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Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium
Cell
Calcium
1:
181-194, 1980
PERTURBATION OF THE PHAGOCYTIC RESPONSE IN P388D1 CULTURED MACROPHAGES BY AGENTS ALTERING CELL CALCIUM Fay K. Kessler, Estelle M. Goodell*, and Richard A. Carchman Department of Pharmacology, MCV/VCU Cancer Center, Medical College of Virginia, Richmond, Virginia 23298 (reprint requests to RAC) *Present address: Surgical Research, Mary Imogene Bassett Cooperstown, New York 13326
Hospital,
ABSTRACT Phagocytosis in adherent P388D1 (Dl) cells was monitored utilizing formalin treated Listeria monocytogenes (Lm) previously labeled with 125iododeoxyuridine. The dependence of thisphagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60-70%) of LIJ phagocytosis by 01 cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of _LJ by D1 cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D1 macrophage model suggests a role for calcium in the phagocytic process. INTRODUCTION The phagocytic activity of macrophages is but one of the functions of this cell type (e.g. antimicrobial and antitumor). The process of phagocytosis by macrophages is a complicated phenomenon requiring the participation of a number of cellular components and/or functions such as metabolism, receptors, and cyclic nucleotides (1). Phagocytosis may be affected by not only cellular events but also the physical and/or chemical nature of the phagocytized particle (2) as well as the presence of opsonic factors (3). Divided into two steps, phagocytosis consists of 181
1. particle attachment and 2. particle ingestion (4). It is the completed two step process which we address in this paper (i.e. attachment + ingestion = phagocytosis). Many laboratories, including our own, have been interested in the role(s) which calcium may play as a modulator of various cellular responses (5, 6, 7, 8) including phagocytosis (9). Our approach in this paper has been to analyse the phagocytic response by utilizing pharmacological probes (RR, D-600, A23187) which have been shown in several different systems to be capable of perturbing cellular calcium metabolism (10, 11, 12, 13, 14, 15, 16). Currently, little is known about calcium dynamics in macrophages. The work of Gennaro et. al. $7) and Schneider et. al. (18) has addressed this problem. Gennaro et. . reported that plasma membrane fractions from purified rabbit alveolar macrophages display a Ca+2-dependent ATPase activity, while Schneider et. al. has shown that treatment of alveolar macrophages with A23187 resulted in a small but significant increase in the re ease of B-glucoronidase and lysozyme. Both authors postulate that Cat;!may play a governing role in the exocytotic processes of macrophages. The P388Dl cells, originally isolated by Dawe and Potter (19) and described as a macrophage like cell line by Koren et. al. (20) were used as the phagocytic protoype in these studies. Several investigators have demonstrated that this cell posesses many macrophage-like capabilities (i.e. Fc receptors, adherence to glass, and phagocytosis) (21, 22, 23) and Goode11 et. al. (24) has reported on the effects of agents which can alter cellular metabolism and cyclic nucleotide levels on the phagocytosis of opsonized sheep erthrocytes by P388D1 cells. By utilizing this macrophage cell line in tissue culture and pharmacological probes known to alter cellular calcium metabolism, we hope to delineate more precisely the role of calcium in regulating phagocytosis. MATERIALS AND METHODS Cells The P388D1 cells were grown in suspension culture at 37 C in a humidified incubator under 5% C02, 95% air. Dulbecco's Modification of Eagle's Minimal Essential Medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (HIFCS), essential and non-essential amino acids, MEM vitamins, and glutamine was used as growth and maintainence medium. Hepatoma tissue culture cells (HTC) were also grown in spinner culture under the same conditions as the D1 cells. Eagle's Minimal Essential Medium (EMEM) similarly supplemented was used as both growth and maintenance medium. The pH of both the cell cultures was maintained at 7.0 to 7.3. Reagents All tissue culture constituents were purchased from Grand Island Biological Company (GIBCO), Grand Island, New York. The ionophore A23187 and D-600 were kindly supplied by Eli Lilly Company, Indianapolis, Ind., and Knollag Laboratories, Ludwigshafen, Germany, respectively. Ruthenium red was purchased from Sigma Chemical Company, St. Loius, MO. and used without further purification. The vehicle for both the ruthenium red and D-600 was DMEM or EMEM. The vehicle for A23187 was 95% ethanol, 182
and subsequent dilutions required by the experimental protocol were made in suplemented DMEM or EMEM. The A23187 and D-600 were stored at 4 C up to one month while the ruthenium red was prepared fresh daily. Iodination
of Listeria monocytogenes
Approximately 108 cells of Listeria ronocytogenes were added to 125 ml of sterile brain heart infusin(Bm broth (Dlfco, Detroit, Mich.). This mixture was then incubated in a 37.C Dubnoff shaker bath with agitation for 6 hours or until the absorbance was between 0.40-0.45 at 618 nm (Bausch and Lomb Spectronic 20). This yielded a cell concentration of 2.5-3.0 x 108 bacterijhml. Twelve ml of sterile flurodeoxyuridine (5 x 10e5 M) and 750 uCi I I iododeoxyuridine (New England Nuclear) were added simultaneously to the mixture which was then incubated for an additional hour. Inactivation of the Lm was accomplished by adding 1.25 ml of buffered formalin to the mixtureand incubating for 30 minutes. The mixture was centrifuged at 100 x g for 30 minutes at 25 C and washed 3 times. After the final wash, the supernatant was discarded and the cells were resuspended in 125 ml of EMEM. Aliquots were then frozen and stored at -20 C in sterile tubes. To establish whether the radioactivity was associated with bacterial macromolecules, lz51 Lm were incubated with 10% trichloroacetic acid. Following centrifugation at 3,000 x g for 20 minutes, 97% of the total radioactivity was found in the acid precipitable fraction indicating that the I251 iododeoxyuridine was indeed incorporated into bacterial macromolecules (e.g. DNA). Determination
