Mechanism of enhanced phagocytic response in protein a treated rat macrophages

Immunology Letters, 34 (1992) 289 296 0165 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved

IMLET 01882

Mechanism of enhanced phagocytic response in protein a treated rat macrophages

Anil Mishra , P.D. Dwivedi, A.S. Verma and P.K. Ray

lnhalation/Immunobiology Division, Industrial Toxicology Research Centre, Lucknow, India

(Received 2 January 1992; accepted 2 October 1992)

1. Summary 2. Introduction

Protein A of S. aureus Cowan I has been shown to stimulate macrophage mediated phagocytosis. The present study was undertaken to un- derstand the mechanism involved in the enhancement of phagocytosis of peritoneal macrophages by protein A. The lucigenin and luminol-dependent chemiluminescence (CL) of rat peritoneal macrophages, after incubation with various concentrations of protein A, flow-cytometric studies using DCFH-DA as a fluorescent compound and phagocytosis of sheep red blood cells (SRBCs) by rat peritoneal macrophages were studied. A significant increase in lucigenin dependent CL due to formation of superoxide anions (O¥2) and in luminol dependent CL due to formation of hydrogen peroxide (H202) was observed in protein A treated macrophages. A significant increase in intracellular hydrogen peroxide (H202) was also observed along with an increase in phagocytosis of SRBCs by protein A treated macrophages. The present findings indicate that protein A helps to increase phagocytosis and triggers respiratory burst of macrophages. Thus, both increased phagocytic response and respiratory burst of macrophages in protein A treated animals may be con- tributing to the antitumor property of protein A reported earlier.

Key words: Protein A; Phagocytic response; Macrophages

Correspondence to." Anil Mishra, Inhalation/Immunobiology Division, Industrial Toxicology Research Centre, Post Box No. 80, M.G. Marg, Lucknow - 226001, India.

The generation of oxygen-derived free radicals represents one of the mechanisms by which phagocytes/macrophages generate high energy free radicals. These are responsible for the generation of 0¥2, "OH and H202 following stimulation by specific substances [1]. The phenomenon of chemiluminescence (CL) shown by high energy oxygen species can be utilized as a measure of the oxidative defense capacity of cell types [2,3].

Protein A is a cell surface glycoprotein (42 kDa) of Staphy lococcus aureus Cowan I. Its binding affinity for the Fc portion of mammalian im- munoglobulins [4] is widely exploited for separa- tion of immunoglobin G and/or its complexes. It is also known for immuno-potentiation [5,6] complement activation, generation of both immediate and delayed type of hypersensitivity [7] and antitumor properties [8-11]. Protein A is also able to abrogate the toxicity of various drugs and carci- nogenic chemicals [12-16]. We have shown earlier that protein A stimulates peritoneal macrophages in both normal and tumor bearing immunoregres- sor animals [17-18]. Phagocytosis is a phenomenon known to be associated with surface receptors of phagocytes i.e., Fc receptor or complement receptors. Ligation of the Fc receptor, signals both the phagocytosis and synthesis and release of re- active oxygen radicals, whereas ligation of complement receptors promotes only phagocytosis but not respiratory burst [19]. Protein A is known to bind with the Fc portion of IgG, which binds to macrophages through Fc receptors. So, it

289

is expected that protein A, after binding to IgG on macrophages, should be able to promote both phagocytosis and respiratory burst. An increased phagocytic response was earlier shown by us in protein A treated animals [15,17]. It was, there- fore, of interest to investigate if protein A can also induce the respiratory burst phenomenon. For this purpose, lucigenin and luminol amplified chemiluminescence of resting and stimulated peritoneal macrophages was studied.

3. Materials and Methods

3.1. Chemicals

5-Amino-2,3-dihydro- 1,4-phthalazinedione (luminol); phorbol,12-myristate,13-acetate (PMA); 9,9-bis-(N-methylacridinium nitrate (lucigenin) and 2,7-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma and Kodak (USA); Protein A from Pharmacia (Sweden); Superoxide dismutase from Boehringer (Germany). All other chemicals were obtained from SISCO (India).

