Inhibition of Classical Complement Activation by Sera from HIV-1-Positive Patients

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 81, No. 2, November, pp. 114–121, 1996 Article No. 0166

Inhibition of Classical Complement Activation by Sera from HIV-1-Positive Patients V. BUREK1

AND

M. GERENCˆER

Department of Clinical Immunology, University Hospital of Infectious Diseases ‘‘Dr Fran Mihaljevic´,’’ University of Zagreb, Zagreb, Croatia

It was shown that gp120/160-coupled CD4/ T cells could be lysed by complement activation, but the target cell lysis was strongly inhibited by the majority of HIV-1-positive sera. Significantly more sera from HIV1-infected patients with CD4/ T cell count higher than 500 ml (N Å 38) as well as from patients with 200–500/ ml (N Å 32) showed strong inhibition of complement activation as compared to sera from those with less than 200 CD4/ T cells/ml (N Å 28) (P Å 0.0064 and 0.0012, respectively). Consequently, highly significant correlation between CDL inhibitory activity and CD4/ T cell count in HIV-1-infected patients was found by Spearman’s rank order analysis (R Å 0.399, P õ 0.001). CH50 titer and functional C1-inhibitor level were significantly lower in inhibitory as compared to the noninhibitory sera (P õ 0.001) and to controls (P õ 0.001). The C3 activation products–C3-circulating immune complexes were not increased in inhibitory sera (P Å 0.014) suggesting that inhibition of complement activation occurred at or before C3 activation level. C4d fragments and antigenic C1-INH concentration were significantly increased in both categories of HIV-1-positive sera, P õ 0.001. These findings indicate that persistent and massive stimulation of complement system with HIV-1 envelope glycoproteins during all stages of the disease induced impairment of classical pathway activation by an inhibitory factor in a majority of patients, which might contribute to the onset of opportunistic infections and AIDS. q 1996 Academic Press, Inc.

INTRODUCTION

For the optimal functioning of the immune system it is essential that upon the maintenance of the immune response and the clearance of the foreign antigen, the system returns to a state of relative quiescence until the next stimulus is introduced. However, the immune

system is chronically activated during HIV-1 infection as detected by spontaneous hyperactivation of B cells, spontaneous lymphocyte proliferation, increased cytokine production, autoimmune phenomena, etc. (1). Complement activation, circulating immune complexes (CICs), and altered complement components profile were also found in AIDS patients (2–7), suggesting that complement system is persistently activated. Massive binding of complement components C1q and C3 to HIV-1 membrane glycoprotein gp41 on free and cellbound virus has been documented in vitro (8–12). However, it is not clear whether complement plays any role in HIV-1 clearance in vivo and whether this long-lasting but not host-protecting activation of complement system by viral proteins may induce increased activation and/or overconsumption of complement regulatory proteins which then impairs complement activity and contributes to the onset of opportunistic infections and AIDS disease. We have confirmed the results from Su¨sal et al. (13) and showed that CD4/ T cells are susceptible to baby rabbit complement (BRC)-induced lysis upon binding of gp120/160 to the CD4 receptor, but we also found that the cell lysis could be inhibited by some HIV-1positive sera. Therefore, we have tested sera from 98 HIV-1-positive patients for their ability to inhibit complement activation caused by CD4-gp120/160 ligation. Inhibitory activity of each serum was also tested in a hemolytic assay when sheep red blood cells labeled with rabbit anti-sheep red blood cell stroma IgG (SRBC/EA7S) were used as targets and normal human serum was used as the source of complement (HC). Additionally, we have determined CH50 titer and C4d complement activation fragments in patients and control sera. The level of functional C1-inhibitor (C1-INH) and the serum concentration of C3-CICs were measured by an enzyme immunoassay (EIA), and antigenic C1-INH concentration by radial immune diffusion (RID) assay. MATERIALS AND METHODS

1

To whom correspondence should be addressed at Department of Clinical Immunology, University Hospital of Infectious Disease ‘‘Dr Fran Mihaljevic´,’’ Mirogojska 8, 10000 Zagreb, Croatia. Fax: /3851-4225-907.