of phagocytosis
of Listeria monocytogenes
by P38801 cells
P388DI cells were removed from suspension culture and centrifuged at 300 x g for 7 - 10 minutes. The pellet was resuspended in fresh DMEM with 10% H FCS ard counted on a Coulter Counter model ZBI. Aliquots of 1 - 2 x 106 cells in 3 ml of supplemented DMEM were transferred to 60 x 15 mm Corning plastic tissue culture dishes and incubated for 24 hours at 37 C. Ruthenium red, A23187, or D-600 were then added to the semiconfluent monolayers and the cells were allowed to preincubate at 37 C ~~~i~~s~ri,~~du:i~~i~~~~r~~112Ssee Results): After the preincubation I Lm (approximately 50,000 counts per minute) w& added to the cells and incubation continued for an additional 30 minutes in the presence of drug. After the completed incubation, representative plates for each drug at all concentrations were stained with trypan blue to determine cell viability. In all cases, cell viability was greater than 95%. After the second incubation, the plates were washed 4 times with 3 ml of phosphate buffered saline (PBS) at pH 7.4, 25 C. The cells were then harvested in 1 ml of PBS using a rubber policeman, and transferred to 12 x 75 mm glass culture tubes. A second ml of PBS was used to wash the plates and then pooled with the first sample. The 2 ml sample was then counted in a Beckman 300 Gamma Counter. The data are expressed in terms of percent stimulation or inhibition of phagocytosis as compared to control values (normalized for cell number). Drug-Listeria
monocytogenes
interaction
Each drug was incubated with 100 ul of Lm and 3 ml of DMEM in a cul7 ture tube for 30 minutes at 37 C. After the incubation the -Lm were cen183
trifuged at 100 x g for 30 minutes and washed twice with fresh DMEM. The pellet was resuspended in 100 ul of DMEM and counted in a Beckman 300 Gama Counter. Equal counts of the drug treated Lm and untreated Lm were added to P388D1 cell monolayers. Phagocytosis was measured asdescribed above. RESULTS Phagocytosis of !$ by P388D1 cells was measured during a 30 minute incubation (Fig. la). Throughout the incubation, substantial amounts of -Lm were phagocytized by P388Dl cells and phagocytosis of &I by P388D1 cells proceeded at a linear rate. By 60 minutes (data not shown), phagocytosis of Lm by P388D1 had reached a plateau. The ratio of &:P388D1 cells was 3571.
2,000
2
20
IO TIME
: u
800
-
600
-
400 ..,7
-
30
(mtnutes)
b
I , , , , , , , , 20
40 TIME
Figuhe
la:
60
60
(minutes)
PhagocyZoaih o 6 "ST Lm by P3B8U~c$?.4 at 37Y (e-e' and 4"i?(0---0!. COK&lW~
??~8807
Ceub
1;te/Le
,&.&.&d
Lm and .theaakd ah da&bed in Ma&.hZ& and MeXhod~. Each point is the mean 2 &andahd ehhOh IS .E.1 04 b @Ax&h.
wia%
Fi.guheIb:
PhagocyXonti 06 If51 Lm ceA22.
Smi-
con@uen-t WC ce..U~ wZ& inculzted width Lm and tietied ah debcni.bed in Ma&kX!h Each plate i.~ the mean 2 Zd Me&to&. S.E. 06 b p&L.&h.
In order to state that the Lm had been phagocytized by the P388111 cells and were not just nonspecifically adhered to the P388D1 cells,A.severa1 experiments were performed. In Figure lb, nonspecific adherence of Lm to the HTC cells, a non-phagocytic cell line in tissue culture is, shown. The adherence of Lm to this cell type was only 3-4% of the Lm that was associated with the P388D1 cells (Fig. la). Since the in Etion phase of phagocytosis is a temperature dependent process (4, 24, 2! ), the ability of P388D cells to phagocytize Lm was measured at 4 C (Fig. la). At this lowered !emperature, P388D 1 cells were able to phagocytize Lm at only one-tenth the rate of phagocytosis at 37 C. After the P388D1 cells had been in contact with the Lm for 30 minutes, gram stains of the cell cultures showed gram positiveTaci1li associated with the cellular cytoplasm. The bacilli were arranged parallel to the lengthwise axis of the P388D cells indicating further that ingestion had occurred (data not shown3 . Unequivocal proof for the ingestion of the -Lm by P388D1 cells must await evaluation by elecron microscopy.
50
10-5M
I 0-7M
10-4M
10-3M -20
Concentrationof Ruthenium Red
Figuhe 2:
RLLthenkm hed
dobe
heApon+c? cuhve.
Semi-
con@wtt P3b8Vj ceR.& wehe expobsd .ta VUJLLOUA conceMom 06 RR boh 2 how. FokYowtig ti incufxtion, tie CC?&!A Wehe expot,ed ;to 12% Lm fjoh 30 m&u&!, and
phocebned
point
a6 dwchibed
above.
Each c&&a
in The mean f S.E. 06 ti~.&HQ.A(2
At concen.&uUonn 06 RR at expi?.Gmn&I. um 1 x IO-5 M and gwua2~ phagocytitos& nign+can.tty d&$&hent (mm conathot vdueh (p~o.011. 185
The histological dye, ruthenium red, produced a dose dependent stimulation of Lm phagocytosis by P388D1 cells (Fig. 2) Maximal stimulation was observed at 10-4 to 10-3 M RR ( 40%). Ruthenium red at 10-4 M was used in subsequent studies because this concentrqtion was on the linear portion of the dose response curve and caused no change in cellular morphology even after prolonged incubations. At this concentration of RR (lo-4 M) a significant (p
90-
20 IO I 2
1 I
I 3
I 4
HOURS
Figwle 3:
i@e,cR" 06 RR (70-d hi)ubiphagocy&h.ih 06 1 Lm by P38bD1 eel%. BeIween 7-2 x IfI6 c&.&?n~pLa&? wehe .&c&at&d wkth RR @h j-4 The cekT,& we~rc then incubated wtih. horn.. Lm 6012 30 tin and phagocqfobib U&A meanwced. E&h paint in the mean t S. E. o fi I6 p.bA?b pin& I 7-4) I 4 expetimeti ) . A.&ZXitime ahe hi.gni.&antiq d&(deh&-nt l(fiom WnRhd The_ 3 and 4 hawk a%me valueh (p~O.071.~
pointi cvre nigni &Lcanf%4 (ti66ehenX ;rJ;hr~ the 1 and 2 hou_h dhug i.hcubatioMn (pcO.01). The calcium channel blocker, D-600 (lo-4 M) produced a significant inhibition (~45%) of phagocytosis by P388Dl cells (Fig. 4). Concentrations of D-600 grgater than 10-4 M were toxic owr the 4 hour drug incubation, while 10' M D-600 was less inhibitory than 10D4 M (data not shown). Figure 4 is a composite of 4 experiments, data points
(p
186
at each time were performed in quadruplicate. Two of the four experiments showed a time dependent inhibition while each of the experiments demonstrated an inhibition of phagocytosis (pdO.01) by D-600.