3.2. Isolation of macrophages

Albino rats (120___ 10 g) were anesthesized and 10 ml of chilled PBS (pH 7.2) was injected into the peritoneal cavity of rats. The swollen belly was massaged vigorously for one minute and the cells were passed in a tube by a 16 G needle. The cells were washed three times with PBS supple- mented with Ca 2+, Mg 2+ and 0.1% glucose. Cells were counted and their viability was asses- sed by trypan blue exclusion method.

3.3. Phagocytosis

The phagocytic activity of peritoneal cells was carried out using opsonized sheep red blood cells (SRBC) according to the method of Koller et al. [20] with minor modifications. Briefly, a known population of activated and nonactivated peritoneal exudate cells ( l×106/ml) was carefully layered over a glass microslide, and incubated for 1 h at 37°C in a humidified atmosphere of 5% CO2. These microslides were kept in petri dishes and washed three times with 5 ml of PBS (pH 7.2) at room temperature. The non-adherent cells

290

were removed by aspirating the fluid with a pas- teur pipet. SRBCs were opsonized with anti- sheep RBC hemolysin (1:500) by incubating at 37°C for 1 h. After opsonization SRBCs were washed (2000 rpm for 15 min) with PBS and a 1% (v/v) SRBC suspension was made for phagocytosis.

The adherent cells on microslides were kept in petri dishes and overlayered with 1 ml of opsonized SRBC suspension and further incubated for 1 h at 37°C. The microslides were washed with PBS, stained with Giemsa stain and examined un- der oil immersion lens in the light microscope. The macrophages were considered positive for phagocytosis if two or more SRBCs were en- gulfed. The percentage of phagocytic cells was calculated.

3.4. Chemiluminescence of macrophages

The lucigenin dependent CL of macrophages was measured at 37°C in Biolumat LB 9505 (Berthold, FRG). The macrophages (0.5x l 0 6

cells) were resuspended in 0.5 ml of PBS buffer, pH 7.0, containing Ca 2+, Mg 2+, 0.1% glucose and 0.8 mM lucigenin or 2 mM luminol, and pre- incubated for 10 min in the Biolumat. At the on- set of the assay, protein A was added to the resting cells maintained in separate tubes at a concentration of 0.125, 0.25, 0.50, 1.0, 2.0 and 4.0 ~g in a total volume of 500 #1. The CL was measured continuously for 30 min at 37°C and expressed as counts integrated over 28 min for 0.5 x 10 6 cells. The CL of the same cells was measured again for 30 min after addition of positive stimuli, PMA (100 ng). A stock solution of PMA in dimethyl sulfoxide at a concentration of 1 mg/ml was pre- pared and was stored in small aliquots at -20°C. It was diluted with PBS (pH 7) immediately be- fore use. Superoxide dismutase (100 #g), catalase (200 pg), and mannitol (10 mM) were added to the incubation mixture of resting cells incubated with 2.0 ktg protein A (the maximal CL was observed at this dose).

3.5. Chemiluminescence of xanthine oxidase reaction

The CL of xanthine oxidase (10 pg) system was

T A B L E 1

Effect o f p ro te in A on the phagocy t ic ac t iv i ty of per i tonea l m a c r o p h a g e s aga ins t sheep red b lood cells in vitro.

Values are m e a n _ SE of six exper iments .

Un t rea t ed Trea ted m a c r o p h a g e s wi th Prote in A (in #g)

mac rophages

(Cont ro l ) 0.5 1.0 2.0 4.0 8.0

% Phagocytos i s 25 .18+ 1.41 34 .32+ 1.94 a 41 .83+ 1.65 b 4 3 . 6 + 2 . 1 8 b 4 6 . 2 5 + 2 . 1 6 b 27.61 + 1.03

~P <0 .05 ; bp <0.001,

used as a cell free control system to assess the effect of protein A on oxidative metabolism. It was measured in a total volume of 0.5 ml in 0.1 M po- tassium phosphatase buffer saturated with oxygen (pH 7.2) containing 0.1 mM xanthine and 0.8 mM lucigenin. Various concentrations of protein A were added in the reaction mixture. The CL was measured with a Biolumat (Berthold, FRG) and estimated as counts/10 s integrals after 1 rain reaction.