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0090-1229/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Patient Sera HIV-1-positive sera from 98 patients were collected in our clinic during the period 1987–1994 and kept

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frozen at 0707C. HIV-1 seropositivity was detected by EIA (Abbott Laboratories) and confirmed by Western blot (Du Pont, Wilmington). CD4/ T-cell count was determined by immunofluorescence (Coulter) and calculated as absolute cell count/ml. The patients were separated in groups according to the CDC classification system (14) as follows: (i) CD4/ T-cell count higher than 500/ml; (ii) CD4/ T-cell count 200–500/ml; (iii) CD4/ Tcell count lower than 200/ml. Control Sera Normal human sera from HIV-1-negative individuals (N Å 12) and in some tests sera from patients with hepatitis B (N Å 4) or with Salmonella typhimurium infection (N Å 2) were used as controls. Target Cells Peripheral blood lymphocytes (PBL) were isolated from the whole blood obtained from healthy donors by density gradient centrifugation. They were stimulated with various mitogens or with 10 units/ml of recombinant interleukin-2 (Boehringer Mannheim, Germany) for 5 days in 10 ml RPMI 1640 medium (Imunoloski Zavod, Croatia), supplemented with 10% human male AB serum (Sigma). CD4/ T-cell enrichment was performed using magnetic beads (Dynal, Germany) by negative selection method after removal of adherent cells. The CD4/ T-cell purity was checked by FACS analysis (Facs Scan, Becton–Dickinson) using dual color CD3/ CD4 kit (Immunotech, France) and it was usually 75– 90%. Natural killer cell resistant CD4/ T-cell line CEM-NKR (15) was used as the positive control. The presence of gp120/160 molecules on the cell membrane was proved by immunofluorescence using FITC-labeled monoclonal antibody (Intracel Corp., U.S.A.). SRBC were obtained from whole sheep blood mixed with Alsever’s solution (1:1) and washed extensively in saline and gelatine veronal buffer (GVB2/; Sigma) prior to use in hemolytic assays. They were coupled with SRBC/EA7S according to the instructions from the manufacturer (Sigma). Complement Normal HC (Sigma) and nontoxic BRC (Serotec) were used as the source of homologous and heterologous complement, respectively. In Chromium51 (Cr51) release assay BRC was used at the dilution 1:25 and in SRBC/EA7S hemolytic assays HC was used at the final dilution 1:300. Complement-Dependent Lysis (CDL) Assay (A) CD4/ target cells. PBL (107) and CD4/ T-cell blasts or CEM-NKR cells were labeled with 0.15 mCi of Cr51 (Amersham, England) for 2 hr at 377C in 0.5 ml

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RPMI 1640 supplemented with 10% human AB serum. After washing in RPMI 1640 medium, 3 1 106 target cells were incubated with 1 mg of recombinant gp120IIIB or 3 mg of gp160IIIB (Intracel Corp., Cambridge, MA) for 45 min at 377C in 0.1 ml medium. The lowest amount of glycoprotein which was able to induce the highest cell lysis in the presence of BRC was employed. Target cells were washed 21 in RPMI 1640 and resuspended in the same medium, and 2 1 104 cells in 0.1 ml/well were incubated in the absence or presence of notinactivated patient or control serum diluted 1:50 and BRC in a U-bottom microtiter plate for 2 hr. Maximum release of Cr51 was obtained by the addition of 0.1% Triton X-100 to the targets. Supernatants were collected using a harvesting system (Skatron, Norway) and counted in a gamma counter. Percentage of lysed cells was calculated as previously described (16). CDL inhibitory activity was determined in each serum using CEM-NKR cells coupled with gp120 as targets and it was calculated for each serum as follows: % inhibition Å 100 0 {(% lysis in the presence of patient serum and complement/% lysis with complement alone) 1 100}. (B) SRBC/EA7S hemolytic assay. CH50 titer was determined in each serum using the microassay method described previously (17), and it was calculated as the reciprocal of the dilution which resulted in 50% hemolysis of target cells. The sera were tested further at the dilution 1:200 for their ability to inhibit hemolysis of SRBC/EA7S target cells. Each serum (1 ml) was added to HC diluted 1:150 in 0.1 ml GVB2/ followed by the addition of 0.1 ml targets in the same buffer. The mixture was incubated for 1 hr at 377C. Normal human sera were used as controls. Testing was performed in triplicates using U-bottom microtiter plates. Following incubation, the plates were centrifuged for 10 min at 800g and 150 ml of supernatant from each well was transferred into a new plate. Maximum hemolysis was obtained by addition of distilled water to targets and absorbance was read at 405 nm. The percentage of hemolysis inhibition was calculated in the same way as for CD4/ T-cell targets. Sera which exhibited 90–100% CDL inhibition in a hemolytic assay and more than 50% in baby rabbit complement induced lysis of gp120-coupled CD4/ T cells under conditions employed in the assay were considered as inhibitory. Detection of Functional C1-INH by EIA We have used a commercially available EIA kit (Quidel, San Diego) following the instructions of the manufacturer. Briefly, the principle of functional C1-INH EIA was as follows: In the first step, standards, controls, and test specimens were incubated with activated C1s labeled with biotin. Upon the formation of C1INH–C1s complexes, each sample was added to micro-