3
2
I
4
HOURS
FLgwte4:
06 v-600 (10m4 MI On phagOCYWL5 1 lm by P38bVj c&Lb C&we
E6 %
E-9 ~MncubzzZed 4 how.
ghand WA% V-600 ,Joh b&cut Each point ih x%e mean + S.E. ad
16 p.tTa&d (4 expe&.&nen~). flu Ltime poin& aheLgti&antiycLL{@eti @wm cona?wt ~igni&anX P.&L&J (p~o.071. No ove&& ;tOnedependency ioh tie V-600 tltipo~beti obnuwed aLthough 2 06 tie 4 expehimeti
did
hhow
hOme
mh.-!y
2inxz
e&x.x2.
The divalent cation ionophore A23187 at concentrations in excess of 2 x 10m6 M caused cellular lysis and were not used in this experimental protocol (data not shown). At the non-toxic concentration of A23187 (2 x 10-6 M) over the 4 hour incubation period, a stimulatory peak in the phagocytic response was observed ("38%); however, this transient response (Fig. 5) rapidly decreased to control values by 3 hours. The differences between the 2 hour point and the control, 1, 3, and 4 hour points were significant at the level ~~0.01. The concomitant addition of RR + O-600 (Fig. 6a) resulted in an interaction between the two drugs such that all the experimental points were statistically indistinquishable from control values. In conrast to the complete antagonism seen with RR + D-600, the combination of A23187 + D-600 resulted in only a partial change of the D-600 response (Fig. 6b). 187
Unlike the response seen with D-600 alone (Fig. 44 the inhibition of phagocytosis (D-600 + A231871 developed slowly (20-25%) over the first two hours and then started to rise, reaching 45% at 4 hours. The depressed response observed at 1 and 2 hours was significantly different from control as well as from the 3 and 4 hour points (p
60 50 5 g
40
.E 3j
30
$
20 IO 3
2
I
4
HOURS
5:
187 (2 x low6 MI on phagoEf&@!! oij cyxo&iA 06 1 Lm by P38bVj cc&~. CCL&& WYW p&e-Lncubateauiith A23187 don be/&en
14%!!
I ad4 how. S.E. 06 12 pW cytib.i.6 a-t the b@%i&kantiy vaeued
Each point .6 the mean + (3 expwimeti). Qhago2 howr dkug i.ncuba.tion wah d&$&Um~
@Om
cOVLIhO~
(p'o.01).
The concomitant addition of the two agents which produced a stimulation of phagocytosis by P388D cells (RR + A23187) is shown in Fig. 6c. A significant stimulation o + phagocytosis was observed; however, the response was not as elevated as was seen with RR alone (Fig. 3) but was more prolonged than with A23187 alone (Fig. 5).
188
a
30 20 IO
0
b
-10 M -30
-*r
b
FQAhe
6a:
Ed&z& 06 RR (JO-4 MI and D-600 (IO-4 MI on ph.agocyti& 06 7251 lm by PSWJ cm. Each data point &T~u&wZ Rhe mea~l t S .E . 06 16 ~&UC% (4 zxptien~). None 06 tithe. da& po,ain& we&e ,(owzd 20 be n.igti&kanfiy d.i~&hhent &orn a%stk kenpectiwe contioL4.
F&he
6b:
E~,@za% 06 A?3787 (2 x lO-6 MI and O-600 IlOw4 MI on ph.agocy;to& 06 Jz51 Lm by Each /lo&t IrepheAe& tie P3bWJ cekt.5. mean t S.E. 05 16 p.ta.te,s (4 ~xptianti)). AU a%ne pointi W~YLR~ ~ignL+antiy di~@~lren.t Qwm cona%oL va&ti (~40.01) and ;tCtc 1 a.nd 2 howt pokth we.&? higni@anaXy LOWeh &an tithe 3 and 4 howl dhug incubatioti (pcO.OJI
Figurre 6c:
Fig.
