In vitro study of Fc-receptor function in autoimmune diseases

Immunology Letters, 9 (1985) 53 56 Elsevier

Imlet 541

IN VITRO STUDY OF Fc-RECEPTOR FUNCTION IN AUTOIMMUNE DISEASES

D. ROCCATELLO J, J. C. BENSA -~, R. COPPO j, C. ROLLINO I, M. D1AZ 2, G. MARTINA I and G. P1CCOLP i Nefroh~gia Medica dell'Universith di Torino. Divisioni Nefrologia e Dialisi. Ospedale S. Giovanni di Torino. hal.v;

2 Centre de l'ran.~hcshm Sanguine tit, Grenohh,. France

(Received 29 June 1984) (Modified version received II October 1984)

(Accepted 15 October 1984]

1. Summary

A simple test for studying in vitro Fc-receptor function of mononuclear phagocytes is described. Immune phagocytosis is analyzed as a dynamic phenomenon by using nearly pure suspensions of monocytes incubated for diverse times with autologous erythrocytes sensitized with highly purified IgG.

In a series of normal volunteers and patients with vasculitis a strict correlation has been found between this in vitro assay and the measure of splenic clearance of IgG-coated red blood cells (RBC), the classical approach for studying in vivo macrophage Fc- receptor function by using sodium chromate 51Cr as tag.

The use of this in vitro assay appears to be valua- ble mainly in cases requiring repeated measurements of Fc-receptor function for monitoring the course of disease or the effects of therapy.

2. Introduction

Methods for measuring the phagocytic activity of the monocyte-macrophage system in man have recently had an upsurge of interest after the demon- stration of an abnormal macrophage function in vasculitis [1] and other diseases [2, 3], in which immune complexes seem to play a pathogenetical role, and for the evidence that the detection of mononuclear phagocyte function monitorizes the effects of

Key words: monocytes phagocytosis Fc receptor function

therapy in autoimmune diseases [1,4]. However, conflicting data have been reported between in vivo [2, 5] and in vitro [6] studies.

In the present report we will describe a simple meth- od for kinetically measuring the Fc-receptor-mediated phagocytosis in vitro.

Results in healthy subjects and patients with vasculitis are compared to the ones obtained by measuring the clearance of lgG-sensitized 51Cr_labelled red blood ceils (RBC), the classical approach for studying phagocytic function in vivo.

3. Materials and Methods

3.1. B l o o d

Twenty ml of fresh venous blood were collected on ethylenediamine tetracetate ( E D T A ) l0 mM final concentration, from 10 healthy volunteers and 12 patients affected by systemic vasculitis with histolog- ical evidence of renal involvement. These patients included 3 cases of periarteritis nodosa (PN), 4 cases of sytemic lupus erythematosus (SLE), 3 cases of Henoch-Schtinlein nephritis (HSN), and 2 cases of cryoglobulinemia (C).

Seven patients (1 PAN, 3 SLE, 2 HSN and 1 C) were studied (before treatment with steroids or im- munosuppressive drugs) in the urinary activity phase (severe proteinuria and/or haematuria) with systemic signs (fever and/or arthralgia and/or cutaneous manifestations or gastrointestinal symptoms). The others were studied during a therapy-induced remis- sion.

0165-2478 / 85 $ 3.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division) 53

3.2. Preparation of monocytes Gradient media for cell separation. A stock solu-

tion was made up by 9 volumes of commercial Per- coll (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and 1 volume of 1.5 M phosphate-buffered saline (PBS). The pH of the stock solution was adjusted to 7.4 with 2 N HCI. By further dilution of the stock solution with 0.15 M PBS, solutions 1 (density 1.085 g/ml) and II (density 1.064 g/ml) were pre- pared as recommended by Pharmacia.

Cell separation. About 7 ml aliquots of blood di- luted 1 : 3 with 0.15 M NaCI were layered on 4 ml of solution 1 in siliconized glass tubes and centrifuged at 800Xg for 20 min at 22°C. The mononuclear cell layer, accumulating at the interface was collected and washed with 0.15 M NaCI by centrifugation at 150Xg for 15 min at 4°C.

The pellet was resuspended in 7.5 ml of cold 0.15 M NaCI and layered on 3 ml of Percoll solution II. Centrifugation at 800Xg for 60 rain at 4°C pro- duced an interface band which was found to contain mainly monocytes. The band was collected and washed in RPMI 1640 medium (Difco Laboratories) supplemented with 3% bovine serum albumin (BSA-RPMI) by centrifugation at 400Xg for 15 min at 4°C.

