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Buffering requirements for cAMP determination by radioimmunoassay in cultured macrophages
Journal of immunological Methods, 154 (1992) 139-141
139
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06451 L e t t e r to t h e editors
Buffering requirements for cAMP determination by radioimmunoassay in cultured macrophages C l a u d e R. R o l a n d a, Kevin J. Martin b and M. Wayne Fiye a a Department of Surgery, Washington Unicersity School of Medicine, and h Department of Nephrology, St. Louis Unit'ersity School of Medicine, St. Louis, MO, USA
(Received 15 July 1991,revised received 16 March 1992.accepted 13 May 1992)
Dear Editors, The role of the adenylate cyclase second messenger system in immune regulation is again (Parker, 1979) coming under increasing scrutiny as evidence accumulates demonstrating the importance of cAMP in the modulation of monokine release (Decker et al., 1984). In order to determine cAMP levels in activated macrophages being held in culture, we have recently employed a modification of Steiner's (1972) method for radioimmunoassay (R1A) of cAMP, which has yielded consistent results when applied to renal parenchymal cells (Stokes et al., 1986). We report here an additional modification required when studying macrophages. 0.5-2 x 106 elicited peritoneal macrophages or splenocytes from the rat were cultured in replicate (six) wells of Costar culture plates (#3424) in a volume of 0.5 or 1.0 ml of culture medium, either RPMI 1640 with 10% fetal calf serum (FCS) (Callery et a~., 1991) or a modified KrebsHenseleit (mKH) solution (Stokes et al., 1986). After having been activated for 15 min with either 2.5 I~g/ml E. coil lipopolysaccharide (Sigma)
Correspondence to: C.R. Roland, Department of Surgery, Room 3347 CSRB, 4939 Audubon Ave.,St. Louis, MO 63110, USA. Tel.: (314) 362-8356. Abbreviations: RIA, radioimmunoassay; cAMP, cyclic adenosine monophosphate; mKH, modified Krebs-Henseleit; MES, 2-(N-morpholino)ethanesulfonicacid; SD, standard deviation; DMPG, 16,16-dimethylprostaglandin E 2.
in the case of the macrophages or with 10 p.g/ml 16,16-dimethyl prostaglandin E2 (DMPG) (Upjohn) in the case of splenocytes, cellular metabolism was terminated with 100 p.I of 1.8 M perchloric acid, followed by neutralization with 100/tl of 3.0 M KHCO 3. The mixture was then buffered with 300 p.I of 20-200 mM 2-(N-morpholino)ethanesulfonic acid (MES), which had been titrated to pH 6.2. The samples were transferred to borosilicate tubes and centrifuged. After measuring the pH of the supernatants, 200/~! were removed for acetylation with 5/~! of a 2:1 mixture of triethylamine and acetic anhydride. A 1/5 dilution was performed by adding 800/~i of 20 mM MES. To 200 /~! of this diluted and acetylated sample, 100/~l of BR-1 primary rabbit antibody against cAMP-succinate (Tamayo et al., 1982) and 200 pA of ['2Sl]succinyi-cAMP-tyrosyl methyl ester (10,000-15,000 cpm/200 /.d) was added for an 18 h incubation at 4°C. Following the incubation, 100 #1 of goat anti-rabbit antibody (ICN) and 1 ml of 3% polyethylene glycol (MW 3000) were added for a second incubation period of 45 rain at 4°C. The samples were then centrifuged at 2000 x g for 20 rain. Gamma emissions over a minute were counted after decanting of the centrifuged tubes. Initial experiments had revealed that cAMP levels in identically treated macrophages using the previously described method (Stokes et al., 1986) were widely inconsistent (data not shown). Since replicate wells treated with perchlorate and KHCO 3 were found to have variable pH values
140 • ~
i
+
ii
,
4-
8,-I
,--
47:
:i
Oi
TABLE 1 THE EFFECT OF CONCENTRATION OF 0.3 ML MES ON pH VARIABILITY IN DIFFERENT CULTURE MEDIA
with 8O
.3
~i
-
MES (mM) 20 100