6a-c:
In aU added cell2
CaoeA, phwunacoLogicaC agenti we,te conconktatiy to nemi-con@Tuent P36tiDJ and incubated be2wze.n J and 4 houm. 189
XSSCUSION The P388Dl cell has been shown to possess many of the properties of normal macrophages (20, 21, 22, 23). In trying to understand the biochemical events involved in macrophage response we have used the P388D1 cell as a model system. Calcium is known to modulate a host of biological responses (7, 8, 26, 27), many of which appear to be involved in various facets of macrophage activity (9, 28, 29). This report explores the effects of agents known to perturb cellular calcium metabolism on phagocytosis in P388DI cells.. These cells rapidly phagocytize I251 Lm during the course of a thirty minute incubation. Initial studies inour laboratory are in agreement with the work of other investigators which showed phagocytosis to be a temperature sensitive process (4, 24, 25). The temperature studies, comparative phagocytosis studies (P388DI vs HTC) and the gram staining all indicate that actual phagocytosis occurred. Therefore, the results observed in subsequent experiments may be attributed to changes in phagocytosis and not attachment of -Lm to P388Dl cells or the tissue culture plasticware. The agents employed in these studies were the calcium antagonists ruthenium red (15, 30) and D-600 (10, 11, 14) and the divalent cation ionophore A23187 (13, 16). Preincubation of each of these agents with Lm did not induce any change in the degree of phagocytosis of Lm by m88DI cells as compared to untreated Lm. This information indicated that none of these drugs exerted an oj%onin-like (or anti-opsonin) effect on the bacteria. Therefore, any changes seen in phagocytosis were due to the effects of the drugs on the cells and not on the Listeria. Ruthenium red has been shown to alter mitochondrial calcium either by inhibiting calcium uptake (15, 30, 31, 32, 33) or by enhancing calcium efflux (31, 32). D-600 can block plasma membrane transport of calcium (10, 11, 14) while no effect by D-600 has been reported on mitochondrial calcium. A23187 increases the calcium permeability of mitochondrial and plasma membranes depending upon the dose administered (13, 16, 34, 35). This study has demonstrated that ruthenium red, D-600, and A23187 were capable of modulating the phagocytic response of the P388DI cells to Lm. The reported effects of these agents on cellular calcium movement COum be involved in their ability to stimulate or inhibit phagocytosis. The possibility that the inhibitory effect of D-600 is related to the blocking of plasma membrane transport of calcium and that drug stimulation of phagocytosis is related to either increased cytoplasmic calcium and/or decreased mitochondrial calcium is at present highly speculative. An increase in calcium efflux has been shown to occur during the phagocytosis of zymoson particles of guina pig polymorphonuclear leukocytes (9). Calcium efflux studies on P388DI cells in the presence of RR and D-600 indicate that these agents modulate intracellular calcium movements (manuscript in preparation). From these results only an inference can presently be drawn about the role of calcium in phagocytosis by P388DI cells, though it does appear that agents known to modulate calcium metabolism at a number of
190
different sites can effectively alter the phagocytic response. It is possible that these agents can be acting through some other mechanism(s) to affect the observed changes in phagocytosis. The results obtained from the measurement of 45Ca++ efflux and/or uptake experiments are difficult to interpret realizing that bhere are probably a number of complex interrelated intracellular calcium compartments involved in cellular calcium metabolism. Despite these problems, additional experiments are currently undentlayutilizing 45Ca++ in order that a clearer understanding of cellular calcium transport and distribution may be revealed as it relates to phagocytosis in this macrophage model.
ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of Mr. Jack Burkhalter in the statistical analysis of the data. We acknowledge the assistance of Dr. William Warner in the preparation of the manuscript. This work was supported in part by ACS grant IM-112, DA-00490, DA-01637. Dr. Carchman is the recipient of a Research Career Development Award, lK04AM00565, from the National Institutes of Health. REFERENCES 1.
Silver;;;in~e;.C.~i;;;~man, R.M. and Cohn, Z.A. (1977). sis. . . . 46, 669-772.
2.
Capo, C., Bourgrand, P., Benolied, A.M. and Depieds, R. (1978). Dependence of phagocytosis on strength of phagocyte particle interaction. Itnnunol.35, 177-182.
3.
van Oss, C.J. (1978). Phagocytosis as a surface phenomenon. Ann. Rev. Microbial. 32, 19-39.
4.
Rabinovitch, M. (1967). The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp. Cell Res. 46, 19-28.
5.
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7.
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Endocyto-
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Baker, P.F., Meves, H. and Ridgway, E.B. (1973). Effects of manganese and other agents on the calcium uptake that follows depolarization of squid axons. J. Physiol. 231, 511-526.
11.
Baker, P.F., Meves, H. and Ridgway, E.B. (1973). Calcium entry in response to maintained depolarization of squid axons. J. Physiol. 231, 527-548.
12.
Chaney, M.P., Demarco, P.V., Jones, N.D. and Occolowitz, J.L. T;;cstr;_&tu;;3;fl~~;187,a divalent cation ionophore. J. . . . .
p75l,, 13.
14.
The effect of A23187 upon Jensen, P. and Ramissen H (1977) Cat+ metabolism in the Human lymphocyte. Biochem. Biophys. Acta 468, 146-156. Jensen, P., Winger, L., Rasmussen, H. and Nowell, P. (1977). The mitogenic effect of A23187 in human peripheral lymphocytes. Biothem. Biophys. Acta 496, 374-383.
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Reed, K.C. and Bygrave, F.L. (1974). The inhibition of mi;;;;;;;. rial calcium transport by lanthanides and ruthenium red. J. 140, 143-155.
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Reed, P.W. and Lardy, H.A. (1972). A23187: A divalent cation ionophore. J. Biol. Chem. 247. 6970-6977.
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Schneider, C., Gennaro, R., de Nicola, G. and Romeo, D. (1978). Regulation by intracellular Ca2+-buffering capaci,ty. Exp. Cell Res. 112, 249-256.
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Gennaro, R., Mottola, C., Schneider, C. and Romeo, D. (1979). Ca2t dependent ATPase activity of alveolar macrophage plasma membrane. Biochem. Biophys. Acta 567, 238-246.
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Koren, H.S., Handwerger, B.S. and Winderlich, J.R. (1975). Identification of macrophage like characteristics in a cultured murine tumor line. J. Irmunol. 114, 894-897.
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Bar, R.S., Kahn, C.R. and Koren H.S. (1977). Insulin inhibition of antibody dependent cytotoxicity and insulin receptors in macrophages. Nature 265, 632-634.
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Lachman, L.B., Blyden, G.T., Hacker, M. and Handschumacher, R.E.' (1976). Characterization of lymphocyte activating factor from mouse tumor cell lines and human lymphocytes. Cell Immunol. 27, 354.
