Inflammation, Vot. 4~ No, 1, 1980

D R U G A N D R H E U M A T O I D FACTOR EFFECTS ON THE UPTAKE OF

I M M U N O G L O B U L I N G AGGREGATES BY N E U T R O P H I L M O N O L A Y E R S

ROBERT TURNER, GRACE COUNTS, HERBERT MASHBURN, WILLIAM TREADWAY, and

LAWRENCE DECHATELET

Departments of Medicine and Biochemistry, The Bowman Gray School o f Medicine of Wake Forest University,

Winston-Salem, North Carolina 27103

Abstract--This study examines aggregate-cell interactions in a newly applied neutrophil monolaycr system, as an in vitro model of rheumatoid inflammation. insoluble and soluble immunoglobulin G (lgG) aggregates were combined with rheumatoid factor (RF) to produce IgG-RF complexes. The presence of RF did not significantly change the uptake of the insoluble aggregates by neutrophils as measured in the monolayer system. Neutrophits exposed to these aggregates showed significantly (P < 0.05) greater uptake than those exposed to soluble aggregates, and the presence or absence of serum did not change these results. Increasing concentrations of radiolabeled aggregates to 1.5 mg/ml and cells to 5 • 106 neutrophils/ml increased cell-associated radioactivity. Addition of eyto- chalasin B to 5 mg/ml progressively depressed cell-associated radioactivity. Gold, but not aspirin, in therapeutic concentrations seemed to suppress aggregate uptake. This system offers a method for quantitatively assaying aggregate uptake which may be an important component of the rheumatoid inflammatory process~

INTRODUCTION

IgG aggregates and IgG-RF complexes have been found in the serum (1), synovial fluid (2), and synovial fluid neutrophils (3) of patients with rheu- matoid arthritis. Previous studies have shown that insoluble IgG aggregates are phagocytized by normal human neutrophils (4) and cause functional changes in these cells (5). Quantitative determination of aggregate uptake by neutrophils was hampered in these studies by difficulties in separating cells and aggregates (5). Neutrophils have been identified adjacent to vascu- lar endothelium in early rheumatoid arthritis (6) and in vivo "pavementing" of these cells on blood vessel walls may be an early important part of most

55 0360-3997/80/0300-0055503.00:0 �9 1980 Plenum Publishing Corporation

56 Turner et al,

i n f l a m m a t o r y p r o c e s s e s (7). A n e u t r o p h i l m o n o l a y e r s y s t e m was , t h e r e -

fo re , u t i l i z ed in t h e s e s t ud i e s to q u a n t i t a t e a g g r e g a t e u p t a k e in t h e p r e s e n c e

a n d a b s e n c e o f h u m o r a l f a c t o r s a n d d r u g s u s e d i n t h e t r e a t m e n t o f r h e u -

m a t o i d a r t h r i t i s .

M A T E R I A L S A N D M E T H O D S

Human Subjects. After obtaining approval from the Institutional Human Experimenta- tion Committee, informed consent was obtained from normal subjects and patients with rheu- matoid arthritis whose sera and neutrophils were utilized in these studies. All studies were carried out according to the Declaration of Helsinki.

Insoluble Aggregate and Complex Preparation. Insoluble aggregates were prepared as previously described (4). A 2 g/100 ml solution of IgG (Human Gamma Globulins, Fraction If, U.S. Biochemical Corporation, Cleveland, Ohio) in 0.9% NaCI (pH 7.4) was allowed to incu- bate overnight at 25 ~ C. The solution was then centrifuged at 10,000g for 20 rain to remove any remaining insoluble material. For use in studies requiring radioacfively labeled IgG, approxi- mately 22 uCi of 1.3 mM ~CrCI3 (New England Nuclear, Boston, Massachusetts, specific ac- tivity approximately 375 Ci/g Cr) was added to each milliliter of the solution and allowed to incubate for 1 hr at 25~ This yielded approximately 1 • 106 epm/ml of the solution. The IgG was heat aggregated at 63 ~ C for 45 rain, cooled to room temperature, and centrifuged at 10,000g for 20 rain. The supernatant was discarded and the precipitate containing insoluble IgG aggre- gates was washed twice with 0.9% NaCI. These precipitates were resuspended to 2.4 mg/ml in the appropriate medium and used as insoluble IgG aggregates, or were resuspended to 6 mg/ml and combined 1:2:5 by volume with 0.9% NaCI and rehumatoid serum, incubated at 25~C for I hr, reprecipitated, and used as insoluble IgG-RF complexes at a concentration of 2.4 mg/ml, The protein concentrations of the insoluble IgG aggregates and insoluble IgG-RF com- plexes were measured using the Lowry technique (8). All aggregate and/or complex prepara- tions contained a mean of 1.2 X 104 cpm/mg of aggregate and/or complex protein.

