American Journal of Hematology 11:347-353 (1981)

Opsonic Activity of Myeloma Immuno- globulins (MM4g) Bruce D. Cheson Division of Hematology-Oncology, University of Utah College of Medicine, Salt Lake City

Patients with MM are at an increased risk for life-threatening bacterial infections, pri- marily by organisms that require opsonization for interaction with granulocytes. In the present study we used a neutrophil chemiluminescence (CL) assay of opsonization to ex- plore the opsonic activity of MM-Ig for zymosan particles. Particles were treated with serum lacking in Ig to explore the contribution of Ig to zymosan opsonization. This ser- um was found to have 69 f 2.6% (x * SEM) of the opsonic activity of normal serum (P < .001). Normal serum concentrations of normal IgG, MM-lgC, and MM-IgA all lacked opsonic activity for zymosan. However, when particles were treated with Nl-IgG, MM-IgG, or MM-IgA as well as complement, normal opsonic activity was generated. In- creasing the concentration of the Ig to stimulate MM serum did not inhibit this opsonic activity. Thus both MM-IgG and MM-IgA can function as normal, nonspecific opsonins.

Key words: multiple myeloma, opsonization, immunoglobulins

INTRODUCTION

Although patients with MM are at an increased risk for life-threatening bacteri- al infections, the precise etiology for this remains unclear. The majority of pa- tients with MM have decreased serum concentrations of naturally occurring anti- bodies and poor antibody response to microbial antigens [ 1-41. Nevertheless, the most frequent pathogens in these patients are those that require factors of the complement system for interaction with granulocytes [5 , 61. Although a variety of bactericidal defects in MM have previously been described, a recent report has demonstrated a deficiency in opsonic activity in MM serum [7]. The most impor- tant serum factors with opsonic activity are immunoglobulins (Ig), particularly IgG, and components of the complement system, particularly C3b [6 , 81. The present study was designed to evaluate the opsonic activity of MM-IgG and MM- IgA in an attempt to characterize further the opsonic abnormality in MM serum.

Received February 26, 1981; accepted June 25, 1981.

Address reprint requests to Bruce D. Cheson, MD, Division of Hematology-Oncology, University of Utah Medical Center, Salt Lake City, Ut. 84132.

0361/8609/81/1104-0347!$02.50 0 1981 Alan R. Liss, Inc.

348 Cheson

MATERIALS AND METHODS

Materials

anced Salt Solution (HBSS) from GIBCO (Grand Island, New York), Macrodex (6% solution of dextran, mol wt 70,000), and Sephacryl S-200 from Pharmacia Fine Chemicals (Piscataway, New Jersey). DEAE Bio-Gel A was obtained from Bio-Rad (Richmond, California).

Zymosan A was obtained from Sigma Chemical Co. (St. Louis), Hanks Bal-

Preparation of Granulocytes Human granulocytes were collected from healthy volunteers as described

previously [9]. After isolation the cells were suspended in HBSS at a concentration of lo7 cells/ml, of which approximately 90% were granulocytes.

Serum Serum was obtained by venipuncture from normal volunteers and patients

with hypogammaglobulinemia (HGS) and stored at - 70°C until use. All samples had normal concentrations of C3 and C4 as determined by radial immunodiffu- sion and normal CH,,. The IgG concentration in HGS was markedly reduced with a range of 102-233 mg/dl. IgA and IgM concentrations in HGS were decreased as well.

Preparation of Igs Normal human IgG was obtained from Miles Laboratories (Elkhart, In-

diana), suspended in HBSS, and further purified over DEAE Bio-Gel A and Seph- acryl S-200 to remove aggregates. Monoclonal MM-IgG and IgA were similarly purified from the serum of three patients with IgG-MM and a single patient with IgA-MM. The clinical characteristics and opsonic defects of these patients have been reported previously (FJ, UB, MA, LT) [7].

