CGin. exp. Immunol. (1979) 36, 46-53.

Circulating immune complexes in serum from patients withdengue haemorrhagic fever

W. RUANGJIRACHUPORN, S. BOONPUCKNAVIG & S. NIMMANITYA* Department of Patho-biology, Faculty ofScience, Mahidol University, and * the Children's Hospital, Rama VI Road, Bangkok 4, Thailand

(Accepted for publication 17 October 1978)

SUMMARY

Circulating immune complexes were detectable in 80% of serum from patients with denguehaemorrhagic fever. The immune complexes were detected for the first time on day twoafter the onset of the fever. The amount of complexes reached the maximum value on day 4 or 5after onset, or when the patients developed shock or subsidence of fever, after which the com-plexes decreased in number.The number ofcomplexes also correlated well with the clinical grading (severity) of the disease,

i.e. the maximum amount was shown in grade III. These complexes may play a part in thepathogenesis of this disease.

INTRODUCTION

During the course of infection, antibodies are formed in the circulation. These antibodies will normallyreact with the specific antigens to form antigen-antibody complexes. The soluble form of the immunecomplexes may play a pathogenic role in immune injury by depositing activating complement in tissues,thereby releasing vasoactive substances and mediating the inflammation (Cochrane & Koffler, 1973;Mannik, Hackenstak & Arend, 1974).There is evidence indicating that circulating immune complexesmay be involved in the pathogenesis of some diseases, such as chronic hepatitis (Nydegger et al., 1974),measles (Charlesworth et al., 1976), malaria (Lambert & Houba, 1974), as well as dengue haemorrhagicfever (DHF) (Thefilopoulos, Wilson & Dixon, 1976).Immune complexes have also been suggested as the initiating factor in the immunopathological

mechanism of shock in dengue haemorrhagic fever (Russel, 1971). Circulating immune complexes inDHF have been detected and found to be related to the clinical grading and stages of the disease (Sobel,Bokisch & Muller-Eberhard, 1975; Theofilopoulos et al., 1976). In addition, immune complexes inDHF were found to be localized in tissue and blood elements (Boonpucknavig et al., 1976a,b).

In the present study, we have attempted to demonstrate the circulating dengue immune complexesby two methods: firstly, by binding immune complexes to molecular Clq and inhibiting IgG-coatedlatex agglutination; and secondly, by binding immune complexes to Raji cell surface receptors andspecifically staining with anti-dengue antibody. The results were evaluated according to the day of fever.the day before and after shock, or the day before and after subsidence of fever, as well as clinical grading.

MATERIALS AND METHODS

Patients. Ninety-four serum specimens from eighty children with dengue haemorrhagic fever, aged between 2 and 1Pyears, were studied. All patients were admitted and treated at the Children's Hospital, Bangkok, Thailand. The diagnosi:was confirmed by initial clinical findings and proven serological data by the haemagglutination inhibition test (HI) at the

Correspondence: Dr S. Boonpucknavig, Department of Pathobiology, Faculty of Science, Mahidol UniversityBangkok, Thailand.

0099-9104/79/0040-0046$02.00 ©) 1979 Blackwell Scientific Publications

46

Immune complex and DHF 47Department of Virology, SEATO Medical Research Laboratory, Bangkok and by the indirect immunofluorescent antibodytechnique (Boonpucknavig et al., 1976c). The patients were devided into four grades depending on the severity of thedisease (Nimmannitya et al., 1969). Following the criteria of Winter et al. (1969), all the patients in this series were classifiedas having secondary DHF.

Detection of circulating immune complexes in serum by inlhibiting IgG-coated latex agglutination with Clq. IgG-coated latex.IgG-coated latex particles were prepared by incubating the suspension of polystrylene latex particles (diameter 0-2 u)with 10 mg/ml ofhuman IgG and 1% serum albumin at 37 C for 1 hr. The mixture was washed twice with 0 02 M phosphatebuffered saline (PBS) pH 7-1 and then adjusted to a concentration of 0 5% suspension and 0.1% sodium azide was addedas preservative (Singer & Plotz, 1956).

Clq. Clq was purified from fresh human serum by precipitation in various ionic strengths ofEDTA buffer as describedelsewhere (Volanakis & Stroud, 1972).

Detection of circulating immune complexes. Equal volumes of various dilution of heat-inactixated serum were incubatedwith diluted Clq for 30 min. 0 025 ml of this mixture was transferred on to a glass slide and mixed with 0 1 ml of IgG-coated latex particles. The slides were then rotated before reading the agglutination. The highest dilution giving positiveagglutination was recorded as the inhibitory titre.