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titer plate precoated with avidin. After washing, horseradish peroxidase(HRP)-conjugated goat anti-human C1-INH was added to each test well. The bound HRP conjugate was detected after addition of chromogenic substrate and the color intensity was measured spectrophotometrically at 405 nm. The concentration of functional C1-INH in a given sample was reported as the percentage of the mean level in standard specimen. The concentration of functional C1-INH less than 40% mean normal was considered significantly decreased. Antigenic concentration of C1-INH was determined by RID assay (Serotec, UK) and calculated as mg/liter). CIC-Raji Cell Replacement EIA CIC-containing IgG and C3 fragments iC3b, C3dg, or C3d (C3-CICs) were detected by EIA (Quidel) using monoclonal antibody which simultaneously binds these C3 activation fragments in a manner analogous to the Raji cell complement receptor-2 binding reaction (18). Results were expressed as micrograms of heat aggregated human gamma globulin equivalents per milliliter (mg Eq/ml). C4d Fragment EIA C4d fragments were determined by EIA (Quidel) and calculated as mg/ml. Serum Fractionation In experiments performed to determine the complement inhibiting factor the serum was passed through the following chromatography columns according to the instructions from the manufacturer: (i) protein A immobilized on 6% agarose (Pierce, U.S.A.) to remove IgG; (ii) D-mannose immobilized on 6% agarose (Pierce) to remove mannose binding protein(s); (iii) wheat germ agglutinin (WGA) Ultrogel (IBF Biotechnics, France) to remove glycoproteins with carbohydrate residuals containing b - D - N - acetylglucosamine (b - D - GlcNAc). Proteins bound to each gel were eluted and tested for CDL inhibiting activity. In order to characterize CDL inhibitor, the serum was preincubated with polyclonal antibodies against C1-INH, C4b-binding protein (C4bBP), Factor H, and Factor I (Serotec, UK) to remove target protein by immune precipitation. Purified Factor I and C1-INH were from Calbiochem (San Diego). Statistics The statistical analysis and graphics were performed using the Statistica program for nonparametric statistics and fitting distributions (StatSoft Inc., Tulsa, OK). RESULTS

The coupling of HIV-1 glycoproteins gp120 and gp160 to CD4 receptors on mitogen or IL-2-activated target

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cells induced cell lysis in the presence of low-toxic BRC (Table 1). The cell lysis was dependent on CD4/ T-cell concentration in the target sample being highest in CD4/ T-cell line CEM-NKR (73.2 and 61.1%, respectively). When target cells and BRC were incubated in the presence of HIV-1-positive sera diluted 1:50, some sera have shown significant inhibition of cell lysis. This phenomenon was tested in sera from 98 HIV-1-positive patients using gp120-coupled CEM-NKR cells as targets and BRC as the source of complement (Fig. 1). When HIV-1-positive patients were separated into three groups according to their CD4/ T-cell count, nonparametric analysis by Kruskal–Wallis analysis of variance by ranks showed significant differences (P õ 0.001), as did median test (overall median Å 52.15; x2 Å 30.256, P õ 0.001). The CDL inhibition values in each group were also compared by Wald–Wolfowitz runs test. Significant differences were found between the group of patients with CD4/ T-cell count higher than 500/ml (N Å 38) as well as with counts of 200– 500/ml (N Å 32) when compared to those having less than 200/ml (N Å 28) (P Å 0.0064 and 0.0012, respectively). Spearman rank order correlation analysis between CDL inhibitory activity and the CD4/ T-cell count in each patient has been found highly positive (R Å 0.3993, P õ 0.001). CDL inhibitory activity was the most frequent in patients with CD4/ T-cell count between 200 and 600/ml and noninhibitors were predominantly sera from patients with less than 200/ml (Fig. 2). Inhibition of complement activation in the presence of HIV-1-positive sera was also confirmed in a hemolytic assay when SRBC/EA7S were used as targets and the normal human serum as the source of complement (Table 1). The number of CDL inhibitory sera in this assay was identical to that obtained in CD4/ T-cell lysis presented in Fig. 1. Accordingly, CH50 titer was found to be significantly lower in CDL inhibitory sera (26.2 { 16.2) as compared to noninhibitory sera (132.5 { 48.4, P õ 0.001). When both categories of sera were compared to normal controls (267 { 55), the differences were highly significant P õ 0.001; Table 2). The functional C1-INH was not detected or was very low in inhibitory sera (% mean normal Å 11.8 { 11.5), and also in noninhibitory sera (% mean normal Å 53.5 { 19.9) (P õ 0.001). Antigenic C1-INH concentration and C4d level were significantly higher in both categories of HIV-1-positive sera when compared to normal controls (P õ 0.001). The level of C3-CICc was significantly increased only in noninhibitory sera (P Å 0.014, Table 2). Heat inactivation of serum at 567C or passing through WGA Ultrogel column removed the CDL inhibitory activity, while passing through the protein A and D-mannose immobilized on agarose did not influence this activity (Table 3). Eluted proteins from WGA gel showed the highest