Opsonization Zymosan particles were opsonized as described previously [7]. Ten mil-

ligrams of zymosan was incubated with normal IgG, MM-IgG, or MM-IgA at 37°C for 30 min in a Dubnoff Metabolic Shaker (Lab-line Instruments, Melrose, Illinois) and washed three times in HBSS. It was then reincubated under the same conditions with a 1:8 dilution of HGS, washed three times in HBSS, and resus- pended to a final concentration of 25 mg/ml.

Opsonization was assayed by measuring the chemiluminescence (CL) of the granulocytes in response to the particles. To initiate the reaction, 1 ml of the cell suspension was added to 10 mg of opsonized zymosan in the absence of nonad- sorbed serum factors. The light generated was quantitated in an Iso-cap 300 liquid scintillation counter (Nuclear Chicago, Searle Industries, Des Plains, Illinois) in the off-coincidence mode as described by Allen and co-workers [lo], with the pre- viously described modifications [7].

Opsonization by Myeloma Immunoglobulins 349

RESULTS

Opsonization of Zymosan by Hypogammaglobulinemic Serum Ten milligrams of zymosan was incubated with either 123 dilute normal ser-

um or 1:8 HGS, and opsonization was measured as the CL in response to these treated particles over a 40-min period. Although HGS is lacking in Ig, it contains normal concentrations of complement components. Therefore, by comparing the activities of normal serum and HGS, it is possible to define the contribution of Ig to opsonic activity. In 22 replicate experiments HGS-treated particles elicited a peak CL of only 69 f 2.6% (mean f SEM) of controls (P c 0.001) (Fig. 1). The deficiency of opsonic activity in the samples with 233 mg/dl was the same as with samples containing 120 mg/dl(68.7% and 70.7% of normal, respectively). Ten additional replicate studies were performed with HCS which contained 260 mg/dl of IgG, and the activity in these sera was 60.1 Vo of normal. These observations suggested that the concentration of Ig was below that which contributes to opson- ic activity. Heat treatment of HGS (56"C, 30 min) eliminated all opsonic activity.

Opsonization of Zymosan by MM-lgs To evaluate the opsonic activity of MM-Igs alone, zymosan was incubated

with 900 ml/dl of pure normal IgG or MM-IgG or with 340 mg/dl of MM-IgA. Light generated in response to any of these particles was similar to that observed with unopsonized particles (Fig. 1). Thus neither the normal nor MM-Igs used in these studies exhibited opsonic activity for zymosan.

In other studies particles were treated first with normal or MM-IgG or MM- IgA and then incubated with HGS. Such Ig treatment resulted in opsonic activity equal to normal serum (Table I). When the order of opsonization was reversed such that particles were treated with HGS prior to IgG, opsonic activity was no different from that observed with HCS alone. This demonstrated that the IgG and IgA are capable of interacting with complement. To exclude the possibility that the opsonic activity of the IgA preparation was related to contamination with small amounts of IgG, we measured the concentrations of these proteins in sam- ples and found IgA of 1,120 mg/dl and IgG 120 mg/dl (approximately 10% IgG). When zymosan was incubated with 90 mg/dl IgG (10% of the original dilution) and then treated with HGS, opsonic activity was 67 f 2.5% of control. Thus, the activity of the IgA preparation could not be ascribed to IgG in the material.

To investigate the effect on zymosan opsonization of concentrations of Ig similar to those observed in MM serum, we repeated the above experiments using a concentration of MM-IgG fivefold greater than in normal serum or MM-IgA threefold greater than in normal serum. As shown in Table I, complete restoration of HGS opsonic activity was also achieved at these high Ig concentrations.

DISCUSSION

In MM tumor cells produce large amounts of a tumor-related Ig, but there have been few studies of the relative opsonic activity of these proteins. Lawrence

350 Cheson

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Fig. 1. Granulocyte CL in response to zymosan particles treated with normal serum (NS), hypo- gammaglobulinemic serum (HGS), normal 1gG (IgGNL), MM-IgG (IgGMM), MM-1gA (IgAMM), or un- opsonized particles (HBSS). HGS activity was 69 a 3% of NS, whereas the CL response to lg-treated particles was no different from unopsonized zymosan.