Soluble Aggregate Preparation. Soluble aggregates were prepared as previously de- scribed (9). Briefly, a 1.5 g/100 ml solution of human IgG in 0.9% NaC1 (pH 7.4) was utilized and for labeling studies, approximately 22.6 #Ci of 5:CrCI~ was added to each milliliter of the solution. After incubating for 1 hr at 25~ the solution was passed over a Sephadex G-10 col- umn (1.2 cm • 30.0 cm) to remove the unbound chromium. Tubes containing peaks of radio- activity and protein were pooled and heated at 63 ~ C for 30 rain. Any insoluble precipitate formed during beating was removed by centrifugation at lO, O00g for 20 rain. The resulting opalescent supernatant was diluted with 0.9% NaC1 to 2.4 rag/ml of soluble IgG aggregates and contained a mean of 5.56 • 104 cpm/mg of aggregates when labeled.

Soluble Aggregate Precipitate Preparation. Soluble IgG aggregates at a concentration of 4.8 rag/ml were combined 1:2 : 5 by volume with 0.9% NaCI and rheumatoid serum, respec- tively. After incubating for 1 hr at 25~ these mixtures were centrifuged at 10,000g for 20 rain and the supernatants removed. The precipitates were washed once, resuspended to 2.4 mg/ml in the appropriate medium and utilized as precipitate IgG-RF complexes containing a mean of 1.2 • 104 cpm/mg of complex protein.

Monolayers. Normal human neutrophils were obtained in 80-90% purity as previously described (4, 9). The cells were suspended in Hanks' balanced salt solution (HBSS) to contain 5 • 10 ~ neutrophils/ml. Five or 2.5 mt of this suspension was placed in 60- or 35-ram petri dishes and the cells allowed to attach by incubation at 37~ for 45 min. The monolayers were then washed twice with HBSS to remove any unattached cells. Solutions containing various concen- trations of up to 20 t~g/ml of cytochalasin B (Aldrich Chemical, Ineorp., Milwaukee, Wisconsin),

Drug and RF Effects on Monolayers 57

up to 2.0 mg/ml of aspirin (acetylsalicylic acid; Sigma Chemical Co., St. Louis, Missouri) or up to 1.0 mg/ml of gold (gold sodium thiomalate; Merck, Sharp, & Dohme, West Point, Pennsyl- vania) all in HBSS were added to some of the monolayers and allowed to incubate at 37~ for 30 min. A doubly concentrated suspension of one of the 51CrCl3-1abeled IgG aggregates was added to all cultures to give a 2.4 rag/ml final aggregate or complex concentration. The neutro- phils and ag~egates or complexes were incubated at 37~ for 30 rain, after which the mono- layers were washed twice with HBSS to remove any loose material. The remaining monolayers were then assayed for radioactivity in a Nuclear Chicago gamma counter. Two-way analysis of variance was performed on all data and Tukey's multiple comparison test was employed in order to test the significance of all possible c<~mparisons of experimental groups.

R E S U L T S

As shown in Table 1, there were no significant differences in the ad- herence of the various aggregates to petri dishes alone. Aggregates showed

significantly (P < 0.05) greater adherence to monolayers threated with 10 p g / m l cytochalasin B than to petri dishes alone. Differing aggregates showed mild var ia t ions in b ind ing to these cells. Precipitate I g G - R F aggregates showed significantly (P < 0.05) less b ind ing to the cytochalasin B-treated cells (mean _+ S E M cpm = 2101 _+ 198), than soluble IgG, insoluble IgG, or insoluble I g G - R F aggregates (4t64 _+ 154, 3835 _+ 380, 4705 _+ 543, re- spectively). Unt rea ted neutrophi ls showed significantly (P < 0.05) more uptake of insoluble and precipitate aggregates than cytochalasin B-treated neutrophils . These untrea ted cells showed marked uptake of insoluble IgG, insoluble I g G - R F , and precipitate I g G - R F aggregates (12280 ___ 1523,

"9535 _+557, 10836 + 590) and significantly (P < 0.05) less uptake (5394 _+

Table 1. Monolayer Studies of Aggregate-Cell Interactions ~

Cytochalasin B-treated Aggregate Petri dishes alone cells Untreated cells

Soluble IgG 1185 ___ 95 4164 + 154 5,394 _+ 214 ~ n = 12

Insoluble IgG 1778 + 245 3835 + 380 12,280 _+ 1523 n = 12

Insoluble IgG-RF 1535 _ 14I 4705 + 543 9,535 + 557 n = 6

Precipitate IgG-RF 1098 _+ 44 210t _+ 198 b 10,836 +_ 590 n = 12

Probability P <~ 0.05 P < 0.05

~Data shown as mean _+ SEM cpm remaining after final washings. bSignificantly (P ~ 0.05) less than other complexes studied with cytochalasin B-treated cells. ~Significantly (P ~ 0.05) less than other complexes studied with untreated cells.