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TABLE 1 Target Cell Lysis (%) Induced by BRC after gp120/160 Coupling to CD4 Receptor on Activated T Cells or by HC after Coupling of Rabbit Anti-Sheep Red Blood Cell Stroma Antibody to SRBC in the Presence or Absence of HIV-1Positive Sera BRC/Noninh Targets:

BRC (% lysis)

PHA blasts Control gp120 gp160 ConA blasts Control gp120 gp160 PWM blasts Control gp120 gp160 II-2 blasts Control gp120 gp160 CD4/ PWM blasts Control gp120 gp160 CEM-NKR cells Control gp120 gp160

% Lysis

BRC/Inh

% Inhibition

% Lysis

0.8 33.1 29

2.5 30.7 25.3

7.3 12.8

1.9 14.9 10.1

55 65.2

00.9 46.5 46

00.3 37.4 40

19.6 13

00.1 16.2 13.5

65.2 70.7

0.1 42.3 43.2

2.1 37.1 45.3

12.3 0

0 10.7 8.8

74.7 79.6

0.3 40.5 43.4

1.2 37.7 43.6

6.9 0

1.3 18.6 15.2

54.1 65

0 71.1 53.5

1.1 69.3 50.4

2.5 5.8

0.7 6.7 5.5

90.6 89.7

2.7 73.2 61.1

3.9 77.3 54.2

0 11.3

1.8 15 5.2

79.5 91.5

HC/Noninh HC (% lysis) 81.4

SRBC EA7S

% Inhibition

% Lysis 93

HC/Inh

% Inhibition 0

% Lysis 8.1

% Inhibition 90

Note. Noninh, complement noninhibitory serum; Inh, complement inhibitory serum; PHA, phytohaemagglutinin; Con A, concanavalin A; PWM, Pokeweed mitogen.

CDL inhibition (52.8%), suggesting that inhibiting factor(s) could be present. Immune precipitation of C1INH, C4bBP, Factor H, and Factor I by polyclonal antibodies did not show any remarkable influence on serum CDL inhibiting activity under conditions employed in the assay. Addition of 1 unit of functional C1-INH or 0.2 mg of Factor I per well showed significant CDL inhibition of complement activity (97.2 and 52.2%, respectively; Table 3). DISCUSSION

Besides evidence already presented that complement alone or together with antibodies directed against HIV1 envelope glycoproteins enhanced virus infectivity (19–21), we have shown that its persistent activation by HIV-1 during the course of infection may have a significant influence on CD4/ T-cell count in infected patients. Furthermore, we have shown that comple-

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ment activation induced increased consumption of complement regulatory protein C1-INH, a serine protease inhibitor. C1-INH regulates the activation of classical pathway of complement as well as of the contact system of intrinsic coagulation, and its increased consumption and/or inactivation indicates the degree of complement activation (22). The decreased level of functional C1INH and the increased level of antigenic C1-INH which we have found in HIV-1 infected patients were linked with the significant impairment of complement function as judged by low CH50 titer, especially in CDL inhibiting sera (Table 2). It was clearly shown that CDL inhibiting activity and decreased CH50 titer were caused by the presence of an active complement inhibiting factor(s), since addition of patient’s serum to normal HC or BRC significantly reduced their activation ability (Table 1). Accordingly, Senaldi et al. (4) have found that concentration of intact complement components C1q, C3, and C4 did not differ significantly in

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plexed with proteinases C1s and C1r were the predominant forms in serum, as it was documented in patients with sepsis complicated by adult respiratory distress syndrom and in patients with typhoid fever (23, 24). The formation of C3/C5 convertase and subsequent C3 activation did not occur like in patients with acquired C1-INH deficiency (25). Accordingly, C3 activation products were significantly increased only in CDL noninhibiting sera, and C4d level was high in all HIV-1positive sera (Table 2). Thus, it could indicate that CDL inhibition occurs at or before C3/C5 convertase level which is regulated by C1-INH at C1 activation level or later by Factor I, Factor H, and cofactors C4bBP, membrane cofactor protein, and soluble complement receptor-1. However, another as yet unidentified factor(s) is more likely to be involved in this phenomenon since removal of C1-INH, C4bBP, Factor I, and Factor H by immunoprecipitation did not influence the inhibitory activity. Incubation of a CDL inhibiting serum with WGA Ultrogel removed its activity, suggesting that complement inhibitory factor is most probably a glycoprotein with b-D-GlcNAc carbohydrate residue (Table 3). Such an increased and persistent activity of complement inhibiting factor(s) could cause the impairment of complement system, but also of contact system of intrinsic coagulation. The complete absence or very low level of C1-INH is known in hereditary as well as in acquired angioedema (24). In hereditary form the syn-

