Journal of Clinical Laboratory Analysis 4:95-98 (1 990)

Elevated Levels of Circulating Immune Complexes in Human Immunodeficiency Virus Infection

Mohan M. Reddy and Michael H. Grieco R.A. Cooke Institute of Allergy, St. Luke’s-Roosevelt Hospital Center, New York, New York

Circulating immune complexes were quan- titated in asymptomatic intravenous drug abusers (IVDA), male homosexuals, and patients with lymphadenopathy or acquired immunodeficiency syndrome (AIDS) by two different methods. Circulating immune coin- plexes were elevated in HIV-seronegative

Key words:

IVDAs and male homosexuals, probably reflecting viral infections such as cytomegal- ovirus (CMV). Circulating immune complexes were also elevated in HIV-seropositive IVDAs and male homosexuals and in patients with lymphadenopathy or AIDS, reflecting infec- tions such as CMV and HIV.

Acquired immunodeficiency syndrome (AIDS), immune complexes, C1 q ELISA, Raji cell replacement assay, human immunodeficiency virus (HIV)

INTRODUCTION

The acquired immunodeficiency syndrome (AIDS) is characterized by multiple immunologic abnormalities (1). Characteristic laboratory features include lymphopenia, decreased CD4 (helpedinducer) lymphocytes and increased CD8 (suppressor/cytotoxic) cells resulting in decreased CD4/CD8 ratio, cutaneous anergy, and impaired T cell function (2). In addition, increased levels of serum thyniosin-alpha, betaz-microglobulin, lysozyme, acid-labile human leukocyte interferon, soluble interleukin-2 receptor, and soluble CD8 levels have been reported in AIDS (3-7).

Immune complexes are produced by the host when anti- bodies bind antigens during the process of immune response. Immune complexes are rapidly phagocytosed and cleared from circulation. However, immune complexes accumu- late and cause significant pathological changes in auto- immune, infectious, and neoplastic diseases (X). We have been studying immunologic differences between human immunodeficiency virus (HIV)-seronegative and HIV- seropositive intravenous drug abusers (IVDAs) and male homosexuals. The HIV-seronegative IVDAs and male homo- sexuals usually have elevated levels of soluble interleukin-2 receptor, soluble CD8, tumor necrosis factor, and betaz- microglobulin, probably reflecting infections such as cyto- megalovirus (CMV) (6,7,9). Since elevated levels of immune complexes have been reported in AIDS, and since auto- immune phenomenon has been suggested in HIV infection (10-1 3), we quantitated immune complexes in HIV-sero- negative and HIV-seropositive IVDAs and male homosexuals and in patients with lymphadenopathy or AIDS by two different methods.

0 1990 Wiley-Liss, Inc.

MATERIALS AND METHODS

Subjects

The patients included 59 HIV-seronegative and 44 HIV- seropositve IVDAs, 37 HIV-seronegative and 38 HIV- seropositive asytmptomatic male homosexuals, 69 patients with uncomplicated generalized lymphadenopathy, and 87 patients meeting the Centers for Disease Control surveillance definition of AIDS. Of these patients, 4.5 had Pneumocystis carinii pneumonia, 27 had Kaposi’s sarcoma, and 1.5 had both P. carinii pneumonia and Kaposi’s sarcoma. Thirty-three healthy HIV-seronegative control subjects were also included in the study.

Methods

Sera obtained from the subjects were stored at - 70°C until used. Circulating immune complexes were quantitated using two different methods.

C1 q enzyme immunoassay

Circulating immune complexes were quantitated by using a CIC enzyme immunoassay (Cytotech, San Diego, CA). Stan- dards (0, 1, 16, and 37 pgEq/ml) and serum samples were added to the C lq-coated microtiter plate wells and incubated. After washing, horseradish peroxidase-conjugated goat anti- human IgG was added and incubated. An enzyme substrate was added after washing. After stopping the color development, the

Received May 17, 1989; accepted June 28, 1989.

Address reprint requests to Mohan M. Reddy, Ph.D., R.A. Cooke Institute of Allergy, St. Luke’s-Roosevelt Hospital Center, New York, NY 10019.

