Immunology Letters, 28 (1991) 1-4 Elsevier

IMLET 01552

Amiloride does not influence the capability of interferon gamma to potentiate superoxide anion and hydrogen peroxide release by

human mononuclear phagocytes

Ma rc o A. Cassatel la 1, Diego Peroni z, Miguel Aste A m e z a g a ~, Feder ica Vicentini 2 and Flavia Bazzoni 1

llnstitute of General Pathology and 2Department of Pediatrics, University of Verona, Verona, Italy

(Received 29 August 1990; revision received 30 November 1990; accepted 2 December 1990)

1. Summary

Since the molecular mechanisms of macrophage activation in response to interferon gamma (IFN-,y) are still not well defined we have investigated wheth- er amiloride, a specific inhibitor of the N a + / H ÷ antiporter, had any effect on the IFN-3,-mediated potentiation of human monocyte and monocyte- derived macrophage capability to produce O£ (respiratory burst). Here, we demonstrate that amiloride neither inhibits the capability of IFN-3, to act.ivate the mononuclear phagocyte respiratory burst nor influences IFN-7 induction of steady-state mRNA levels for 2 components of the superoxide anion-generating enzyme system. On the contrary, we show that IFN-7-enhanced expression of the HLA-DRa gene is significantly inhibited by amilo- ride (see also ref. 5). These data indicate that Na ÷ / H ÷ antiporter stimulation by IFN-7 is not in- volved in the mechanism of activation of macro- phage oxidative metabolism.

2. Introduction

The ability to produce O~- and H202 in response to external stimuli is one of the most important func-

Key words." IFN-7; Macrophage activation; Respiratory burst; NADPH oxidase; Amiloride

Correspondence to: Dr. Marco A. Cassatella, Istituto di Patolo- gia Generale, Strada Le Grazie 4, 37134 Verona, Italy.

tions exerted by macrophages and neutrophils, and is responsible for the killing of intracellular patho- gens and tumor cells [1]. This capability has been shown to be enhanced by IFN-7 [2, 3], which was identified as the lymphocyte-derived molecule act- ing as macrophage activating factor [2]. IFN-3, exerts immunoregulatory, antiviral and antiproliferative effects on different cell types, but the molecular and biochemical basis of its action is still largely un- known. Studies performed by our group on the mechanism of IFN-'y activation of the capability of macrophages to produce toxic oxygen-derived molecules showed that this phenomenon was ac- companied by changes in the kinetic parameters of NADPH oxidase, the superoxide anion-generating enzyme [4]. Recently, Prpic et al. [5] reported that, in murine peritoneal macrophages, IFN-7 initiates rapid exchanges of Na ÷ and H ÷ by means of the Na ÷ / H ÷ antiporter and that these ion fluxes are sensitive to the specific inhibitor amiloride. This drug decreased significantly the expression of specific genes induced by IFN-7 in those cells, such as JE and I-A3. On the basis of these latter observa- tions we decided to investigate whether activation of human macrophage respiratory burst by IFN-7 was dependent on Na ÷ / H ÷ exchange.

3. Materials and Methods

3.1. Isolation and cultivation o f human monocytes and monocyte-derived macrophages (MDM)

This was done as described in ref. 4. Tissue culture

0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V.

plastic 96- or 24-well trays (Flow Laboratories) were used to cultivate monocytes and macrophages and to perform functional assays. To test the effect of amiloride, monocyte or MDM cultures were in- cubated for 10 min with different concentrations of the drug; then 100 U/ml rlFN-3, were added and in- cubation of monolayers were prolonged for the times indicated in the Results. Adherent cells were washed several times with PBS before performing functional assays.

3.2. Superoxide anion release and hydrogen perox- ide production

The methods used to measure the release of these toxic oxygen metabolites were described in detail in ref. 6 for superoxide anion and in ref. 7 for hydrogen peroxide.

3.3. Northern blot analysis

This was done exactly as previously described [8]. m R N A for the heavy chain subunit of cytochrome b (X-CGD) was detected by hybridization with the fragment purified from the plasmid provided by S. H. Orkin (Children's Hospital, Harvard Universi- ty, Boston) [9]; m R N A for the 47-kDa cytosolic component of N A D P H oxidase (p47-phox), was de- tected with the fragment isolated from the cDNA provided by K. J. Lomax (Gene Therapy Unit, NIH, Bethesda) [10]; m R N A for the u-chain of HLA-DR (DR-o0 was revealed with the fragment purified from the cDNA obtained from P. Wettstein (Wistar Insti- tute, PA) [11].

3.4. Miscellaneous

Proteins were assayed in cell lysates, after over- night treatment with 1 M NaOH, by the Lowry meth- od [12]. Phorbol myristate acetate (PMA), Concan- avalin A (Con A) and amiloride were purchased from Sigma (St. Louis, MO, U.S.A.). rlFN-3, (lot no. K9009A1, 2 mg/ml, 2 x 107 U/mg) was produced by Genentech Inc. (San Francisco, CA) and kindly provided by Boehringer Ingelheim, Vienna, Austria. Other chemicals were of the highest purity available from commercial sources. All the reagents and solu- tions were prepared with clinical pyrogen-free water.