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Mizel, S.B., Oppenheim, J.J. and Rosenstreich, D.L. (1978). Characterization of lymphocyte activating factor (LAF) produced by the macrophage cell line P388Dl. I. Enhancement of LAF production by activated T lymphocytes. J. Immunol. 120, 1497-1503.
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Goodell, E.M., Bilgin, S. and Carchman, R.A. (1978). Biochemical characteristics of phagocytosis in the P388Dl cell. Exp. Cell Res. 114, 57-62.
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Hoff, S.F., Huang, Y.O., Wisnieski, B. and Fox, C.F. (1976). Temperature dependent events during phagocytosis of latex beads by cultured mouse LM cells. J. Cell Biol. 70, 127a. 192
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Atkinson, J.P. and Parker, C.W. (1977). Modulation of macrophage Fc receptor function by cytochalasin sensitive structures. Cell Immunol. 33, 353-363.
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Stossel, T.P. (1973). Studies of phagocytosis: kinetic effects of cations and heat labile opsonin. J. Cell Biol. 58, 346-356.
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Moore, C.L. (1971). Specific inhibition of mitochondrial Cat+ transport by ruthenium red. Biochem. Biophys. Res. Comm. 42, 298305.
31.
Rossi, C.S., Vashington, F.D. and Carafoli, E. (1973). The effect of ruthenium red on the uptake and release of Cat+ by mitochondria. Biochem. Biophys. Res. Comm. 50, 846-852.
32.
Sordahl, L.A. (1975). Effects of magnesium, ruthenium red, and the antibiotic ionophore A23187 on initial rates of calcium uptake and release by heart mitochondria. Arch. Biochem. Biophys. 167, 104115.
33.
Vashington, F.D., Gazzotti, I., Tiozza, R. and Carafoli, E. (1972). The effect of ruthenium red on Cat+ transport and respiration in rat liver mitochondria. Biochem. Biophys. Acta 256, 43-54.
34.
Babcock, D.F., First, N.L. and Lardy, H.A. (1976). Action of ionophore A23187 at the cellular level. J. Biol. Chem. 251, 38813886.
35.
Scarpa, A. (1975). Kinetics and energy coupling of Cat+ transport in mitochondria. In Calcium Transport in Contraction and Secretion (E. Carafoli, F. Clementi, W. Drabikowsky, and A. Margreth, eds.) p. 65-76. North Holland Publishing Co., New York, N.Y.
Received
17 Dee 79:
revised
version
received
193
25 Mar 80:
accepted
26 Mar 80
Calcium
1:
181-194, 1980
PERTURBATION OF THE PHAGOCYTIC RESPONSE IN P388D1 CULTURED MACROPHAGES BY AGENTS ALTERING CELL CALCIUM Fay K. Kessler, Estelle M. Goodell*, and Richard A. Carchman Department of Pharmacology, MCV/VCU Cancer Center, Medical College of Virginia, Richmond, Virginia 23298 (reprint requests to RAC) *Present address: Surgical Research, Mary Imogene Bassett Cooperstown, New York 13326
Hospital,
ABSTRACT Phagocytosis in adherent P388D1 (Dl) cells was monitored utilizing formalin treated Listeria monocytogenes (Lm) previously labeled with 125iododeoxyuridine. The dependence of thisphagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60-70%) of LIJ phagocytosis by 01 cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of _LJ by D1 cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D1 macrophage model suggests a role for calcium in the phagocytic process. INTRODUCTION The phagocytic activity of macrophages is but one of the functions of this cell type (e.g. antimicrobial and antitumor). The process of phagocytosis by macrophages is a complicated phenomenon requiring the participation of a number of cellular components and/or functions such as metabolism, receptors, and cyclic nucleotides (1). Phagocytosis may be affected by not only cellular events but also the physical and/or chemical nature of the phagocytized particle (2) as well as the presence of opsonic factors (3). Divided into two steps, phagocytosis consists of 181
1. particle attachment and 2. particle ingestion (4). It is the completed two step process which we address in this paper (i.e. attachment + ingestion = phagocytosis). Many laboratories, including our own, have been interested in the role(s) which calcium may play as a modulator of various cellular responses (5, 6, 7, 8) including phagocytosis (9). Our approach in this paper has been to analyse the phagocytic response by utilizing pharmacological probes (RR, D-600, A23187) which have been shown in several different systems to be capable of perturbing cellular calcium metabolism (10, 11, 12, 13, 14, 15, 16). Currently, little is known about calcium dynamics in macrophages. The work of Gennaro et. al. $7) and Schneider et. al. (18) has addressed this problem. Gennaro et. . reported that plasma membrane fractions from purified rabbit alveolar macrophages display a Ca+2-dependent ATPase activity, while Schneider et. al. has shown that treatment of alveolar macrophages with A23187 resulted in a small but significant increase in the re ease of B-glucoronidase and lysozyme. Both authors postulate that Cat;!may play a governing role in the exocytotic processes of macrophages. The P388Dl cells, originally isolated by Dawe and Potter (19) and described as a macrophage like cell line by Koren et. al. (20) were used as the phagocytic protoype in these studies. Several investigators have demonstrated that this cell posesses many macrophage-like capabilities (i.e. Fc receptors, adherence to glass, and phagocytosis) (21, 22, 23) and Goode11 et. al. (24) has reported on the effects of agents which can alter cellular metabolism and cyclic nucleotide levels on the phagocytosis of opsonized sheep erthrocytes by P388D1 cells. By utilizing this macrophage cell line in tissue culture and pharmacological probes known to alter cellular calcium metabolism, we hope to delineate more precisely the role of calcium in regulating