58 Turner et al.

]'able 2. Monolayer Studies of Serum Effects on Aggregate-Cell Interactions"

Soluble IgG aggregates (X cmp -+ S E M )

Insoluble IgG aggregates (X cpm + S E M )

Data calculated for (+) serum ( - ) serum (+) serum ( - ) serum

Cytochalasin B-treated cells 4514 + 302 3908 + 207 5,107 +_ 523 4,237 -e 433 Untreated cells 5386 _+ 605 5336 • 301 15,455 • 878 14,482 ___ 674 Untreated-eytochalasin B 872 + 463 1428 • 376 10,348 _ 959 10,244 _+ 543 Probability P < 0.05 P < 0.05

~ = 6 for all experiments.

214) of soluble IgG aggregates. These studies were repeated, utilizing solu- ble and insoluble IgG aggregates in the presence and absence of fresh serum. As shown in Table 2, soluble aggregates again were taken up less avidly than insoluble aggregates by both cytochalasin B and untreated cells. There were no differences in the uptake of either of these types of complexes in the presence or absence of fresh serum. Differences in uptake between un- treated and cytochalasin B-treated cells were compared as a possible mea- sure of phagocytosis and again were higher for insoluble than soluble IgG aggregates without demonstrable serum effects on the uptake data.

The effects of varying the amounts of insoluble IgG aggregates pre- sented to monolayers prepared from suspensions containing constant num- bers of neutrophils (5 • 106 neutrophils/ml) were studied as shown in Fig- ure 1. It is apparent that increasing aggregate concentrations from 0.01 to 1.5 mg/ml result in increasing amounts of cell associated radioactivity.

55

5O

45

~ 40

~k 30

2O

~ 1 I I I I I 0,5 1.0 1.5 2.0 2.5 3.0

Insoluble IgG Conc-mg/ml

Fig. 1. Effects of varying insoluble IgG aggregate concentrations on neutrophil monolayer aggregate uptake, n = 6, X 4- S E M shown for all concentrations studied.

Drug and RF Effects on Monolayers 59

55

5O

~, 40

35

�9 30

5 i I I I I I

5xlO 2 5xlO $ 5xlO 4 5xlO 5 5xlO c~ 5xlO 7

Neutrophil Conc- Cells Imt

Fig. 2. Effects of varying neutrophil concentrations on monolayer uptake of radioactive heat- aggregated immunoglobulin G. n = 6, X + S E M shown for all data.

These differences were significant at the 0.05 level. The effects of varying neutrophil monolayer concentrations in suspensions with constant (2.4 mg/ml) aggregate concentrations were then studied (Figure 2). As shown in the figure, uptake increases to 5 • 106 neutrophils/ml and there is no sig- nificant increase in uptake at higher concentrations to 5 • 107 neutrophils/ml. All subsequent studies utilized suspensions containing 2.4 mg/ml of ag- gregates and monolayers prepared from suspensions containing 5 • 106 neutrophils/ml. Effects of varying concentrations of cytochalasin B on the system were then studied (Figure 3). There is significant inhibition of up- take of the tagged aggregates to 5 #g/mt. There is no significant difference

55

5O

45

4c

:~ 35

2C

tC

1=6

I I l I ........ L_ 0 I.P5 2.5 5.0 LO.O I~.0

Cytocholasin B Conc-#glm[

Fig. 3. Effects of varying concentrations of eytochalasin B on neutrophil monolayer uptake of

immunoglobulin G aggregates. X + S E M shown for all data.

60 Turner et al.

i 2~ n~

IC

I I I I 0 0.002 0.02 0.2 2.0

Aspirin Conr

Fig. 4. Effect of aspirin on enutrophil mono]ayer uptake of heat-aggregated immunoglobulin G. n = 6, X 4- S E M shown for all data,

in cell-associated radioactivity between cells exposed to l0 #g/ml of cyto- chalasin B and those exposed to 5 or 15/zg/ml of this agent. The effects of aspirin on the system were then assayed (Figure 4). It is apparent from Fig- ure 4 that aspirin exhibits a slight but significant effect on aggregate uptake. Statistical significance is reached with this agent at the 2.0 mg/ml concen- tration. As shown in Figure 5, gold exhibits a somewhat greater but more variable effect on aggregate uptake with statistically significant suppression of uptake over untreated cells reached at the 1 mg/ml concentration.