3.6. Determination of DFC-fluorescence of peritoneal macrophages by flow cytometry

The intracellular H202 was determined by flow cytometry using intraceUular oxidation of D C H F to the highly fluorescent DCF according to Bass et al. [21]. Macrophages (1 x 106) were preincuba- ted in 1 ml of Ca 2+ and Mg 2+ free PBS buffer containing 10 #M D C F H - D A in a horizontal-agi- tation water bath at 37°C. After preincubation, protein A (0.5, 1.0, 2.0 and 4.0 #g) was added. The resting and stimulated cells were incubated simultaneously. After 15 and 30 minutes of incubation, 1.0 ml of aliquots were taken for flow cytometric analysis ((ICP)l l , Phywe, Gottingen, FRG).

4. Results

4.1. Effect of protein A on phagocytosis of SRBCs by peritoneal macrophages

Phagocytosis of SRBCs was significantly increased ( P = 0.001) in the protein A treated group as compared to the untreated group. The effect was dose dependent with 0.5, 1, 2 and 4/~g dose

causing about 36, 65, 76 and 85% enhancement, respectively. However, at a higher dose (8 pg) further enhancement was not observed, only a mar- ginal increase was found as compared to control (Table 1).

4.2. Effect of protein A on luminol and lucigen& dependent CL

The effect of protein A on resting and stimulated peritoneal rat macrophages is shown in Figs. 1-4. Protein A at a dose between 0.125 /~g and 4.0 #g, resulted in three- to four-fold increase in lucigenin dependent CL (Figs. 1 and 2) and five- fold increase in luminol dependent CL (Figs. 3 and 4) of resting and PMA stimulated macropha-

400,

3 ~

~ $ 0 0

~ lSO

150" E 1001

S O

2

Control (100 % ) i o.r,x 10 Counts/rain. (Meon of six experfments)

t- i! !::. - . " , .

* . ' " . - . . .. .: ...

i .° .- . . . . ' . . .

, .i .* "°*

: :: i:: i. ? 0 0.25 0.5 1.0 2.0 /..0

Proteln'A' Cone. (Fig)

Fig. 1. Lucigenin dependent C L in res t ing cells.

291

/.Off

350'

~ 3 0 0

Z¢~ 2 5 0 '

200 ¥

150. 4:

~ 1 0 0

$ so

6 Control (100%)-3.2x10 Counts/ram. (Meon of six experiments)

0 025

i l i:!

!:t !:! • :1 i i ~

: . : :i 0.5 1.0 2.0

. ' ! .i

/..0 P Protein A Conc.[/ug)

F i g . 2 . Lucigenin dependent CL in PMA stimulated cells.

ges. At higher doses of protein A, there was no further increase in CL. Macrophages, stimulated with PMA exhibited a higher degree of CL in the

292

/.SO-

/.00-

350-

~ $ O 0 - w

2S0-

200"

"8 1S0-

100- $ ¢ J

so-

3 ControH 100%)~ 0.8x10 Counts/rain• (Meon of six experiments)

0 0.2S

~ J l J f J

f J f J

f J

. ~ f J

F ~ . ~ J f J

f J ~ J

! J f J

/ J

fz ' O.S 1.0 2.0

Protein ~,' Conc.(pg)

~ J f J

r J ~ f j

f J ~ j

~ J f j

f J f ~ ~ J

~ J

~ J

~ J

f J ~ J r J f J

F i g . 3 . Luminol dependent CL in resting cells•

5 Control (lO0%h~l..SxtO Co¢lnts/m,n 500 (Meon ot six explriments)

/.50 ~ _

~00 ~

.~ 350' ~ ,-~

/ F j

f J ¢: /

,00 . .

r J

0 0.25 0.5 1•0 2.0 /..0 Protein 'A' Conc.(/ug }

F i g . 4 . Luminol dependent CL in PMA stimulated cells.

presence of protein A concentrations up to 2.0 pg than the stimulated control (without protein A) group. In the absence of cells, however, protein A did not increase CL of lucigenin alone (data not shown)• The viability of resting and stimulated cells after incubation with protein A was near- ly 100% as measured by trypan blue dye exclusion test.