phagocytosis. MATERIALS AND METHODS Cells The P388D1 cells were grown in suspension culture at 37 C in a humidified incubator under 5% C02, 95% air. Dulbecco's Modification of Eagle's Minimal Essential Medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (HIFCS), essential and non-essential amino acids, MEM vitamins, and glutamine was used as growth and maintainence medium. Hepatoma tissue culture cells (HTC) were also grown in spinner culture under the same conditions as the D1 cells. Eagle's Minimal Essential Medium (EMEM) similarly supplemented was used as both growth and maintenance medium. The pH of both the cell cultures was maintained at 7.0 to 7.3. Reagents All tissue culture constituents were purchased from Grand Island Biological Company (GIBCO), Grand Island, New York. The ionophore A23187 and D-600 were kindly supplied by Eli Lilly Company, Indianapolis, Ind., and Knollag Laboratories, Ludwigshafen, Germany, respectively. Ruthenium red was purchased from Sigma Chemical Company, St. Loius, MO. and used without further purification. The vehicle for both the ruthenium red and D-600 was DMEM or EMEM. The vehicle for A23187 was 95% ethanol, 182
and subsequent dilutions required by the experimental protocol were made in suplemented DMEM or EMEM. The A23187 and D-600 were stored at 4 C up to one month while the ruthenium red was prepared fresh daily. Iodination
of Listeria monocytogenes
Approximately 108 cells of Listeria ronocytogenes were added to 125 ml of sterile brain heart infusin(Bm broth (Dlfco, Detroit, Mich.). This mixture was then incubated in a 37.C Dubnoff shaker bath with agitation for 6 hours or until the absorbance was between 0.40-0.45 at 618 nm (Bausch and Lomb Spectronic 20). This yielded a cell concentration of 2.5-3.0 x 108 bacterijhml. Twelve ml of sterile flurodeoxyuridine (5 x 10e5 M) and 750 uCi I I iododeoxyuridine (New England Nuclear) were added simultaneously to the mixture which was then incubated for an additional hour. Inactivation of the Lm was accomplished by adding 1.25 ml of buffered formalin to the mixtureand incubating for 30 minutes. The mixture was centrifuged at 100 x g for 30 minutes at 25 C and washed 3 times. After the final wash, the supernatant was discarded and the cells were resuspended in 125 ml of EMEM. Aliquots were then frozen and stored at -20 C in sterile tubes. To establish whether the radioactivity was associated with bacterial macromolecules, lz51 Lm were incubated with 10% trichloroacetic acid. Following centrifugation at 3,000 x g for 20 minutes, 97% of the total radioactivity was found in the acid precipitable fraction indicating that the I251 iododeoxyuridine was indeed incorporated into bacterial macromolecules (e.g. DNA). Determination
of phagocytosis
of Listeria monocytogenes
by P38801 cells
P388DI cells were removed from suspension culture and centrifuged at 300 x g for 7 - 10 minutes. The pellet was resuspended in fresh DMEM with 10% H FCS ard counted on a Coulter Counter model ZBI. Aliquots of 1 - 2 x 106 cells in 3 ml of supplemented DMEM were transferred to 60 x 15 mm Corning plastic tissue culture dishes and incubated for 24 hours at 37 C. Ruthenium red, A23187, or D-600 were then added to the semiconfluent monolayers and the cells were allowed to preincubate at 37 C ~~~i~~s~ri,~~du:i~~i~~~~r~~112Ssee Results): After the preincubation I Lm (approximately 50,000 counts per minute) w& added to the cells and incubation continued for an additional 30 minutes in the presence of drug. After the completed incubation, representative plates for each drug at all concentrations were stained with trypan blue to determine cell viability. In all cases, cell viability was greater than 95%. After the second incubation, the plates were washed 4 times with 3 ml of phosphate buffered saline (PBS) at pH 7.4, 25 C. The cells were then harvested in 1 ml of PBS using a rubber policeman, and transferred to 12 x 75 mm glass culture tubes. A second ml of PBS was used to wash the plates and then pooled with the first sample. The 2 ml sample was then counted in a Beckman 300 Gamma Counter. The data are expressed in terms of percent stimulation or inhibition of phagocytosis as compared to control values (normalized for cell number). Drug-Listeria
monocytogenes
interaction
Each drug was incubated with 100 ul of Lm and 3 ml of DMEM in a cul7 ture tube for 30 minutes at 37 C. After the incubation the -Lm were cen183
trifuged at 100 x g for 30 minutes and washed twice with fresh DMEM. The pellet was resuspended in 100 ul of DMEM and counted in a Beckman 300 Gama Counter. Equal counts of the drug treated Lm and untreated Lm were added to P388D1 cell monolayers. Phagocytosis was measured asdescribed above. RESULTS Phagocytosis of !$ by P388D1 cells was measured during a 30 minute incubation (Fig. la). Throughout the incubation, substantial amounts of -Lm were phagocytized by P388Dl cells and phagocytosis of &I by P388D1 cells proceeded at a linear rate. By 60 minutes (data not shown), phagocytosis of Lm by P388D1 had reached a plateau. The ratio of &:P388D1 cells was 3571.
2,000
2
20
IO TIME
: u
800
-
600
-
400 ..,7
-
30
(mtnutes)
b
I , , , , , , , , 20
40 TIME
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la:
60
60
(minutes)
PhagocyZoaih o 6 "ST Lm by P3B8U~c$?.4 at 37Y (e-e' and 4"i?(0---0!. COK&lW~
??~8807
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Lm and .theaakd ah da&bed in Ma&.hZ& and MeXhod~. Each point is the mean 2 &andahd ehhOh IS .E.1 04 b @Ax&h.
wia%
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PhagocyXonti 06 If51 Lm ceA22.
Smi-
con@uen-t WC ce..U~ wZ& inculzted width Lm and tietied ah debcni.bed in Ma&kX!h Each plate i.~ the mean 2 Zd Me&to&. S.E. 06 b p&L.&h.