96 Reddy and Grieco

absorbance was measured at 405-nm wavelength. The con- centration of immune complexes in the patient samples was determined by using a calculator and deriving the best fit line employing the method of least squares (linear regression curve) and expressed as micrograms of heat-aggregated human gamma globulin equivalents per milliliter (kgEq/ml). If the measured concentration of immune complexes in a serum sample was greater that the concentration of the highest standard, the serum sample was further diluted and repeated. The coefficient of variation obtained in our laboratory was 1 1 %.

Raji-cell replacement enzyme immunoassay

Circulating immune complexes were also measured using a CIC Raji-cell replacement enzyme immunoassay (Cytotech, San Diego, CA). Raji cells are derived from the B lympho- cyte culture cell line and bear CR2 complement receptors that bind the fragments of activated C3. The Raji-cell assay is based on the ability of the Raji-cell CR2 receptors to bind immune complexes containing C3 fragments. The Cytotech Raji-cell replacement immunoassay also measures circulat- ing immune complexes containing C3 fragments by using an immobilized monoclonal antibody in a manner analogous to the Raji-cell CR2 binding reaction. Standards (0, 3, 22, and 58 kgEq/ml) and serum specimens were added to microtiter plate wells coated with monoclonal antibody to human C3 fragments and incubated. After the incubation, a wash cycle removed the unbound serum proteins. A horseradish peroxidase-conjugated mouse antihuman IgG was added, using a procedure similar to the C l q enzyme immunoassay described above. The coefficient of variation obtained in our laboratory was 13%.

R ES U LTS The level of circulating immune complexes in healthy

controls was 3.8 ? 0.8 (mean ? SE) and 1.1 5 0.3 kgEq/ml compared with 8.4 ? 0.7 and 2.6 ? 0.5 pgEq/ml in HIV- seronegative IVDAs by C l q enzyme immunoassay and Raji- cell replacement enzyme immunoassay, respectively (Table 1). The difference is statistically significant. By contrast, the HIV-seronegative male homosexuals had a significantly ele- vated level of immune complexes, 1 1 .O 2 0.9 KgEqiml, com- pared with the control level of 3.8 ? 0.8 by C l q enzyme immunoassay and a significantly decreased level, 0.3 ? 0.1 pgEq/ml, compared with the control value of 1 . 1 k 0.3 by Raji-cell replacement enzyme immunoassay. The HIV- seropositive asymptomatic IVDAs and male homosexuals and the patients with lymphadenopathy or AIDS had significantly elevated levels of circulating immune complexes detected by C I q enzyme immunoassay and also by Raji-cell replacement enzyme immunoassay. The relative distribution of individ- ual values is depicted in Figure 1; the range of values was broad in these groups. There was no documentable relation- ship between the serum immune complex levels in the AIDS

patients and the presence of infections such as P. curinii, CMV, Epstein-Barr virus (EBV), and/or Kaposi’s sarcoma.

We examined several correlations using results obtained from asymptomatic IVDAs and male homosexuals and patients with lymphadenopathy or AIDS. The immune complexes obtained by C 1 q enzyme immunoassay and Raji-cell replace- ment assay correlated well (r = 0.25; p < 0.01). Immune complexes obtained by Clq enzyme immunoassay and Raji- cell replacement assay also correlated well with soluble CD8 levels (r = 0.18, p < 0.01; r = 0.34, p < 0.001). There was no correlation between immune complexes and betaz- microglobulin, soluble interleukin-2 receptor, and neop- terin levels.

DISCUSSION

Several immune parameters have been reported as abnor- mal in patients with AIDS, including the proportion and abso- lute levels of phenotypic markers for CD4 (T helper/inducer) and CD8 (T suppressoricytotoxic) lymphocytes, with a decrease of the CD4/CD8 ratio and increased levels of serum thymosin-alpha, serum and urine B2-microglobulin, serum lysozyme, circulating acid-labile human leukocyte interferon, soluble interleukin-2 receptors, and soluble CD8 (1-7). The results of the present study demonstrate that levels of circu- lating immune complexes are also elevated in HIV-seropositive asymptomatic IVDAs and in patients with lymphadenopathy or AIDS.