In preliminary studies this procedure resulted in cell suspensions that contained more than 1.8X 106 monocytes (with a final yield of 48_+9.5%) as judged by monoclonal antibodies (Ortho-mune Ortho Di- agnostic Sy. Raritan, N J) by using the immunofluo- rescence technique with FITC-conjugated specific antibody (goat to mouse lgG (Bionetics)). Suspen- sions included 78-87% OKMI, 8-15% OKT3 and 5-11% Leu 12 (Becton Dickinson) positive cells. These cells were >90% viable by ethidium bromide exclusion test for both patients and controls.

3.3. Assay for phagocytic activity of monocytes Autologous RBC sensitized with highly purified

anti-Rh(D) lgG (Centre de Transfusion Sanguine, Lyon) (34 #g/ml of packed RBC) were used as target cells. After three washes, three 0.5 ml aliquots of a 0.5% suspension of sensitized RBC in BSA-RPMI medium were mixed with three suspensions containing 0.3 X l06 monocytes and then incubated in a humid atmosphere at 37 °C (respectively

54

for i5, 30 and 45 min). One drop of each mixture, after the addition of a solution of acridine orange, was placed on a slide glass. One hundred consecu- tive cells and the total number of sensitized RBC ingested were counted. Our preliminary studies con- firmed Newman's observation [7], though on a heterologous system using differently sensitized erythrocytes, that Fc-mediated phagocytosis is a nearly linear phenomenon. Thus, the tangent to the angle (tgc0 subtended by the regression line obtained from the 3 considered points (at 15, 30, 45 min of incubation) has been assumed to be the phagocytosis index.

Detecting more than 3 points during the first 45 rain of incubation only makes the use of cells more wasteful without any substantial advantage.

3.4./n vivo clearance of lgG-sensitized RBC All patients and 5 normal subjects were also de-

tected for splenic macrophage immune clearance. The entire procedure was performed with sterile, py- rogen-free materials by using the Crome-Mollison protocol [8], recently developed by Frank et al. [2]. Briefly, 15 ml of citrated blood were collected from patients and normal subjects. After centrifugation at 750Xg for 5 min at room temperature and the remo- val of plasma and huffy coat, the erythrocytes were washed twice with 0.15 M NaC1. Two ml of packed cells were incubated for 25 min at 37°C with 100 #C of 51Cr. The 51Cr-labelled erythrocytes were then washed 3 times with 0.15 M NaCI and 1.5 ml of packed cells were incubated for 45 min at 37°C with an appropriate amount of purified anti-Rh(D)IgG and finally washed 3 times. One ml of packed cells, adjusted to a volume of 10 ml with 0.15 M NaCI, was reinjected. Blood samples taken after reinjection at 10, 20, 30, 45, 60, 90 min were counted in a gam- ma counter and the clearance half-time ( T,L, ) of the sensitized erythrocytes was calculated by relating the counts/min in sequential samples to the counts/min in the initial 10 min sample, after correction of each sample on the basis of haematocrit levels.

3.5. Statistical tests For statistical analysis the Mann-Whitney two

tailed U-test and the correlation coefficient were em- ployed.

4. Results

4.1. Assay for phago~Ttic activit), of monot:vtes The values of the phagocytosis index obtained

from normal controls ranged from 0.8 to 1.37 tga. Values lower than the 90% confidence limit of 0.83 tgc~ were considered pathological. Results in healthy subjects and patients are shown in Fig. 1.

Of interest, only the 7 patients showing a clinically active disease also had a defective Fc-receptor mediated phagocytosis.

The difference between values obtained from active and inactive patients was highly significant (P<0.004).

4.2. Correlation between in vivo and in vitro assays for studying Fc-receptor function

A significant inverse correlation was found between in vivo clearance of 5] Cr-labelled IgG-sensitized RBC (expression of macrophage Fc-receptor function) and in vitro Fc-receptor mediated phagocytosis (r=-0.68, P<0.01) (Fig. 2).

5. Discussion

There is always the need to make sure that what is observed in vitro has some counterpart in vivo and is not an artifact. Most described techniques for measuring phagocytosis of mononuclear phagocytes are limited by so many variables that some authors [9] conclude that in vitro phagocytosis cannot be re-

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controls ac t ive inact ive patients patients

Fig. I. Results of in vitro assay for phagocytic activity in healthy subjects and patients.