4. Results and Discussion

To study the effect of amiloride on IFN-2/activa- tion of macrophage respiratory burst we used "in vitro" cultured 1- or 2-day-old human monocytes and 6- or 7-day-old MDM. We first tested whether amiloride alone was able to induce production of O~- in 24 h cultured monocyes. As shown in Ta- ble 1, doses of amiloride ranging from 0.05 mM to 2 mM were unable to trigger the production of any significant amount of O~-; on the contrary, other known stimuli of the respiratory burst, such as PMA and Con A [3, 4] were effective. We then checked whether exposure of 6-day-old MDM to amiloride for 2 0 - 4 8 h had any possible toxic effect on these cells. Analysis of Trypan blue dye exclusion by light microscopy revealed that all macrophages were via- ble after exposure for 48 h to doses of amiloride up to 0 .2-0 .3 mM.

Table 2, showing the results of a typical experi- ment, demonstrates that doses of amiloride below 0.25-0.3 mM did not influence the production of O~- or H20 2 by 7-day-old MDM. It is also evident that doses of amiloride up to 250/~M did not inhibit, at any of the time points tested, the capability of rlFN--y to potentiate the H20 2 generation in re- sponse to 100 ng/ml PMA. Similar results were ob- tained when 10 ng/ml PMA were used or when O~- generation was measured (not shown). Experiments performed with the same donors revealed that also the enhancement of MDM respiratory burst after

TABLE 1

Effect of amiloride as triggering stimulus of monocyte respirato- ry burst.

nmol O2-/h/mg protein

Resting 9.8 PMA 100 ng/ml 125.0 Con A 100/zg/ml 32.3 Amiloride 0.05 mM 10.6 Amiloride 0.25 mM 12.5 Amiloride 0.50 mM 10.5 Amiloride 1.00 mM 9.1 Amiloride 2.00 mM 9.1

Mean values of duplicate assays of one experiment representative of 3 performed are reported. Assays were performed on 24-h cul-

tured monocytes as described in ref. 6.

TABLE 2

Effect of amiloride on IFN-3, potentiation of human monocyte- derived macrophage respiratory burst.

nmol H202/h/mg protein

Resting PMA 100 ng/ml

Incubation for 24 h with: Medium 0.6 84.7

+ Amiloride 50/~M 1.1 90.6 + Amiloride 250 #M 7 89.4

IFN-y (I00 IU/ml) 7.2 236 + Amiloride 50/zM 10.9 261 + Amiloride 250/~M 4.5 223

Incubation for 48 h with: Medium 3.3 42

+ Amiloride 50/zM 1.7 55 + Amiloride 250 ttM 5.3 51

IFN- 7 5.5 182 + Amiloride 50/zM 10 195 + Amiloride 250/~M 11 201

The table reports the mean values of triplicate assays of a representative experiment out of 7 performed. Amiloride was ad- ded to the 7-day-old MDM 10 min before rlFN- 3, addition. As-

says were done exactly as described in ref. 7.

2 o

o~ E .%

ten O

m o

70

5 0

lO

, i

ii: ii

P M A 10 n g / m l

C o n A l O 0 , u g / r n l

MEDIUM IFN-GAMMA AMILORIDE IFN-GAMMA 4" IOO/JM A M I L O R I D E

Fig. 1. Effect of amiloride on the capability of rlFN-~, to enhance O£ production by 2-day-old monocytes in response to Con A and PMA. Amiloride was added to the cultures 10 min before ad- dition of 100 U/ml rlFN--}, and monolayers incubation was prolonged for a further 20 h. Assays were done as described in ref. 6. The mean values of one representative experiment out of

3 performed are reported.

! Z M.. w

o=. o=. o O

X- CGD

p47 -phox

1 ~ DR -a

Fig. 2. Effect of amiloride on IFN--},-induced expression of X- CGD, p47-phox and Dr-ct mRNA. RNA was isolated from monocyte-derived macrophages (MDM) after 7 days of culture. 100/~M amiloride and 100 U/ml rlFN-3, were added at day 6 to part of the cultures. 10 #g of total RNA were loaded on each gel lane. Filters were sequentially hybridized with X-CGD, p47-phox and Dr-c~ cDNA probes. The figure shows one experiment

representative of 3 performed.

l ipopolysacchar ide t rea tment [13] was not blocked

by the presence o f ami lor ide (not shown). The effect

o f ami lor ide on the capabi l i ty o f rlFN-3, to potent i - ate O~- genera t ion by monocytes in response to

P M A and C o n A is shown in Fig. 1. Monocy te acti-

va t ion by 24-h t rea tment with rlFN-3, was less pro-

nounced than the one observed with macrophages ,

but, also with these cells, po ten t ia t ion o f the respira-

tory burst was not inf luenced by 100/zM amiloride.