Our results showed that circulating levels of immune com- plexes increased significantly in HIV-seropositive asymptom- atic IVDAs and HIV-seropositive male homosexuals compared with healthy controls. However, HIV-seronegative IVDAs also had significantly elevated circulating immune complexes mea- sured by both methods compared with normal controls, prob- ably reflecting infections such as CMV, EBV, and hepatitis B. On the other hand, HIV-seronegative asymptomatic male homosexuals had increased levels of circulating immune com- plexes as measured by C l q enzyme immunoassay and decreased circulating immune complexes as measured by the Raji-cell replacement assay. The reason for this discrepancy is not clear, although different immune complex assays detect different components of the immune complexes (8).

Autoimmune phenomena have been considered in AIDS, since hyperglobulinemia and polyclonal B cell activation are some of the characteristic laboratory features of AIDS (10,l l) . Immune complexes and T cell antibodies have been observed in the sera of patients with AIDS (12- 16). Although immune complexes have been found in many HIV-infected patients, there is no evidence of significant dysfunction of the immune system leading to clinical autoimmunity (10). On the other hand, it has been proposed that early HIV infection parallels graft-versus-host reaction, in which B cell hyperactivity, auto- antibody production, selective T cell dysfunction, and loss of CD4 T cell function without loss of CD4 T cells occur

Immune Complexes and HIV Infection 97

TABLE 1. Circulating Immune Complexes as Obtained by Two Methods in IVDAs, Male Homosexuals, and Patients With Lymphadenopathy or AIDS

Circulating immune complexes OLgEqimU

Raji-cell Number of HIV C I q enzyme replacement

Groups subjects antibody immunoassay assay

Normal controls 33 Negative 3.8 2 0.8 Asymptomatic IVDAs 59 Negative 8.4 t 0.7 2.6 5 0.5

Asymptomatic IVDAs 54 Pos I t ive

1 . 1 f 0.3

p value* < 0.0005 < 0.01

p value* < 0.0005 < 0.000s

p value* < 0.0005 < 0.02s

16.2 t 2.0 8.7 _t 1.0

p valuet < 0.0005 < 0,0005 0.3 _t 0.1

Asymptomatic male homosexuals 38 Positive 14.5 t 1.3 4.6 4 1 . 1 p value* < 0.000s < 0.005 p value$ < 0.025 < 0.0005

Asymptomatic male homosexuals 37 Negative I 1 .o t 0.9

People with lymphadenopathy 69 Positive 9.7 t 1.0 7.3 2 0.8 p value* < 0.0005 < 0.0005

2.5 2 0.5 People with AlDS 87 Positive 8.3 2 0.6 p value* < 0.000s < 0.05

Values are means _t SE *The p value compared with normal controls according to the student f test. tThe p value compared with HIV-seronegative IVDAs. $The p value compared with HIV-seronegative male homosexuals.

In W x W 2

n a "

38

28

26

z4t . 22 r

H I V - V E IVDA HOMOSEXUALS I VDA HOMOSEXUALS SYNDROME H I V t V E H I V - V E H I V - V E n i v t ~ ~ H I V t YE H I V t VE

Fig. 1. Levels of immune complexes in serum of asymptomatic intrave- nous drug abusers and male homosexuals and patients with lymphadenopa-

thy or AIDS, obtained by Clq enzyme immunoassay (solid circles) and Raji-cell replacement enzyme immunoassay (open circles).

98 Reddy and Grieco

( 1 I ) . HIV, as well as CMV and EBV, are polyclonal B cell activators ( 1 1). The risk groups of HIV-seronegative IVDAs and male homosexuals, although negative for HIV, had ele- vated levels of circulating immune complexes. The cause of this elevation may be the variety of infections that these indi- viduals contract, such as infections with CMV and EBV. The chronic exposure and reinfection could lead to a persistently elevated level of circulating immune complexes.

ACKNOWLEDGMENTS

We thank Mary Reilingh, Mark Sydlo, and Gary Jelich for excellent technical assistance.

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