; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoicacid. ~
~~
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2134
TNFcu-induced Ca2+Oscillation and
Cl- Current Neutrophils in
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solution of the surfactant Pluronic F-127 were added concurrently with fluo-3 AM to the incubating medium. To complete loading of the cells with the probe, cells were incubated for 45 min in the moist chamber. Representative examples of rhTNFa action are shown in the figures that are typical of at least five replicates prepared from different cell preparations.In all assays, control samples of cell suspension that had been treated similarly but not loaded with the dye were set aside to measure autofluorescence. Electrophysiological Recordings-We used the whole-cell configuration of the patch-clamp technique (37) to record from single neutrophils of nearly equal sizes (10to 12 pm). Pipettes were pulled to neutrophil functions (29,30), we considered studying calcium/ provide electrode resistance of 4 to 6 megaohms in whole-cell recordcalmodulin-dependentproteinkinase(CaMkinase).Thus, ing. We electronically compensated series resistance, which was obbecause of the importance of a Ca2+role in activating neutro- tained after capacitance compensation by direct readout from the patch amplifier. When the largest, activated currents, passing through phil C1- channels and the importance of C1- channels, in uncompensated 5 megaohms (in response to a 160 mV step) were -2 general, in activation (31), volume regulation (32), secretion and division (35)in many nonexcitable cells, nA,wewould incur only 6% error. Families of whole-cell currents (33), killing (34), were elicited from a holding potential of -60 mVby voltage pulses we examinedthe effect of rhTNFa on the activationof whole- delivered in 20- or 25-mV steps, from -100 to +I20or +150 mV at a cell Ca2+-activated C1- current in human neutrophils, and we frequency of 0.1 Hz. We considered for experimentation only those determined the participationof CaM kinase inthe process of cells that we witnessed for >5 min with stable baselines. Recording was performed by an Axopatch-IC (Axon Instruments, Foster City, activation. We report here that Ca2+ changes mediate the effects of rhTNFa and that CaM kinase plays a role in the CA) patch-clamp amplifier with a 10-gigaohm feedback resistor and mechanism underlying rhTNFa-induced activation of Ca2+- active low pass filter. Records were digitized at 1 and 10 kHz and were filtered at 5 kHz. We performed data acquisition with pClamp dependent C1- channels and possiblytheassociated Ca2+ Clampex software (Axon Instruments) running on a computer (IBM changes in human neutrophils. PC/AT) that interfaced with the amplifier by means of an analog-todigital converter (a Labmaster board). Measurements and plotting EXPERIMENTAL PROCEDURES were carried out by pClamp Clampfit and Sigma Plot software (Jandel Scientific, Corte Madera, CA). Reagents-We purchased rhTNFa having specific activity of 5 X Recording Solutions-To record C1- current from neutrophil, we IO6 units/mg from BioSource International, Camarillo, CA. Purity was >98%, as determined by electrophoresis and amino acid amino used choline or N-methyl-D-glucamine (NMDG) as major cations (in terminus sequencing analysis; endotoxin content was