FIG. 1. CDL inhibition values in HIV-1-positive sera from patients separated into three groups according to CD4/ T-cell count and tested using gp120-coupled CD4/ T-cell line CEM-NKR cells as targets and baby rabbit serum as the source of complement. Gp120coupled target cell lysis in BRC alone was 59.1%, and the mean CDL inhibition value in the presence of normal control sera was 2.8 { 3.0 (N Å 12). The overall distribution of values was analyzed by Kruskal–Wallis Analysis of Variance by Ranks (P õ 0.001) and by Median test (overall median Å 52.15; x2 Å 30.256, P õ 0.001). The values between the groups were compared by Wald–Wolfowitz Runs test. Both groups with CD4/ T-cell count above 200/ml showed significantly higher CDL inhibition values as compared to the group with CD4/ T-cell count lower than 200/ml (P Å 0.0064 and 0.0012, respectively).

HIV-1-infected patients as compared to healthy controls. The sera which showed an absence or very low concentration of functional C1-INH and a significantly increased concentration of antigenic C1-INH exhibited the highest CDL inhibitory activity. This might suggest that cleaved and inactivated C1-INH or C1-INH com-

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FIG. 2. Distribution of the CDL inhibitors and noninhibitors among HIV-1-positive patients in relation to their CD4/ T-cell count. Correlation between CDL inhibiting activity and CD4/ T-cell count was calculated by Spearman’s Rank Order Analysis: R Å 0.399, P õ 0.001.

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TABLE 2 Determination of CH50 Titer, Functional and Antigenic C1-Inhibitor, C4d Fragments, and C3-CICs in Control Sera and in Complement Inhibiting and Noninhibiting Sera from HIV-1-Infected Patients F-C1-INH (% of normal)

CH50 I N Mean SD P

49 26.2 16.2

NI 49 132.5 48.4 õ0.001

A-C1-INH (mg/liter)

I

NI

I

NI

I

NI

I

19 11.8 11.5

30 53.5 19.9

31 618.1 158.3

10 635.7 179.5

8 16.65 1.83

14 15.97 2.72

10 6.4 3.9

õ0.001

ns

14

8

11

267.0

105.5

SD

55.0

12.3

P

õ1

õ0.001

Control N Mean

C3-CICs (mg Eq/ml)

C4d (mg/ml)

ns

NI 12 12.8 7.3 ns

12

8

250.1

5.59

4.6

105.2

2.05

1.4

õ0.001

0.001

õ0.001

ns

0.014

Note. The values obtained in control and in CDL inhibiting and noninhibiting sera were compared by Wald–Wolfowitz Runs test. Significant P values are given in the table. F-C1-INH, functional C1-INH; A-C1-INH, antigenic C1-INH; I, complement inhibitory sera; NI, complement noninhibitory sera; N, number of tested sera; SD, standard deviation; ns, not significant.

thesis of C1-INH in the liver is deficient due to the genetic defect, whereas in acquired form the deficiency is caused by an increased catabolism of C1-INH. Acquired C1-INH deficiency has been found in patients with monoclonal expansion of surface Ig (B-cell neoplasia) when increased activation of complement by idiotype–antiidiotype complexes caused depletion of functional C1-INH. (25). The impairment of functional C1-INH in contact system of intrinsic coagulation, particularly regarding activated Factor XII (26), was also documented in AIDS patients in the form of thrombotic thrombocytopenic purpura (27). C3-CICs (iC3b, C3dg, and C3d) which have been significantly increased only in complement noninhibiting sera from HIV-1-infected patients could induce potentially destructive events in the host including anaphylatoxin release, cell lysis, leukocyte stimulation, and activation of macrophages and other immunocompetent cells (28). When CICs become fixed to cell membranes, destruction of the normal cell can occur not only by antibody-dependent complement-mediated lysis, but also by antibody-dependent cellular cytotoxicity, phagocytosis, and/or cytotoxic T cells (29). Assuming that activated CD4/ T cells coupled with gp120 may be susceptible to the attack of complement system as it was demonstrated on Table 1 and by Su¨sal et al. (13), CDL inhibition could play a significant role in protection of CD4/ T cells from complement induced lysis (Fig. 1). Unfortunately, the same mechanism could protect HIV-1, which means that virus has