i
i ) I
.4
140 200 RPM! 1640 withFCS ND 5.62+_1.166.01+_0.2 6.04+_0.16 mKH 4.21+1.526.78+0.81 6.32+0.09 6.24+-0.05
.6
.6
. • .a 1LO 1.1 1.2 1,3 1,4 1.6 t.e 1.7 t.n t.a 2.0 VOlWlm (A~) of 20 I~A ME8 lldd4KI
.T
Fig. I. pH variability in six replicate wells diminishes as increasingamountsof 20 mM MES are added to a macrophage culture treated with HCIO4 and KHCO3 in preparation for cAMP radioimmunoassay.Addition of 0.3 ml of 20 mM MES has been the establishedmethod of buffering renal cell cultures (Stokes et al., 1986). after the addition of 0.3 ml of 20 mM MES buffer, increasing amounts of 20 mM MES were added to six replicate wells containing 500,000 LPS-stimulated macrophages, which had been treated with perchlorate and KHCO 3 (Fig. 1). There was a dose-dependent diminution of variability in pH values among wells with the addition of greater volumes of 20 mM MES. When 0.3 ml of 20 mM MES was added, the replicate wells had an average pH of 4.2 with a standard deviation of 2.05. Maximal improvement was achieved at 1.9 ml of 20 mM MES, which buffered the replicate wells to pH 6.35 _+0.11. It was estimated that, at a volume of 0.3 ml, 100-200 mM of MES might be needed to buffer the supernatant in the culture wells of lysed macrophages. Accordingly, replicate wells containing stimulated peritoneal macrophages in either 0.5 mi of RPMI 1640 with 10% FCS, culture
medium used for macrophages (Callery et al., 1991), or mKH solution, culture medium used for renal parenchymal cells (Stokes et al., 1986), were treated with perchloric acid and were neutralized with KHCO 3. 0.3 ml of either 100, 140, or 200 mM MES were added to each well. The effect of increasing MES concentration on pH, as shown in Table I, was to raise the pH to the pKa (6.15) of MES and to reduce variability among wells. containing either type of medium. To establish that correction of pH variability would correspond to reproducible cAMP values and to compare buffering requirements of macrophages and splenocytes, replicate wells containing 2 x 106 LPS-activated peritoneal macrophages or DMPG-stimulated splenocytes in 1.0 ml of RPMI 1640 with 10% FCS were neutralized with 20 or 200 mM MES after treatment with perchloric acid and KHCO 3. Table II shows that the reduction of variability in pH with the higher concentration of MES corresponded to a reduction in variability among replicate cAMP values obtained from the peritoneal macrophages. The data also show that cells from the spleen, which are comprised predominantly of lymphocytes, do not appear to have the same buffering
TABLE 11 A COMPARISON OF THE BUFFERING REQUIREMENTS OF STIMULATED PERITONEAL MACROPHAGES AND SPLENOCYTES
pH SD/mean cAMP(pmol/ml) SD/mean
Splenocytes 20 mM MES 6.45 +0.36 0.057 6.0 +_0.23 0.039
200 mM MES 5.94 +0.27 0.046 5.88 +_0.48 0.082
Peritoneal macrophages 20 mM MES 200 mM MES 6.38 +0.97 6.12 _+0.22 0.152 0.035 2.01 +_0.27 2.16 +_0.15 0.134 0.070
141 requirement as do macrophages. This is consistent with previous work in which the buffering capacity of 20 mM MES was sufficient to buffer the lysates of other non-macrophage cells. In order to determine the effects of pH and an increased concentration of MES buffer on the efficiency of cAMP extraction, cAMP (Sigma) was added, for a final concentration of 4 p m o l / m l , to wells containing medium only. Immediately thereafter, the wells were treated with perchloric acid and KHCO 3. Following buffering with either 20 or 200 mM MES, 2 0 0 / t l were acetylated for cAMP RIA. Recovery of cAMP was between 66-68.5% with no significant difference observed between the recovery after neutralization with 20 mM MES (2.64 + 0.33 p m o l / m l ; 66% recovery) or 200 mM MES (2.74 + 0.41 p m o l / m l ; 68.5% recovery). These results suggest that