Since IFN-,y is known to increase in macrophages m R N A steady state levels for two componen t s o f the N A D P H oxidase system, X - C G D and p47-phox

[14-16], o ther than class II h is tocompat ib i l i ty com-

plex genes [17], we decided to investigate whether

ami lor ide had inf luence on the expression o f these

genes. Nor the rn blots o f Fig. 2 show the results o f

a typical experiment . Increases o f X - C G D and p47- phox m R N A transcripts induced by 20 h t rea tment

with rIFN-~, were n o t i n h i b i t ed by 100/~M ami lo - ride. In con t ras t , increase o f HLA-DRc~ gene expres- s ion due to rIFN-~, was s ign i f i can t ly i nh ib i t ed by ami lo r ide , cons i s t en t wi th the results o f P rp ic et al. [5].

In conclusion, we have demonstrated that amilo- ride, used at concentrations known to block the Na + / H + antiporter [18], does not inhibit the IFN- ~, enhancement of mononuclear phagocytes' capa- bility to produce toxic oxygen metabolites. Our results are not in contrast with the observations reported by Prpic et al. [5], who showed that IFN-~, induces rapid, amiloride-sensitive, increase in cyto- solic pH and in the influx of 22No+ into murine peritoneal macrophages and that these changes ap- pear to participate in the IFN-~,-induced increases in mRNA accumulation for JE and I-A/] genes. In fact, we also found that, in human MDM, induction of HLA-DRo~ mRNA steady-state levels by IFN-~, was sensitive to amiloride. Thus, IFN-7 can activate different macrophage functions by using distinct and specific intracellular pathways and signals. Fur- ther studies are necessary to elucidate completely the signal transduction and the molecular mechanisms used by IFN-~, to activate myelomonocytic cells.

Acknowledgements

We are gra tefu l to Prof . E Rossi a n d G. Be r ton for the i r cr i t ical r ead ing o f this m a n u s c r i p t a n d to Fab i o Pol i a n d Feder ica Calze t t i for the i r excellent t echni - cal assis tance. Th i s work has b e e n s u p p o r t e d by g ran t s f rom C.N.R . ( c o n t r i b u t i o n No. 89.01923.04) a n d f r o m the Min i s t e r i o P u b b l i c a I s t r u z i o n e ( fondi 40°70). M . A . A . is s u p p o r t e d by a fe l lowship f rom the Th i r d Wor ld A c a d e m y o f Sciences (TWAS) . T h e gene rous c o n t r i b u t i o n o f the I t a l i an A s s o c i a t i o n for C a n c e r Research (AIRC) , wh ich also s u p p o r t s FN., is g ra te fu l ly acknowledged .

References

[1] Bellavite, P. (1988) Free Radical Biol. Med. 4, 225. [2] Nathan, C. F., Murray, H. W., Wiebe, M. E. and Rubin,

B. Y. (1983) J. Exp. Med. 158, 670. [3] Berton, G., Zeni, L., Cassatella, M. A. and Rossi, F. (1986)

Biochem. Biophys. Res. Commun. 138, 1276. [4] Cassatella, M. A., Della Bianca, V., Berton, G. and Rossi,

F. (1985) Biochem. Biophys. Res. Commun. 132, 908. [5] Prpic, V., Yu, S. F., Figueiredo, F., Hollenbach, P. W., Gaw-

di, G., Herman, B., Uhing, R. J. and Adams, D. O. (1989) Science 244, 469.

[6] Berton, G., Cassatella, M. A., Cabrini, G. and Rossi, F. (1985) Immunology 54, 371.

[7] Cassatella, M. A., Berton, G., Agostini, C., Zambello, R., Trentin, L., Cipriani, A. and Semenzato, G. P. (1989) Immu- nology 66, 451.

[8] Cassatella, M. A., Hartman, L., Perussia, B. and Trinchieri, G. (1989) J. Clin. Invest. 83, 1570.

[9] Royer-Pokora, B., Kunkel, L. M., Monaco, A. P., Goff, S. C., Newburger, P. E., Bahener, R. L., Cole, F. S., Cur- nutte, J. T. and Orkin, S. H. (1986) Nature 322, 32.

[10] Lomax, K. L, Leto, T. L., Nunoi, H., Gallin, J. I. and Malech, H. L. (1989) Science 245,409.

[11] Korman, A. J., Knudsen, P. J., Kaufman, J. and Strominger, J. L. (1982) Proc. Natl. Acad. Sci. USA 79, 1844.

[12] Lowry, O. H., Rosebrough, H. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265.

[13] Pabst, M. and Johnston Jr., R. B. (1980) J. Exp. Med. 151, 101.

[14] Newburger, P. E., Ezekowitz, R. A. B., Whitney, C. and Or- kin, S. H. (1988) Proc. Natl. Acad. Sci. USA 85, 5215.

[15] Cassatella, M. A., Bazzoni, F., Flynn, R. M., Dusi, S., Trin- chieri, G. and Rossi, E (1990) J. Biol. Chem., in press.

[16] Abramson, S. L., Lomax, K. J., Malech, H. L. and Gallin, J. I. (1990) Clin. Res. 236A.

[17] Celada, A. M., Klemsz, M. J. and Maki, R. A. (1989) Eur. J. Immunol. 19, 1103.

[18] Swallow, C. S., Grinstein, S. and Rotstein, O. D. (1990) Bin- chim. Biophys. Acta 1022, 203.