The CL of stimulated macrophages measured in the presence of a) lucigenin is due to the reaction of O~-2 with lucigenin [21-23] and b) luminol is due to the H202 myeloperoxidase reaction [24-25]. A control experiment using protein A and the CL of lucigenin with 02 produced by xanthine oxidase was performed according to Totter et al. [26]. Protein A neither changed the enzymatic activity nor the CL produced by xanthine oxidase using xanthine as substrate. The superoxide mediated CL of xanthine oxidase was almost un- changed in the presence of 1, 2 and 4/~g of protein A i.e., only 110, 126 and 132%, respectively. Since these changes are not of the magnitude seen in protein A treated cells, it indicates that protein A is not acting simply by interfering with the reaction of lucigenin and superoxide anion. The in- creasing effect of protein A on the lucigenin and

luminol dependent CL appears to be due to the direct effect of protein A on the macrophages.

To determine whether the effect of protein A on the CL of macrophages is specific or other proteins are also able to produce similar results, experiments were performed with BSA. CL of resting cells changes in the presence of 1 and 2 ~tg of BSA by a factor of 1.36 and 1.61, respectively, compared to resting control cells. CL of PMA stimulated cells changes in the presence of 1 and 2 /~g BSA by a factor of 1.14 and 1.63, respectively. This indicates that the significant elevation in CL of macrophages in the presence of protein A may be a specific property of protein A.

To elucidate as to which oxygen species is involved in protein A dependent CL generation, enzymatic and chemical scavengers of oxygen metabolites, including superoxide dismutase (SOD), catalase and mannitol [27] were added to protein A treated cells. Enzymatic and chemical scavengers inhibited the CL of macrophages in the presence of protein A (Fig. 5). Superoxide dismutase inhibits more than 90% of CL of stimulated control cells, as well as the CL of cells stimulated in the presence of protein A. The other oxygen radical scavengers such as catalase and mannitol have 30% and 20% reduction ability, respectively, of CL of protein A treated macrophages.

¢00"

+ 11~'

~ 300"

~lO0" .=

I "

4 Control(100%)10.Txl0 Count~/mln (Moon of tour expriment=)

CoI|I

~"==:':'1 toni ,i. so D

~ ¢oUak~Cetalose

I ¢ell--onintol

] Celrd.Protein A

[ ] Ce~l+Protein A+SOO

] Cells+Protein/U-Cototalo

[ ] '~ Celts+ Protein Ad-Monintol

iii +i! 2/4tl Protein 'A' ¢on¢.

Fig. 5. Lucigenin dependent CL with free radical inhibitors.

2 7 5 . Control=tO0%=_29.27+/. . .9 r e l o t i v e

2 2 5 -

17S"

c I

u

7 s o o

"~ 25

o o.s 1.o

f |uore scene e

• : : . i I . . ' . . ' 1 ~ ' . . '

• . . ' 1 i . . '

. . • ~ , ' ' .

;..;I ; : .

2.O ~.0 Protein A Concentrotion (Fg I

Fig. 6. Protein A and DCF fluorescence of resting macrophages.

4.3. Fluorescence of resting and stimulated peritoneal macrophages with flow cytometry

Normal unstimulated macrophages, incubated with 5 /~M DCFH-DA, showed a low level of fluorescence intensity, whereas macrophages pre- incubated with D CF H -D A when stimulated with different concentrations of protein A (i.e., 0.5, 1.0, 2.0 and 4.0/~g) showed about two- to three- fold increase in cellular fluorescence which correlated with an increased production of intracellular H202 (Figs. 6 and 7).

300' Confrol=lO0"/.- = 43.16_ 4. 5.6 re la t ive f luorescence

250"

200

u ~, 15o e~

-- 100

.<

0 0 0.5

Pro te in

-../ .•.•. .....

liiii• ...°. ...- . .+ u]

. , . .

• . . + . . . + . . .

• . . . . .

. . . . . . ..

" . . 7 • . . . . . .

i i : • • , . + . . . . .

. . . . . . . . , . . . . . . . • . . . . . , .

• . • , . . . .