the requirement for strong buffering capability is related to the ceils themselves or a factor produced by the cells in culture rather than recovery of cAMP in acellular medium. In addition, the higher concentration of MES does not adversely affect the recovery of cAMP. MES buffer is a zwitterionic N-substituted aliphatic amino acid first described as a buffer for biological systems by Good (1966). This buffer has been effective when used after perchlorate and KI-ICO 3 treatment of renal parenchymal cells prior to RIA of cAMP (Stokes et al., 1986). With the application of this method to peritoneal and hepatic macrophages, the use of MES at the concentration (20 mM) previously reported (Stokes et al., 1986) was ineffective in buffering the contents of the culture wells causing widely variant cAMP levels as determined by RIA despite identical treatment of all wells. The results of these experiments indicate that an approximately one log increase in MES concentration is required to reliably buffer lysates of stimulated macrophages in culture. On the other hand, the recovery of exogenously applied cAMP to tubes in the absence of cells does not appear to be sensitive to the ambient pH within the range of MES buffer concentrations tested. It is tempting to speculate that the increased buffering capacity of MES required in a macrophage culture is related to the well-established ability of macrophages to release products
such as nitric oxide (Hibbs ,~'t al., 1987) and oxygen-derived radicals (Bauusta et al., 1990). Since MES has been shown to uncouple electron transport in phosphorylation (Good et al., 1966) and to be an electron donor for photoreduction (Yamazaki et al., 1970), it is possible that macrophage products which affect oxidation-reduction reactions adversely affect the ability of MES to buffer at concentrations sufficient to buffer renal parenchymal cells and splenocytes.
Acknowledgements We thank Theresa Belgeri for secretarial assistance.
References Bautista, A.P., Meszaros.K., Bojta, J. and Spitzcr, J.J. (1990) Supcroxide anion generation in the liver during the early stage of endotoxemiain rats. J. Leuk. Biol. 48, 123. Callery, M.P., Mangino,M.J. and Flye, M.W.(1991) A biological basis for limited Kupffer cell reactivity to portal-derived endotoxin.Surgery ! 10, 221. Decker, K. and Birmelin, M. (1984) Synthesisof prostanoids and cyclicnucleotidesby phagocytosingrat Kupffercells. Eur. J. Biochem. 142, 219. Good, N.E., Winget,G.D., Winter, W., Connolly,T.N., lzawa, S. and Singh, R.M.M. (1966) Hydrogenbuffersfor biological research. Biochemistry5, 467. Hibbs,Jr., J.B., Vavrin,Z. and Taintor, R.R. (1987) L-Arginine is required for expression of the activated macrophage effector mechanismcausing selectivemetabolic inhibition in target cells. J. Immunol. 138, 550. Parker, C.W. (1979)The role of intracellular mediatorsin the immune response,in: S. Cohen, E. Pick and J. Oppenheim (Eds.), Biology of Lymphokines. Academic Press, New York, p. 541. Steiner, A.L., Parker, C.W. and Kipnis,D.M. (1972) Radioimmunoassay for cyclic nucleotides: 1. Preparation of antibodies and iodinated cyclic nucleotides. J. Biol. Chem. 247, 1106. Stokes, Jr., T.J., McConkey, lr., C.L. and Martin, K.J. (1986) Atriopeptin 111 increases cGMP in glomeruli but not in proximal tubules of dog kidney. Am. J. Physiol.250, F27. Tamayo, J., Bellosin-Font.E., Sicard, G., Anderson, C. aild Martin, K.J. (1982) Desensitization to parathyroid hormone in the isolated perfused canine kidney: Reversalof altered receptor-adenylate cyclase system by guanosine triphosphate in vivo. Endocrinology111, 1311. Yamazaki, R.K. and Tolbert, N.E. (1970) Pbotoreactionsof flavin mononucleotideand a flavoproteinwith zwinerionic buffers. Biochim.Biophys.Acta 197, 90.