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adapted itself to make use of the host complement system by interacting with complement components to its advantage. Since C1q more effectively binds gp41 remaining on viral membrane after release of noncovalently bound gp120 (11), HIV-1 protection from CDL seems to be more effective than that of CD4/ T cells. This implies that CD4/ T-cell depletion might depend directly on the percentage of activated and gp120-coupled cells as well as on the degree of complement activation/inhibition and not on HIV-1-infected cells. In the early phase of infection, the proportion of gp120-coupled CD4/ cells is relatively small (30) and there is still a tendency for CD4/ cell count recovery (fluctuations seen in CD4/ cell count and virus load in asymptomatic and ARC patients). As the disease progresses, higher proportions of gp120-coupled CD4/ cells are present and more CD4/ cells might be depleted by complement activation. This was confirmed in a 10-year study by Daniel et al. (31). They have shown that CD4/ T-cell count was significantly lower in HIV-1-infected hemophiliacs with complement-fixing IgM, complement-fixing IgG / IgM, and especially with gp120-IgM/IgG complement-fixing complexes on the surface of CD4/ cells. The presence of later complexes also correlated with progression to terminal disease. Similarly, Wang et al. (32) presented data on T-cell depletion in CD4 transgenic mice by gp120 and gp120-specific antibodies from AIDS patients. These findings strongly support our results and the proposed mechanism of complement induced CD4/ T-cell elimination.

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CDL Inhibitory Activity in HIV-1-Positive Sera after Heat Inactivation for 30 min at 567C, after Passage Through Different Chromatographic Columns, and after Immune Precipitation of Complement Regulatory Proteins C1-INH, C4b-BP, Factor H, and Factor I None (% lysis) BRC dil. 1:25 Control Inh. serum No. 161 Not treated Heat inactivated Protein A column WGA column D-Mannose column Serum No. 161 eluate Protein A column WGA column D-Mannose column Inh. serum No. 322 Not treated Anti-C1-INH Anti-C4bBP Anti-Factor H Anti-Factor I Ninh. serum No. 152 Not treated Heat inactivated Protein A column WGA column D-Mannose column C1-INH (1 unit) Factor I (0.2 mg)

gp120 (% lysis)

% Inhibition

ACKNOWLEDGMENT 1.6

67.1

1.4 0.3 1.1 0.7 0.2

8.5 48.2 5.2 71.8 5.4

87.3 28.2 92.3 0 92

3.7 1.7 3

60.8 31.7 46.8

9.4 52.8 30.3

00.4 2.4 0.8 01.6 8.9

9.6 1.4 00.2 01.5 17.5

85.7 97.9 100 100 73.9

1.9 2.1 1.8 1.2 1 0 0.5

68.5 59.4 71.9 72.4 63.9 1.7 32.1

0 11.5 0 0 4.8 97.2 52.2

Note. Gp120-coupled CD4/ T-cell blasts were used as targets in the presence of serum or serum fractions at final dilution 1:50 and BRC. Purified C1-inhibitor and Factor I were used to show their CDL inhibitory activity compared to HIV-1 positive sera. CDL inhibition (%) was calculated as described under Materials and Methods.

In an early phase of complement activation, C1-INH, C4bBP, and Factor I play the major inhibiting role, but in terminal phase the cell lysis is controled by complement regulatory proteins present on the cell membrane, like decay accelerating factor (DAF), protectin (CD59), and homologous restriction factor. However, acquired changes in the membrane expression of these proteins were reported in HIV-1-infected individuals (33–35) which could increase susceptibility of CD4gp120-coupled cells to CDL. Therefore, the presence of high CDL inhibiting activity in sera from HIV-1-infected patients could be beneficial in protection of gp120-coupled CD4/ cells from lysis, but it would also cause significant impairment of functional complement activity against HIV-1 as well as against bacteria, mycoplasmas, fungi, or other viruses which contributed to the onset of AIDS. Unfortunately, it is yet not clear when CDL inhibiting activity appears in the course

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of HIV-1 infection and how long it exists in infected patients. Thus, further investigation of this phenomenon in HIV-1-infected individuals, particularly in those with a long asymptomatic period, could explain the mechanism which enables HIV-1 to avoid its elimination despite massive host immune response. It could also explain the mechanism of CD4/ T-cell protection and/or depletion during onset of the disease.