In order to state that the Lm had been phagocytized by the P388111 cells and were not just nonspecifically adhered to the P388D1 cells,A.severa1 experiments were performed. In Figure lb, nonspecific adherence of Lm to the HTC cells, a non-phagocytic cell line in tissue culture is, shown. The adherence of Lm to this cell type was only 3-4% of the Lm that was associated with the P388D1 cells (Fig. la). Since the in Etion phase of phagocytosis is a temperature dependent process (4, 24, 2! ), the ability of P388D cells to phagocytize Lm was measured at 4 C (Fig. la). At this lowered !emperature, P388D 1 cells were able to phagocytize Lm at only one-tenth the rate of phagocytosis at 37 C. After the P388D1 cells had been in contact with the Lm for 30 minutes, gram stains of the cell cultures showed gram positiveTaci1li associated with the cellular cytoplasm. The bacilli were arranged parallel to the lengthwise axis of the P388D cells indicating further that ingestion had occurred (data not shown3 . Unequivocal proof for the ingestion of the -Lm by P388D1 cells must await evaluation by elecron microscopy.
50
10-5M
I 0-7M
10-4M
10-3M -20
Concentrationof Ruthenium Red
Figuhe 2:
RLLthenkm hed
dobe
heApon+c? cuhve.
Semi-
con@wtt P3b8Vj ceR.& wehe expobsd .ta VUJLLOUA conceMom 06 RR boh 2 how. FokYowtig ti incufxtion, tie CC?&!A Wehe expot,ed ;to 12% Lm fjoh 30 m&u&!, and
phocebned
point
a6 dwchibed
above.
Each c&&a
in The mean f S.E. 06 ti~.&HQ.A(2
At concen.&uUonn 06 RR at expi?.Gmn&I. um 1 x IO-5 M and gwua2~ phagocytitos& nign+can.tty d&$&hent (mm conathot vdueh (p~o.011. 185
The histological dye, ruthenium red, produced a dose dependent stimulation of Lm phagocytosis by P388D1 cells (Fig. 2) Maximal stimulation was observed at 10-4 to 10-3 M RR ( 40%). Ruthenium red at 10-4 M was used in subsequent studies because this concentrqtion was on the linear portion of the dose response curve and caused no change in cellular morphology even after prolonged incubations. At this concentration of RR (lo-4 M) a significant (p
90-
20 IO I 2
1 I
I 3
I 4
HOURS
Figwle 3:
i@e,cR" 06 RR (70-d hi)ubiphagocy&h.ih 06 1 Lm by P38bD1 eel%. BeIween 7-2 x IfI6 c&.&?n~pLa&? wehe .&c&at&d wkth RR @h j-4 The cekT,& we~rc then incubated wtih. horn.. Lm 6012 30 tin and phagocqfobib U&A meanwced. E&h paint in the mean t S. E. o fi I6 p.bA?b pin& I 7-4) I 4 expetimeti ) . A.&ZXitime ahe hi.gni.&antiq d&(deh&-nt l(fiom WnRhd The_ 3 and 4 hawk a%me valueh (p~O.071.~
pointi cvre nigni &Lcanf%4 (ti66ehenX ;rJ;hr~ the 1 and 2 hou_h dhug i.hcubatioMn (pcO.01). The calcium channel blocker, D-600 (lo-4 M) produced a significant inhibition (~45%) of phagocytosis by P388Dl cells (Fig. 4). Concentrations of D-600 grgater than 10-4 M were toxic owr the 4 hour drug incubation, while 10' M D-600 was less inhibitory than 10D4 M (data not shown). Figure 4 is a composite of 4 experiments, data points
(p
186
at each time were performed in quadruplicate. Two of the four experiments showed a time dependent inhibition while each of the experiments demonstrated an inhibition of phagocytosis (pdO.01) by D-600.
3
2
I
4
HOURS
FLgwte4:
06 v-600 (10m4 MI On phagOCYWL5 1 lm by P38bVj c&Lb C&we
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16 p.tTa&d (4 expe&.&nen~). flu Ltime poin& aheLgti&antiycLL{@eti @wm cona?wt ~igni&anX P.&L&J (p~o.071. No ove&& ;tOnedependency ioh tie V-600 tltipo~beti obnuwed aLthough 2 06 tie 4 expehimeti
did
hhow
hOme
mh.-!y
2inxz
e&x.x2.
The divalent cation ionophore A23187 at concentrations in excess of 2 x 10m6 M caused cellular lysis and were not used in this experimental protocol (data not shown). At the non-toxic concentration of A23187 (2 x 10-6 M) over the 4 hour incubation period, a stimulatory peak in the phagocytic response was observed ("38%); however, this transient response (Fig. 5) rapidly decreased to control values by 3 hours. The differences between the 2 hour point and the control, 1, 3, and 4 hour points were significant at the level ~~0.01. The concomitant addition of RR + O-600 (Fig. 6a) resulted in an interaction between the two drugs such that all the experimental points were statistically indistinquishable from control values. In conrast to the complete antagonism seen with RR + D-600, the combination of A23187 + D-600 resulted in only a partial change of the D-600 response (Fig. 6b). 187
Unlike the response seen with D-600 alone (Fig. 44 the inhibition of phagocytosis (D-600 + A231871 developed slowly (20-25%) over the first two hours and then started to rise, reaching 45% at 4 hours. The depressed response observed at 1 and 2 hours was significantly different from control as well as from the 3 and 4 hour points (p
60 50 5 g
40
.E 3j
30
$
20 IO 3
2
I
4
HOURS
5:
187 (2 x low6 MI on phagoEf&@!! oij cyxo&iA 06 1 Lm by P38bVj cc&~. CCL&& WYW p&e-Lncubateauiith A23187 don be/&en
14%!!
I ad4 how. S.E. 06 12 pW cytib.i.6 a-t the b@%i&kantiy vaeued
Each point .6 the mean + (3 expwimeti). Qhago2 howr dkug i.ncuba.tion wah d&$&Um~
@Om
cOVLIhO~
(p'o.01).
The concomitant addition of the two agents which produced a stimulation of phagocytosis by P388D cells (RR + A23187) is shown in Fig. 6c. A significant stimulation o + phagocytosis was observed; however, the response was not as elevated as was seen with RR alone (Fig. 3) but was more prolonged than with A23187 alone (Fig. 5).