139
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06451 L e t t e r to t h e editors
Buffering requirements for cAMP determination by radioimmunoassay in cultured macrophages C l a u d e R. R o l a n d a, Kevin J. Martin b and M. Wayne Fiye a a Department of Surgery, Washington Unicersity School of Medicine, and h Department of Nephrology, St. Louis Unit'ersity School of Medicine, St. Louis, MO, USA
(Received 15 July 1991,revised received 16 March 1992.accepted 13 May 1992)
Dear Editors, The role of the adenylate cyclase second messenger system in immune regulation is again (Parker, 1979) coming under increasing scrutiny as evidence accumulates demonstrating the importance of cAMP in the modulation of monokine release (Decker et al., 1984). In order to determine cAMP levels in activated macrophages being held in culture, we have recently employed a modification of Steiner's (1972) method for radioimmunoassay (R1A) of cAMP, which has yielded consistent results when applied to renal parenchymal cells (Stokes et al., 1986). We report here an additional modification required when studying macrophages. 0.5-2 x 106 elicited peritoneal macrophages or splenocytes from the rat were cultured in replicate (six) wells of Costar culture plates (#3424) in a volume of 0.5 or 1.0 ml of culture medium, either RPMI 1640 with 10% fetal calf serum (FCS) (Callery et a~., 1991) or a modified KrebsHenseleit (mKH) solution (Stokes et al., 1986). After having been activated for 15 min with either 2.5 I~g/ml E. coil lipopolysaccharide (Sigma)
Correspondence to: C.R. Roland, Department of Surgery, Room 3347 CSRB, 4939 Audubon Ave.,St. Louis, MO 63110, USA. Tel.: (314) 362-8356. Abbreviations: RIA, radioimmunoassay; cAMP, cyclic adenosine monophosphate; mKH, modified Krebs-Henseleit; MES, 2-(N-morpholino)ethanesulfonicacid; SD, standard deviation; DMPG, 16,16-dimethylprostaglandin E 2.
in the case of the macrophages or with 10 p.g/ml 16,16-dimethyl prostaglandin E2 (DMPG) (Upjohn) in the case of splenocytes, cellular metabolism was terminated with 100 p.I of 1.8 M perchloric acid, followed by neutralization with 100/tl of 3.0 M KHCO 3. The mixture was then buffered with 300 p.I of 20-200 mM 2-(N-morpholino)ethanesulfonic acid (MES), which had been titrated to pH 6.2. The samples were transferred to borosilicate tubes and centrifuged. After measuring the pH of the supernatants, 200/~! were removed for acetylation with 5/~! of a 2:1 mixture of triethylamine and acetic anhydride. A 1/5 dilution was performed by adding 800/~i of 20 mM MES. To 200 /~! of this diluted and acetylated sample, 100/~l of BR-1 primary rabbit antibody against cAMP-succinate (Tamayo et al., 1982) and 200 pA of ['2Sl]succinyi-cAMP-tyrosyl methyl ester (10,000-15,000 cpm/200 /.d) was added for an 18 h incubation at 4°C. Following the incubation, 100 #1 of goat anti-rabbit antibody (ICN) and 1 ml of 3% polyethylene glycol (MW 3000) were added for a second incubation period of 45 rain at 4°C. The samples were then centrifuged at 2000 x g for 20 rain. Gamma emissions over a minute were counted after decanting of the centrifuged tubes. Initial experiments had revealed that cAMP levels in identically treated macrophages using the previously described method (Stokes et al., 1986) were widely inconsistent (data not shown). Since replicate wells treated with perchlorate and KHCO 3 were found to have variable pH values
140 • ~
i
+
ii
,
4-
8,-I
,--
47:
:i
Oi
TABLE 1 THE EFFECT OF CONCENTRATION OF 0.3 ML MES ON pH VARIABILITY IN DIFFERENT CULTURE MEDIA
with 8O
.3
~i
-
MES (mM) 20 100
i
i ) I
.4
140 200 RPM! 1640 withFCS ND 5.62+_1.166.01+_0.2 6.04+_0.16 mKH 4.21+1.526.78+0.81 6.32+0.09 6.24+-0.05
.6
.6