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This work was supported in part by the Ministry of Science of Republic Croatia (Grant 3-01-111). REFERENCES 1. Lifson, A. R., Rutherford, G. W., and Jaffe, H. W., The natural history of human immunodeficiency virus infection. J. Infect. Dis. 158, 1360–1367, 1988. 2. Morrow, W. J. W., Wharton, M., Stricker, R. B., and Levy, J. A., Circulating immune complexes in patients with AIDS contain the AIDS associated retrovirus. Clin. Immunol. Immunopathol. 40, 515–522, 1986. 3. Carini, C., Perricone, C., Fratazzi, C., Fontana, L., de Carolis, C., D‘Amelio, R., Sirianni, M. C., and Aiuti, F., Complement activation is associated with the presence of specific human immunodeficiency virus (HIV-1)-anti HIV immune complexes in patients with acquired immunodeficiency syndrom related complex or lymphadenopathy syndrome. Scand. J. Immunol. 30, 347–353, 1989. 4. Senaldi, G., Peakman, M., McManus, T., Davies, E. T., Tee, D. E. H., and Vergani, D., Activation of the complement system in human immunodeficiency virus infection: relevance of the classical pathway to pathogenesis and disease severity. J. Infect. Dis. 162, 1227–1232, 1990. 5. Spear, G. T., Landay, A. L., Sullivan, B. L., Dittel, B., and Lint, T. F., Activation of complement on the surface of cells infected by human immunodeficiency virus. J. Immunol. 144, 1490–96, 1990. 6. Saarloos, M.-N., Lint, T. F., and Spear, G. T., Efficasy of HIVspecific and antibody-independent mechanism for complement activation by HIV-infected cells. Clin. Exp. Immunol. 99, 189– 195, 1995. 7. Jarvis, J. N., Taylor, H., and Iobidze, M., Complement activation and immune complexes in early congenital HIV infection. J. Acquir. Immune Def. Syndr. Hum. Retrovir. 8, 480–485, 1995. 8. Ebenbichler, C. F., Thielens, N. M., Vornhagen, R., Marschang, P., Arlaud, G. J., and Dierich, M. P., Human immunodeficiency virus type 1 activates the classical pathway of complement by direct C1 binding through specific sites in the transmembrane glycoprotein gp41. J. Exp. Med. 174, 1417–1424, 1991. 9. Spear, G. T., Jiang, H., Sullivan, B., Gewurz, H., Landay, A. L., and Lint, T. F., Direct binding of complement component C1q to human immunodeficiency virus (HIV) and human T lymphotrophic virus 1 (HTLV-1) coinfected cells. AIDS Res. Hum. Retrovir. 7, 579–585, 1991. 10. Thieblemont, N., Haeffner-Cavaillon, N., Weis, L., Maillet, F., and Kazatchkine, M. D., Complement activation by gp160 glycoprotein of HIV-1. AIDS Res. Hum. Retrovir. 9, 229–233, 1993. 11. Thielens, N. M., Bally, I. M., Ebenbichler, C. F., Dierich, M. P., and Arlaud, G. J., Further characterization of the interaction between the C1q subcomponent of human C1 and the transmem-

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AP: Clin

COMPLEMENT INHIBITION IN AIDS

12.

13.

14.

15.

16.

17. 18.

19.

20.

21.

22.

23.

24.

brane envelope glycoprotein gp41 of HIV-1. J. Immunol. 151, 6583–6592, 1993. Prohaszka, Z., Hidvegi, T., Ujhelyi, E., Stiber, H., Dierich, M. P., Su¨sal, C., and Fu¨st, G., Interaction of complement and specific antibodies with external glycoprotein 120 of HIV-1. Immunology 85, 184–189, 1995. Su¨sal, C., Kirschfink, M., Kro¨pelin, M., Daniel, V., and Opelz, G., Complement activation by recombinant HIV-1 glycoprotein gp120. J. Immunol. 152, 6028–6034, 1994. Centers for Disease Control. 1993 Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR 41, 1–18, 1993. Howell, D. N., Andreotti, P. E., Dawson, J. R., and Cresswell, P., Natural killing target antigens as inducers of interferon: Studies with an immunoselected natural killing resistant human T lymphoblastoid cell line. J. Immunol. 134, 971–980, 1985. Gerencˆer, M., Valpotic´, I., Jukic´, B., Tomasˆkovic´, M., and Basˆic´, I., Qualitative analysis of cellular immune functions in Equine Infectious Anemia show homology with AIDS. Arch. Virol. 104, 249–257, 1989. Liu, C-C., and Young, J. D-E., A semi-automated microassay for complement activity. J. Immunol. Methods 114, 33–41, 1988. Theofilopoulos, A. N., Wilson, C. B., and Dixon, F. J., The Raji cell radioimmune assay for detecting immune complexes in human sera. J. Clin. Invest. 57, 169–176, 1976. Robinson, W. E., Jr., Montefiori, D. C., and Mitchell, W. M., Complement-mediated antibody-dependent enhancement of HIV-1 infection requires CD4 and complement receptors. Virology 175, 600–604, 1990. Bakker, L. J., Nottet, H. S. L. M., de Vos N. M., de Graaf, L., Van Strijp, J. A. G., Visser, M. R., and Verhoef, J., Antibodies and complement enhance binding and uptake of HIV-1 by human monocytes. AIDS 6, 35–41, 1993. Thieblemont, N., Haeffner-Cavaillon, N., Ledur, A., L‘Age-Stehr, J., Ziegler-Heitbrock, L., and Kazatchkine, M. D., CR1 (CD35) and CR3 (CD11b/CD18) mediate infection of human monocyte and monocytic cell lines with complement-opsonized HIV independently of CD4. Clin. Exp. Immunol. 92, 106–113, 1993. Porcel, J. M., Peakman, M., Senaldi, G., and Vergani, D., Methods assesing complement activation in the clinical immunology laboratory. J. Immunol. Methods 157, 1–9, 1993. Nuijens, J. H., Eerenburg-Belmer, A. J. M., Huijbregts, C. C. M., Schreuder, W. O., Felt-Bersma, R. J. F., Abbink, J. J., Thijs, L. G., and Hack, C. E., Proteolytic inactivation of plasma C1 inhibitor in sepsis. J. Clin. Invest. 84, 443–450, 1989. Oltvai, Z. N., Wong, E. C. C., Atkinson, J. P., and Tung, K. S. K., C1-inhibitor: Molecular and immunologic basis of hereditary and acquired angioedema. Lab. Invest. 65, 381–388, 1991.