100

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• p <0,01

• :

o~, ~o 1:5 phagocytosis index { t go4 )

0 normal subjects

• pat i ents

Fig. 2. Correla t ion between in vivo clearance of sensitized erythrocytes by splenic macrophages and in vitro assay for phagocyt-

ic activity of monocytes . • Patients, © healthy subjects.

garded as a specific assay, but is useful only if used in conjunction with other confirmatory assays.

Traditionally, monocytes have been isolated by their propensity to adhere to glass or plastic surfaces and therefore most reported assays of phagocytosis detect this function on adherent monocytes [6, 10, 1 l]. Obviously monolayers may be over- crowded causing difficulty in distinguishing particles inside or outside of phagocytes. Moreover the number of adherent cells cannot be foreseen and con- stantly different ratios between phagocytes and indi- cator panicles are obtained.

We used nearly pure suspensions of monocytes which are more suitable for functional assays. In- deed an exact ratio between phagocytes and indica- tor particles - which is crucial for obtaining repro- ducible rates of phagocytosis and clearly distinguishable ingested particles are easily achieved. Moreover the kinetic analysis of phagocytosis also eludes the variables generally related to in vitro assays: slight differences in count, concentration in the medium, viability and purity of cells or in the avidity of monocytes, which might lead to different amounts of ingested RBC after a determined time of incubation even for identical rates of phagocytosis.

This procedure makes an appropriate use of cells, since assay volumes are small, and quantitation

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of phagocytosis does not require long hours of work at the microscope.

In comparison to methods that measure the up- take of radiolabelled particles in vitro, our technique avoids the use of dangerous and expensive materials with the added advantage of being free of artifactu- al changes in phagocyte-associated radiolabel or ex- cretion of radiolabel into the assay medium.

Of interest, the values obtained by using this technique correlate to the ones of macrophagic clearance of sensitized radiolabelled RBC in vivo. Therefore this assay may be suggested as a non-invasive meth- od for detecting mononuclear phagocyte immune function and for monitoring the effects of therapy, which needs frequent measurements, impossible with in vivo procedures because of the latency of elimina- tion of chromate and the risks of repeated exposure to radiation.

Acknowledgement

The authors wish to thank Miss Amalia Turletti for her excellent secretarial help.

References

[I] Lockwood, C. M., Worlledge, S., Path, F. R. C., Nicholas, A., Cotton, C. and Peters, D. K. (1979) N. Engl. J. Med. 300, 524 530.

[2] Frank, M. M., Hamburger, M. l., Lawley, T. J., Kimberly, R. P. and Plotz, P. H. (1979) N. Engl. J. Med. 300, 518 523.

[3] Hamburger, M. I., Gorevic, P. D., Lawley, T. J., Franklin, E. C. and Frank, M. M. 0979) Trans. Am. Assoc. Physi- cians92, 104 112.

[4] Hamburger, M. 1., Gerardi, E. N., Fields, -I'. F. and Ben- nett, R. S. (1981) Artif. Organs 5,264 268.

[5] Kimberly, R. P., Parris, T. M., lnman, R. D. and McDou- gal, J. S. (1983) Clin. Exp. lmmunol. 51,261 268.

[6] Kavai, M., Zsindely, A., Sonkoly, 1., Major, M., Demj/m, 1. and Szegedi, G. Y. (1983) Clin, Exp. Immunol. 51,255 260,

[7] Newman, S. J., Musson, R. A. and Henson, P. M. (1980) J. lmmunol. 125, 2236 2244.

[8] Crome, P. and Mollison, P. L. (1964) Br. J. Haematol. 10, 137 154.

[9] Shaw, D. R. and Griffin, F. M. (1981) in: Methods for Studying Mononuclear Phagocytes (D. O. Adams, P. J. Edelson and H. Koren, Eds.), pp. 511 528, Academic Press, New York.

[10] Davin, J. C., Foidart, J. B. and Mahieu, P. R. (1982) Proc. Eur. Dial. Transplant. Assoc. 19, 590 596.

[I I] Ooi, Y. M. and Ooi, B. S. (1983) Kidney Int. 23,851 854.

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In vitro study of Fc-receptor function in autoimmune diseases