TABLE 1. Comparison of the Opsonic Activity of Normal IgC, MM-lgC, and MM-IgA Percent normal opsonic activity

Normal concentrationa High concentrationb HGS 69 f 3% - IgGNL + HGS 106 f 4% 121 f 7% IgGMM + HGS 111 f 3% 95 f 6%

algG = 900 mg/dl, IgA = 340 mg/dl. bIgG = 4,500 mg/dl, IgA = 1,020 mg/dl. Zymosan particles were opsonized in either a normal serum concentration of lg prior to exposure to HGS or to a fivefold greater concentration of IgG or a threefold greater concentration of IgA. Each protein was studied at least in triplicate. The values expressed for IgG-MM are the mean for the three patient proteins evaluated. The IgA is from a single patient. The values are expressed as mean f SEM.

IgAMM -+ HGS 95 f 2% 99 f 2 %

et a1 [ 1 11 and Spiegelberg et a1 [ 121 have previously demonstrated that neutrophils have receptors for MM-IgG as well as MM-IgA. Therefore, particles treated with these proteins should be capable of neutrophil activation. Indeed, MM-IgG and MM-IgA bound to nonphagocytosable surfaces stimulate neutrophil degranulation and activation of the cellular respiratory burst [13-151. Although latex beads coat- ed with various subclasses of MM-IgG undergo phagocytosis [ 161, the effective- ness of these proteins compared with normal human IgG has not been evaluated.

Numerous investigators have evaluated opsonization by secretory IgA [17-251, yet documentation of opsonic activity has been limited [21]. However, there have been few studies of the opsonic activity of human serum IgA. Hallgren

Opsonization by Myeloma Immunoglobulins 351

and Stalenheim [17] demonstrated that protein A-IgG complexes treated with nor- mal serum IgG or IgA were readily phagocytosed by neutrophils, whereas those opsonized with MM-IgA were not. The reasons for this discrepancy are unclear but may reflect competition among the various subtypes of IgG and IgA for the neutrophil antibody receptors [l 11.

In the present study we used a CL assay to compare the opsonic activity of either MM-IgG or MM-IgA with normal, polyclonal IgG. This assay has been shown to correlate well with other measures of specific and nonspecific opsoniza- tion [26-281. We demonstrate that MM-IgG and MM-IgA both interact with com- plement to function as effective opsonins for zymosan particles. IgA has been shown to be capable of activating the alternative complement pathway [29], a ne- cessary system for zymosan opsonization [30]. Thus the present findings are in keeping with the observations of Kaplan et a1 [21], who demonstrated that normal colostral IgA required complement to be an effective opsonin for human erythro- cytes.

It has been postulated that a deficiency of residual Ig in MM is responsible, in part, for the marked increase in bacterial infections observed in those patients. However, the present data demonstrate that, along with complement, MM-Igs can generate a potent, nonspecific opsonin system. Indeed, nonspecific opsonization may be all that is required for some strains of S . pneumoniae, the most common pathogen in MM [ 5 , 61. Many in vitro and in vivo defects have been described in MM, including abnormalities in chemotaxis, skin window migration, granulocyte adherence, bacterial killing, complement concentration, and opsonization [7, 31-35]. However, Ig plays only a small role, if any, in these phenomena.

Defective opsonization by MM serum could result from an abnormality in either MM-Ig or complement, which represent the most important factors of the heat-stable [8] and heat-labile ( 5 , 61 opsonin systems, respectively. Previous studies [7] have demonstrated that the defect in opsonization is correctable with the addition of intact normal serum. However, when heat-activated normal serum is used, a significant opsonic defect remains despite normal concentrations of complement components in the MM serum. The present studies demonstrate that MM-Ig can interact with normal complement to form an effective opsonin system. These observations, in conjunction with the previously noted defects, suggest the likelihood of a qualitative abnormality of the heat-labile opsonin system in MM serum. This hypothesis remains to be evaluated.

ACKNOWLEDGMENTS

cine Dean’s Research Advisory Committee, and by NIH grant 5R 10 CA13238.

assistance in M-protein purification.

This work was supported in part by the University of Utah College of Medi-

The author wishes to thank Dr. Allen Edmundson and Kathryn Ely for their

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