25. Geha, R. S., Quinti, I., Austen, K. F., Cicardi, M., Sheffer, A., and Rosen, F. S., Acquired C1-inhibitor deficiency associated with antiidiotypic antibody to monoclonal immunoglobulins. N. Engl. J. Med. 312, 534–539, 1985. 26. de Agostini, A., Lijnen, H. R., Pixley, R. A., Colman, R. W., and Schapira, M., Inactivation of factor XII active fragment in normal plasma. Predominant role of C1-inhibitor. J. Clin. Invest. 73, 1542–1560, 1984. 27. Platanias, L. C., Paiusco, D., Bernstein, S., and Murali, M. R., Thrombotic thrombocytopenic purpura as the first manifestation of human immunodeficiency virus infection. Am. J. Med. 87, 699–700, 1989. 28. McDougal, J. S., and McDuffie, F. C., Immune complexes in man: Detection and clinical significance. Adv. Clin. Chem. 24, 1–60, 1985. 29. Erdei, A., Fu¨st, G., and Gergely, J., The role of C3 in the immune response. Immunol. Today 12, 332–337, 1991. 30. Daniel, V., Su¨sal, C., Prodeus, A. P., Weimer, R., Zimmermann, R., Huth-Ku¨hne, A., and Opelz, G., CD4/ lymphocyte depletion in HIV-infected patients is associated with gp120-immunoglobulin-complement attachment to CD4/ cells. Vox. Sang. 64, 31– 36, 1993. 31. Daniel, V., Su¨sal, C., Weimer, R., Zipperle, S., Kro¨pelin, M., Zimmermann, R., Huth-Ku¨hne, A., and Opelz, G., Sequential occurrence of IgM, IgM/IgG and gp120-IgM/IgG complement complexes on CD4/ blood lymphocyte depletion in HIV/ hemophilia patients: Results of a 10-year study. Immunol. Lett. 47, 97–102, 1995. 32. Wang, Z.-Q., Orlikowsky, T., Dudhane, A., Mitter, R., Blum, M., Lacy, E., Riethmu¨ller, G., and Hoffmann, M. K., Deletion of T-lymphocytes in human CD4 transgenic mice induced by HIVgp120 and gp120-specific antibodies from AIDS patients. Eur. J. Immunol. 24, 1553–1557, 1994. 33. Lederman, M. M., Purvis, S. F., Walter, E. I., Carey, J. T., and Medof, M. E., Heightened complement sensitivity of acquired immunodeficiency syndrome lymphocytes related to diminished expression of decay-accelerating factor. Proc. Natl. Acad. Sci. USA 86, 4205–4209, 1989. 34. Weiss, L., Okada, N., Haeffner-Cavaillon, N., Hattori, T., Faucher, C., Kazatchkine, M. D., and Okada, H., Decreased expression of the membrane inhibitor of complement-mediated cytolysis CD59 on T-lymphocytes of HIV-1 infected patients. AIDS 6, 379–385, 1992. 35. Jarvis, J. N., Taylor, H., Long, P. M., Gutta, P. V., Pousak, T., and Fine, N., Diminished expression of cell-surface complement regulatory proteins in HIV-infected children and with HIV infection of peripheral blood mononuclear cells in vitro. J. Acquir. Immune Def. Syndr. Hum. Retrovir. 9, 249–256, 1995.

Received February 3, 1995; accepted with revision June 7, 1996

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