188
a
30 20 IO
0
b
-10 M -30
-*r
b
FQAhe
6a:
Ed&z& 06 RR (JO-4 MI and D-600 (IO-4 MI on ph.agocyti& 06 7251 lm by PSWJ cm. Each data point &T~u&wZ Rhe mea~l t S .E . 06 16 ~&UC% (4 zxptien~). None 06 tithe. da& po,ain& we&e ,(owzd 20 be n.igti&kanfiy d.i~&hhent &orn a%stk kenpectiwe contioL4.
F&he
6b:
E~,@za% 06 A?3787 (2 x lO-6 MI and O-600 IlOw4 MI on ph.agocy;to& 06 Jz51 Lm by Each /lo&t IrepheAe& tie P3bWJ cekt.5. mean t S.E. 05 16 p.ta.te,s (4 ~xptianti)). AU a%ne pointi W~YLR~ ~ignL+antiy di~@~lren.t Qwm cona%oL va&ti (~40.01) and ;tCtc 1 a.nd 2 howt pokth we.&? higni@anaXy LOWeh &an tithe 3 and 4 howl dhug incubatioti (pcO.OJI
Figurre 6c:
Fig.
6a-c:
In aU added cell2
CaoeA, phwunacoLogicaC agenti we,te conconktatiy to nemi-con@Tuent P36tiDJ and incubated be2wze.n J and 4 houm. 189
XSSCUSION The P388Dl cell has been shown to possess many of the properties of normal macrophages (20, 21, 22, 23). In trying to understand the biochemical events involved in macrophage response we have used the P388D1 cell as a model system. Calcium is known to modulate a host of biological responses (7, 8, 26, 27), many of which appear to be involved in various facets of macrophage activity (9, 28, 29). This report explores the effects of agents known to perturb cellular calcium metabolism on phagocytosis in P388DI cells.. These cells rapidly phagocytize I251 Lm during the course of a thirty minute incubation. Initial studies inour laboratory are in agreement with the work of other investigators which showed phagocytosis to be a temperature sensitive process (4, 24, 25). The temperature studies, comparative phagocytosis studies (P388DI vs HTC) and the gram staining all indicate that actual phagocytosis occurred. Therefore, the results observed in subsequent experiments may be attributed to changes in phagocytosis and not attachment of -Lm to P388Dl cells or the tissue culture plasticware. The agents employed in these studies were the calcium antagonists ruthenium red (15, 30) and D-600 (10, 11, 14) and the divalent cation ionophore A23187 (13, 16). Preincubation of each of these agents with Lm did not induce any change in the degree of phagocytosis of Lm by m88DI cells as compared to untreated Lm. This information indicated that none of these drugs exerted an oj%onin-like (or anti-opsonin) effect on the bacteria. Therefore, any changes seen in phagocytosis were due to the effects of the drugs on the cells and not on the Listeria. Ruthenium red has been shown to alter mitochondrial calcium either by inhibiting calcium uptake (15, 30, 31, 32, 33) or by enhancing calcium efflux (31, 32). D-600 can block plasma membrane transport of calcium (10, 11, 14) while no effect by D-600 has been reported on mitochondrial calcium. A23187 increases the calcium permeability of mitochondrial and plasma membranes depending upon the dose administered (13, 16, 34, 35). This study has demonstrated that ruthenium red, D-600, and A23187 were capable of modulating the phagocytic response of the P388DI cells to Lm. The reported effects of these agents on cellular calcium movement COum be involved in their ability to stimulate or inhibit phagocytosis. The possibility that the inhibitory effect of D-600 is related to the blocking of plasma membrane transport of calcium and that drug stimulation of phagocytosis is related to either increased cytoplasmic calcium and/or decreased mitochondrial calcium is at present highly speculative. An increase in calcium efflux has been shown to occur during the phagocytosis of zymoson particles of guina pig polymorphonuclear leukocytes (9). Calcium efflux studies on P388DI cells in the presence of RR and D-600 indicate that these agents modulate intracellular calcium movements (manuscript in preparation). From these results only an inference can presently be drawn about the role of calcium in phagocytosis by P388DI cells, though it does appear that agents known to modulate calcium metabolism at a number of
190
different sites can effectively alter the phagocytic response. It is possible that these agents can be acting through some other mechanism(s) to affect the observed changes in phagocytosis. The results obtained from the measurement of 45Ca++ efflux and/or uptake experiments are difficult to interpret realizing that bhere are probably a number of complex interrelated intracellular calcium compartments involved in cellular calcium metabolism. Despite these problems, additional experiments are currently undentlayutilizing 45Ca++ in order that a clearer understanding of cellular calcium transport and distribution may be revealed as it relates to phagocytosis in this macrophage model.
ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of Mr. Jack Burkhalter in the statistical analysis of the data. We acknowledge the assistance of Dr. William Warner in the preparation of the manuscript. This work was supported in part by ACS grant IM-112, DA-00490, DA-01637. Dr. Carchman is the recipient of a Research Career Development Award, lK04AM00565, from the National Institutes of Health. REFERENCES 1.
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2.
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The effect of A23187 upon Jensen, P. and Ramissen H (1977) Cat+ metabolism in the Human lymphocyte. Biochem. Biophys. Acta 468, 146-156. Jensen, P., Winger, L., Rasmussen, H. and Nowell, P. (1977). The mitogenic effect of A23187 in human peripheral lymphocytes. Biothem. Biophys. Acta 496, 374-383.
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Reed, P.W. and Lardy, H.A. (1972). A23187: A divalent cation ionophore. J. Biol. Chem. 247. 6970-6977.
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Scarpa, A. (1975). Kinetics and energy coupling of Cat+ transport in mitochondria. In Calcium Transport in Contraction and Secretion (E. Carafoli, F. Clementi, W. Drabikowsky, and A. Margreth, eds.) p. 65-76. North Holland Publishing Co., New York, N.Y.
Received
17 Dee 79:
revised
version
received
193
25 Mar 80:
accepted
26 Mar 80