Detection of circulating immune complexes by the Raji cell immunofluorescent assay. Normal human IgG. This was preparedfrom pooled normal serum by 40% saturated ammonium sulphate precipitation. The precipitate was dissolved in 0 01 Mphosphate buffer pH 7-4 and fractionated on DEAE cellulose G50 in this buffer (Levy & Sober, 1960). The purified humanIgG gave a single line against anti-whole human serum when tested by immunoelectrophoresis.

Rabbit anti-human IgG. Animals were injected in the footpad with 1-2 mg/ml purified human IgG with Freund'scomplete adjuvant. They were boosted with the same dose 1 or 2 weeks later. The antisera were collected at weekly intervalsand gave a single line against normal human serum by immunoelectrophoresis.

F(ab')2 anti-IgG Fc. F(ab')2 of anti-human IgG Fc was prepared by pepsin digestion as described by Nisonoff et al.(1960).

Fluorescein-conljugated antibody. Anti-human IgG and anti-dengue antibody were conjugated with fluorescein isothio-cyanate by the standard method described elsewhere (Nairn, 1976).

Lymphoblastoid cell line. Raji cells which are derived from Burkitt's lymphoma were cultured in RPMI culture medium.(Pulvertaft, 1964; Theofilopoulos et al., 1974b). The cell viability was determined by Trypan blue exclusion, and cellsurface receptors for IgG (Fc) and for complement were determined by exposing the cells to fluorescein-conjugated humanaggregated IgG (Theofilopoulos, Dixon & Bokish, 1974a) and fluorescein-conjugated complement coated salmonella groupD (Gelfand et al., 1976), respectively.

Assay. The Raii cell culture was washed three times with Eagle's minimal essential medium (MEM) and adjusted to5 x 10' cells/ml in MEM. 0-2 ml of cell suspension were incubated with 0-025 ml of 10 mg/ml human IgG at 37°C for 30min. The cells were washed twice with MEM and then reacted with 0-025 ml of 5 0 mg/ml of F(ab')2 rabbit anti-humanIgG (Fc) at 4 C for 30 min to eliminate the undesirable fluorescein background caused by 7S IgG binding to the (Fc) recep-tors for IgG. Subsequently, the cells were washed twice with MEM and incubated at 4 C for 30 min with 0-025 ml offluorescein-conjugated anti-human IgG or fluorescein-conjugated human anti-dengue antibody. They were washed threetimes with MEM and smeared onto glass slides. The smears were fixed with 95% alcohol, washed twice in PBS and mountedwith buffered glycerine. Positive stained cells were counted per 500 cells with a fluorescent microscope.

Preparation of soluble immune complexes. Dengue antigens. Haemagglutinating dengue antigens were prepared from infectedsuckling mice brain with dengue-2 (New Guinea C.) by sucrose acetone extraction (Clarke & Casals, 1958).

Dengue antibody. Dengue antibody was obtained from a globulin fraction of hyperimmune human anti-dengue serumby fractionation through DEAE cellulose G50 (Levy & Sober, 1960).

Soluble dengue antigen-antibody complexes. Equal volumes of various HI titres of dengue antibody were mixed withdengue antigen for 1 hr at 37TC, constantly shaken and left overnight at room temperature. The dengue immune com-plexes were precipitated with 4% concentration of polyethylene glycol (mol. wt 4000) and redissolved in 0-1 Ni borate bufferedsaline pH 8 4. The protein concentration of the dengue immune complexes was determined at OD 280 nm.

Soluble aggregated human gamma globulin. Human IgG in PBS was centrifuged at 120,000 g for 30 min in orderto remove the aggregated and damaged globulin. The supernatant was used to form soluble aggregated IgG by heating at63°C for 15 min, followed by precipitating with 40% saturated ammonium sulphate. The precipitate was dissolved anddialysed against PBS. Soluble aggregated human globulin was obtained by centrifugation at 79,000 g for 10 min toremove the insoluble aggregated human gamma globulin. The protein concentration of heat aggregated human gammaglobulin (AHG) was determined at OD 280 nm (Fletcher & Lin, 1977).

Absorption ofsera in Raji cells. 0-1 ml each of undiluted serum from five samples was absorbed three times with 6x 106xvashed Raji cell pellets at 37TC for 30 min. The reaction of unabsorbed serum by the Raji cell immunofluorescent assay andby inhibition IgG-coated latex agglutination was compared with that after the absorption.

48 W. Ruangjirachuporn, S. Boonpucknavig & S. NimmanityaRESULTS

Detection of circulating immune complexesInhibition IgG-coated latex agglutination method. Ninety-four serum specimens were examined. Eighty-

four per cent of these specimens yielded inhibitory titres greater than 1:4. In normal controls theinhibitory titre was 1:4 or less. Two out of five negative sera showed endogenous agglutination. Therelationships between the positive results and the clinical course were as follows.