. • .a 1LO 1.1 1.2 1,3 1,4 1.6 t.e 1.7 t.n t.a 2.0 VOlWlm (A~) of 20 I~A ME8 lldd4KI
.T
Fig. I. pH variability in six replicate wells diminishes as increasingamountsof 20 mM MES are added to a macrophage culture treated with HCIO4 and KHCO3 in preparation for cAMP radioimmunoassay.Addition of 0.3 ml of 20 mM MES has been the establishedmethod of buffering renal cell cultures (Stokes et al., 1986). after the addition of 0.3 ml of 20 mM MES buffer, increasing amounts of 20 mM MES were added to six replicate wells containing 500,000 LPS-stimulated macrophages, which had been treated with perchlorate and KHCO 3 (Fig. 1). There was a dose-dependent diminution of variability in pH values among wells with the addition of greater volumes of 20 mM MES. When 0.3 ml of 20 mM MES was added, the replicate wells had an average pH of 4.2 with a standard deviation of 2.05. Maximal improvement was achieved at 1.9 ml of 20 mM MES, which buffered the replicate wells to pH 6.35 _+0.11. It was estimated that, at a volume of 0.3 ml, 100-200 mM of MES might be needed to buffer the supernatant in the culture wells of lysed macrophages. Accordingly, replicate wells containing stimulated peritoneal macrophages in either 0.5 mi of RPMI 1640 with 10% FCS, culture
medium used for macrophages (Callery et al., 1991), or mKH solution, culture medium used for renal parenchymal cells (Stokes et al., 1986), were treated with perchloric acid and were neutralized with KHCO 3. 0.3 ml of either 100, 140, or 200 mM MES were added to each well. The effect of increasing MES concentration on pH, as shown in Table I, was to raise the pH to the pKa (6.15) of MES and to reduce variability among wells. containing either type of medium. To establish that correction of pH variability would correspond to reproducible cAMP values and to compare buffering requirements of macrophages and splenocytes, replicate wells containing 2 x 106 LPS-activated peritoneal macrophages or DMPG-stimulated splenocytes in 1.0 ml of RPMI 1640 with 10% FCS were neutralized with 20 or 200 mM MES after treatment with perchloric acid and KHCO 3. Table II shows that the reduction of variability in pH with the higher concentration of MES corresponded to a reduction in variability among replicate cAMP values obtained from the peritoneal macrophages. The data also show that cells from the spleen, which are comprised predominantly of lymphocytes, do not appear to have the same buffering
TABLE 11 A COMPARISON OF THE BUFFERING REQUIREMENTS OF STIMULATED PERITONEAL MACROPHAGES AND SPLENOCYTES
pH SD/mean cAMP(pmol/ml) SD/mean
Splenocytes 20 mM MES 6.45 +0.36 0.057 6.0 +_0.23 0.039
200 mM MES 5.94 +0.27 0.046 5.88 +_0.48 0.082
Peritoneal macrophages 20 mM MES 200 mM MES 6.38 +0.97 6.12 _+0.22 0.152 0.035 2.01 +_0.27 2.16 +_0.15 0.134 0.070
141 requirement as do macrophages. This is consistent with previous work in which the buffering capacity of 20 mM MES was sufficient to buffer the lysates of other non-macrophage cells. In order to determine the effects of pH and an increased concentration of MES buffer on the efficiency of cAMP extraction, cAMP (Sigma) was added, for a final concentration of 4 p m o l / m l , to wells containing medium only. Immediately thereafter, the wells were treated with perchloric acid and KHCO 3. Following buffering with either 20 or 200 mM MES, 2 0 0 / t l were acetylated for cAMP RIA. Recovery of cAMP was between 66-68.5% with no significant difference observed between the recovery after neutralization with 20 mM MES (2.64 + 0.33 p m o l / m l ; 66% recovery) or 200 mM MES (2.74 + 0.41 p m o l / m l ; 68.5% recovery). These results suggest that the requirement for strong buffering capability is related to