2 . 0 t..O

Fig. 7. Protein A and DCF fluorescence of macrophages.

A Concln l ro l ion

PMA stimulated

293

5. Discussion

Macrophages and polymorphonuclear cells play an essential role in host defense. A variety of microbial and tumoricidal systems are present in phagocytes, some dependent on oxygen and oth- ers effective in its absence [28]. Chemilumines- cence procedure has increasingly been used to measure phagocytic function. An oxidative response occurs in phagocytic cells that are actively phagocytizing particles resulting in the emission of light called chemiluminescence [29]. The oxidative burst is closely linked to bactericidal and tumoricidal activity of phagocytic cells [30]. Our results suggest that rat peritoneal macrophages are stimulated by protein A to show an increased phagocytosis along with an increased level of luminol and lucigenin dependent CL. Protein A induced increase in CL of resting and stimulated macrophages was dose dependent (linearity was seen from 0.5 to 4/~g). However, at a higher concentration of protein A, the linearity was not maintained which may be related to large dose inhibition phenomenon as observed in various bio- logical reactions.

In the previous reports [11,12] we have shown that protein A could increase both the number and function of macrophages in both normal and tumor regressor animals. Prasad et al. [17] observed an increase in phagocytosis of opsonised SRBCs by macrophages obtained from protein A treated Swiss mice. This suggests that protein A not only stimulates the macrophages but also po- tentiates its other functional properties. Produc- tion of toxic oxygen metabolites by phagocytes measured by CL has been shown to be linearly related to the killing of bacterial species. It has also been reported that peak CL response of macrophages directly correlated with the percentage of killed Candida cells [31]. Our results of phagocytosis of opsonized SRBCs correlates very well with the above said finding of Ballart et al. [31]. Even though protein A enhances both phagocytosis and oxidative burst there is no need to believe that there exists any correlation between these two phenomena. These observations may have re- levance in the increased tumoricidal properties induced by protein A [11,12].

The activity of phagocytic cells such as mono-

294

cytes or macrophages results in a burst of oxidative activity on appropriate stimulation. The resulting free oxygen radicals, merely 0 ; 2 and H202, take part in tumoricidal activity [32]. Pro- tein A, at a low concentration shows an activating effect on resting as well as stimulated macrophages in vitro. The inhibition of the activating effect of protein A on macrophages by superoxide dismutase, catalase and mannitol indicates that superoxide anions, (O;2), "OH and H202, are involved in protein A mediated stimulation of macrophages. This study for the first time shows that protein A while stimulating phagocytosis of macrophages, also activates the respiratory burst in macrophages. Oxygen free-radical generation could be considered as a sensitive indicator of protein A induced stimulation of oxidative activity.

Acknow~dgemen~

We are extremely thankful to Dr. A.P. Joshi, Dr. S.V. Gangal (CFB, Delhi), Dr. R.K. Agarwal (INMAS, Delhi), Dr. K.C. Mahajan and Dr. D.S. Joshi (BARC, Bombay) for providing the labora- tory facilities. One of the authors (AM) is also thankful to CSIR, New Delhi for awarding the Pool Officership.

References

[1] Klebbanoff, S.J. (1980) Am. Intern. Med. 93, 480. [2] Allen, R.C., Stjernholm, R.L., Reed, M.A., Harper, R.B.,

Gupta, S., Sleele, R.A. and Waring, W.W. (1977) J. Infect. Dis. 136, 510.

[3] Wueff, K. (1983) in: Methods of Enzymatic Analysis. (H.B. Bergrneyer, Ed.) 3rd edn., pp. 340-368, Verlag Chemie, Welheim.

[4] Forsgren, A. and Sjoquist, J. (1967) J. Immunol. 99, 19. [5] Catalona, W.J., Ratliff, T.C. and McCool, R.E. (1981)

Nature 291, 77. [6] Romagnani, S., Amadon, A., Gudizi, M.G., Biagiotti, R.,

Maggi, E. and Ricci, M., (1978) Immunology 35, 471. [7] Forsgren, A., Ghetie, V., Lindmark, R. and Sjoquist, J.