? g 5

I I ,I I 0 0.001 0.01 O, I 1.0

Gold Sodium Thiomalete Conc-rng/ml

Fig, 5, Effect of gold on neutrophil monolayer uptake of ~mmunoglobulin G aggregates, n = 6, X �9 S E M shown for all data.

Drug and RF Effects on Monolayers 61

DISCUSSION

In this .study a monolayer system previously utilized for measuring rates of phagocytosis (11) was modified for use with neutrophils and radio- labeled soluble, insoluble, and precipitate IgG aggregates (4, 9, I0). With this system (Table 1, untreated cells column) all particulate aggregates are avidly taken up by neutr0phils to a significantly (P ~ 0.05) greater extent than soluble IgG aggregates studied under similar conditions. This is true whether the aggregates are insoluble to begin with or are formed by pre- cipitation and is also true whether or not they contain RF. Cytochatasin B has been shown to inhibit phagocytosis but not adherence of particles to phagocytes (12-14) and was utilized in our system in an attempt to disso- ciate the adherence and phagocytic phases of aggregate-neutrophil inter- action. Although the precipitate IgG-RF complexes seemed to bind less well to the neutrophils than the insoluble IgG aggregates or IgG-RF com- plexes (Table 1, cytochalasin B column), our studies do not differentiate between partial and complete inhibition of phagocytosis at this concentra- tion of cytochalasin B. Therefore, no firm conclusion can be drawn at present from these findings. Previous studies (4) have suggested that serum was necessary for phagocytosis of insoluble IgG aggregates. However, the mono- layer studies reported here (Table 2) clearly demonstrate that serum has no effect on uptake of these aggregates by untreated cells or on adherence of these aggregates to cytochalasin B-treated cells. The presence of serum in the system similarly did not affect the binding or uptake of soluble IgG aggregates.

The data shown in Figure 1 and Figure 2 establish the limits of this system, showing that uptake increases to 1.5 rag/ml aggregates and 5 X 10 ~ neutrophils/ml. The aggregate and cell concentrations used in subsequent studies utilized these maximal conditions. The cytochalasin B dose-response studies (Figure 3) showed significant changes to 5 #g/mE There were no significant differences between the uptake of cells exposed to 10/zg/ml of cytoehalasin B and the uptake of cells exposed to 5 gg/ml or 15/~g/ml of this agent. The 10 #g/ml dose used in the experiments shown in Table 1, therefore, seems to have been capable of producing maximum suppression of aggregate uptake in our system. Our concentrations were similar to those inhibiting phagoeytosis but not adherence in previous studies (12-14), and our dose-response curve for cytochalasin B is in keeping with that previously described by others (15). The effect of aspirin on phagocytosis has been variable in previous studies (16, 17). Our studies show (Figure 4) significant depression of this function at 2 mg/ml but an insignificant depression at lower (0.2 mg/ml) doses which are therapeutically attainable in vivo. All such results are difficult to interpret because, although aspirin may not be

62 Turner et al.

hydrolyzed prior to absorption from the gastrointestinal tract (18), it is quickly hydrolyzed after absorption (19). Our findings are, however, in keeping with in vivo (2) and subsequent in vitro (17) studies suggesting no significant effect of therapeutically attainable concentrations of this agent on phagocytosis.

Serum levels of gold in patients treated chronically with this agent may range between 0.00! and 0.01 mg/ml (21). A study suggesting inhibition of phagocytosis of starch granules by rabbit neutrophils has been performed with these concentrations of this agent (22). A "skin window" study (23) has suggested that gold treatment may suppress the in vivo phagocytic ac- tivity of rheumatoid inflammatory cells. Gold has been shown to be concen- trated.in the lysosomes of phagocytic ceils (24), and one study (25) has esti- mated that lysosomal granule concentrations range from 2 to 9 times the 1 mg/ml utilized as the highest concentration (Figure 5) for our studies with this agent. There is significant inhibition of aggregate uptake with this concentration of gold in our system, which used human neutrophils and IgG aggregates. Our studies would therefore reinforce the suggestion that suppression of phagocytosis may be an important mechanism of ac- tion of this agent in rheumatoid arthritis. Further studies are indicated to determine directly the serum and neutrophil levels of nonsteroidal anti- inflammatory agents and long-acting antirheumatic drugs so that more appropriate in vitro systems can be developed for testing these agents prior to their utilization in patients with rheumatoid arthritis.

Acknowledgments--This study was supported in part by a grant from the Easter Seal Research Foundation of the National Easter Seal Society for Crippled Children and Adults and by grant number A1-10732 from the National Institute of Allergy and Infectious Disease.

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Drug and RF Effects on Monolayers 63

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