the ceils themselves or a factor produced by the cells in culture rather than recovery of cAMP in acellular medium. In addition, the higher concentration of MES does not adversely affect the recovery of cAMP. MES buffer is a zwitterionic N-substituted aliphatic amino acid first described as a buffer for biological systems by Good (1966). This buffer has been effective when used after perchlorate and KI-ICO 3 treatment of renal parenchymal cells prior to RIA of cAMP (Stokes et al., 1986). With the application of this method to peritoneal and hepatic macrophages, the use of MES at the concentration (20 mM) previously reported (Stokes et al., 1986) was ineffective in buffering the contents of the culture wells causing widely variant cAMP levels as determined by RIA despite identical treatment of all wells. The results of these experiments indicate that an approximately one log increase in MES concentration is required to reliably buffer lysates of stimulated macrophages in culture. On the other hand, the recovery of exogenously applied cAMP to tubes in the absence of cells does not appear to be sensitive to the ambient pH within the range of MES buffer concentrations tested. It is tempting to speculate that the increased buffering capacity of MES required in a macrophage culture is related to the well-established ability of macrophages to release products
such as nitric oxide (Hibbs ,~'t al., 1987) and oxygen-derived radicals (Bauusta et al., 1990). Since MES has been shown to uncouple electron transport in phosphorylation (Good et al., 1966) and to be an electron donor for photoreduction (Yamazaki et al., 1970), it is possible that macrophage products which affect oxidation-reduction reactions adversely affect the ability of MES to buffer at concentrations sufficient to buffer renal parenchymal cells and splenocytes.
Acknowledgements We thank Theresa Belgeri for secretarial assistance.
References Bautista, A.P., Meszaros.K., Bojta, J. and Spitzcr, J.J. (1990) Supcroxide anion generation in the liver during the early stage of endotoxemiain rats. J. Leuk. Biol. 48, 123. Callery, M.P., Mangino,M.J. and Flye, M.W.(1991) A biological basis for limited Kupffer cell reactivity to portal-derived endotoxin.Surgery ! 10, 221. Decker, K. and Birmelin, M. (1984) Synthesisof prostanoids and cyclicnucleotidesby phagocytosingrat Kupffercells. Eur. J. Biochem. 142, 219. Good, N.E., Winget,G.D., Winter, W., Connolly,T.N., lzawa, S. and Singh, R.M.M. (1966) Hydrogenbuffersfor biological research. Biochemistry5, 467. Hibbs,Jr., J.B., Vavrin,Z. and Taintor, R.R. (1987) L-Arginine is required for expression of the activated macrophage effector mechanismcausing selectivemetabolic inhibition in target cells. J. Immunol. 138, 550. Parker, C.W. (1979)The role of intracellular mediatorsin the immune response,in: S. Cohen, E. Pick and J. Oppenheim (Eds.), Biology of Lymphokines. Academic Press, New York, p. 541. Steiner, A.L., Parker, C.W. and Kipnis,D.M. (1972) Radioimmunoassay for cyclic nucleotides: 1. Preparation of antibodies and iodinated cyclic nucleotides. J. Biol. Chem. 247, 1106. Stokes, Jr., T.J., McConkey, lr., C.L. and Martin, K.J. (1986) Atriopeptin 111 increases cGMP in glomeruli but not in proximal tubules of dog kidney. Am. J. Physiol.250, F27. Tamayo, J., Bellosin-Font.E., Sicard, G., Anderson, C. aild Martin, K.J. (1982) Desensitization to parathyroid hormone in the isolated perfused canine kidney: Reversalof altered receptor-adenylate cyclase system by guanosine triphosphate in vivo. Endocrinology111, 1311. Yamazaki, R.K. and Tolbert, N.E. (1970) Pbotoreactionsof flavin mononucleotideand a flavoproteinwith zwinerionic buffers. Biochim.Biophys.Acta 197, 90.