(1983) in: Staphylococci and Staphylococcal Infections (C.S.F. Easmon and C. Adlam, Eds.) Vol. 2, pp. 429~,80, Academic Press, London.

[8] Ray, P.K., Cooper, D.R., Bassett, J.G. and Mark, R. (1979) Fed. Proc. 38, 44.

[9] Ray, P.K., Idiculla, A., Mark, R., Rhoads, J.E., Thomas, H., Bassett, J.G. and Cooper, D.R. (1981) Cancer 49, 1800.

[10] Ray, P.K. and Bandyopadhyay, S. (1983) lmmunol. Commun. 12, 453.

[11] Ray, P.K., Bandyopadhyay, S., Dohadwala, M., Cancha- napan, P. and Mobini, J. (1984) Cancer Immunol. Immunother. 18, 29.

[12] Ray, P.K., Dohadwala, M., Bandyopadhyay, S., Cancha- napan, P. and McLaughlin, D. (1985) Cancer Chem. Pharmacol. 14, 59.

[13] Dohadwala, M. and Ray, P.K. (1985) Cancer Chem. Pharmacol. 14, 135.

[14] Srivastava, S.P., Singh, K.P. Saxena, A.K., Seth, P.K. and Ray, P.K. (1987) Biochem. Pharmacol. 36, 4055.

[15] Singh, K.P., Saxena, A.K., Dwivedi, P.D., Zaidi, S.1.A. and Ray, P.K. (1987) Immunopharmacol. Immunotoxicol. 9, 281.

[16] Singh, K.P., Saxena, A.K., Zaidi, S.I.A., Dwivedi, P.D., Srivastava, S.P., Seth, P.K. and Ray, P.K. (1988) J. Appl. Toxi. 8, 407.

[17] Prasad, A.K., Singh, K.P., Saxena, A.K. and Ray, P.K. (1987) Immunotoxicol. lmmunopharmacol. 9, 541.

[18] Dwivedi, P.D., Verma, A.S., Mishra, A., Singh, K.P., Saxena, A.K., Dutta, K.K., Prasad, A.K., Mathur, N. and Ray, P.K. (1989) Toxicol. Lett. 49, 1.

[19] Wright, S.D. and Silverstein, S.C. (1983) J. Exp. Med. 158, 2016.

[20] Koller, K.D., Roan, J.G. and Brouner, S.A. (1980) J.

Environ. Pathol. Toxicol. 3,307. [21] Bass, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P.,

Seeds, M.C. and Thomas, M. (1983) J. Immunol. 130, 1910.

[22] Weiss, S.J. and Lobuglio, A.F. (1982) Lab. Invest. 47, 5. [23] Dahlgreen, C., Amiansson, H. and Magnusson, K.E.

(1955) Infect. Immun. 47, 326. [24] Slevens, P. and Hong. D. (1984) Microcham. J. 30, 480. [25] Kharazhi, A., Hoiby, M., Doring, G. and Valerius, N.H.

(1984) Infect. lmmun. 44, 587. [26] Dechatelet, L.R., Long, G.D., Shirley, P.S., Bass, D.A.,

Thomas, M.S., Henderson, P.W. and Cohen, M.S. (1982) J. Immunol. 129.

[27] Toller, J.R., Medina, V.J. and Scoseria, J.L. (1960) J. Biol. Chem. 235, 238.

[28] Gabig, T.G. and Babior, B.M. (1981) Ann. Rev. Med. 32, 313.

[29] Allen, R.C., Stjerholm, R.L., Reed, M.A., Harper, T.B., Gupta S., Steele, R.H. and Waring, W.W. (1977) J. Infect. Dis. 136, 510.

[30] Garbner, J.V., Mills, E.L., Gray, B.H. and Quie, P.G. (1977) J. Lab. Clin. Med. 89, 153.

[31] Ballart, I.J., Estevez, M.E., Diez, R.A. and Sen, L. (1987) J. Immunol. Methods 97, 263.

[32] Adams, D.O., Johnson, W.J., Fiorito, E. and Nathan, C.F. (1981) J. Immunol. 127, 1973.

295

Documents

Mechanism of enhanced phagocytic response in protein a treated rat macrophages