Day offever. Circulating immune complexes were first detected on day 2 of fever (inhibitory titreof 1: 8) and then slightly increased to a maximum value on day 5 (inhibitory titre of 1: 128) and declinedthereafter (Fig. 1).

Day ofshock or subsidence offever. Circulating immune complexes first appeared 3 days before shockor subsidence of fever (inhibitory titre of 1: 8) and increased to a maximum inhibitory titre of 1: 128on the day of shock or the day of subsidence of fever and then decreased as shown in Fig. 2.

Clinical grades. The correlation between the inhibitory titre of the immune complexes and differentclinical gradings of the severity of the disease are shown in Fig. 3. There was a gradual increase in

1:128 0

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FIG. 1. Distribution of inhibition titres of latex agglutination on the days of fever from ninety-four serumspecimens from patients with DHF. (o) Inhibitory titre of each specimen.

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FIG. 3. Distribution of inhibitory titres of latex agglutination with different clinical gradings from fifty-sixpatients with DHF.

inhibitory titre from grade I to grade IV. At a maximum value of grade IV the inhibitory titre was 1: 128.Raji cell immunofluorescent assay. The percentages of positive stained Raji cells with conjugated anti-

human IgG and conjugated anti-dengue antibody after exposure to serum from patients were 74-68 and58-33, respectively. The correlation between the positive results and the clinical course were as follows.

Day offever. Fig. 4 shows circulating immune complexes in the serum of patients with denguehaemorrhagic fever which first appeared on day 2 of fever with 1-03% positive staining Raji cells forhuman IgG and 0.34% for dengue antigen. The percentages of positive staining Raji cells increased to amaximum value of 8.5% for human IgG and 1-86% for dengue antigen on day 5; thereafter the valuedecreased.

Day ofshock or subsidence offever. The percentages of Raji cells with positive staining for humanIgG and dengue antigen on the day before and after shock, or the day before and after subsidence offever are shown in Fig. 5. The percentage of positive stained Raji cells for human IgG increased from 3days before shock or before subsidence of fever (1-25%) and reached a maximum value of 7 45% onday 1 after shock or subsidence of fever, whereas the percentage of Raji cells with positive staining fordengue antigen increased from 3 days before shock or subsidence of fever (0.12%) and reached amaximum value (1-86%) on day 1 before shock or subsidence of fever.

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Days of feverFIG. 4. Percentages of Raji cells stained with conjugated anti-human gamma globulin (rn) and conjugated anti-dengue antibody (ED) from ninety-four serum specimens from patients with DHF on the day of fever.

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W. Ruangjirachuporn, S. Boonpucknavig a S. Nimmanitya

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4I

3

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FIG. 6. Relationship between clinical gradings of the disease and percentages of Raji cells binding immunecomplexes stained with conjugated anti-human gamma globulin (z) and conjugated anti-dengue antibody(8) from fifty-six patients with DHF.

Clinical grades. Fig. 6 shows the relationship between the percentages of positive stained Raji cellsfor human IgG and for dengue antigen and the clinical grading of the disease. There was a steadyincrease in the percentage of positive cells while the severity of the disease changed from grade I to

grade III. However, the percentages decreased when the severity was at grade IV. Since there were

only three cases of grade IV, the results were consequently difficult to interpret properly.

Comparative studiesThe results of the detection of serum with heat-aggregated human IgG, and with dengue immune

complexes in various antigen-antibody ratios and in various concentrations are summarized in Tables1 and 2. The detection of circulating immune complexes in the sera from patients with dengue haemor-

TABLE 1. Agglutination inhibition activity (dilution titres) and percentage of positive Raji cells with various con-centrations of heat-aggregated human gamma globulin

Concentration of heat-aggregated gamma globulin (mg/ml):

5 1-25 0-312 0-156 0-078 0 039 0-018 04009 0004 0*002

Inhibitory activity + + + + + + - - - -

Percentage of Raji cells positive 19-12 11-85 9 31 8-52 6 91 3-48 1P69 0-48 0 13 0

+ = Positive detection of immune complexes by inhibition of IgG-coated latex agglutination.- = Negative detection of immune complexes by inhibition of IgG-coated latex agglutination.

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52 W. Ruangjirachuporn, S. Boonpucknavig Lg S. Nimmanityarhagic fever was expressed as the amount of preformed dengue immune complexes or heat-aggregatedIgG. In order to detect the heat-aggregated human IgG by the latex agglutination test or the Rajicell assay, the concentration was at least 39 pg/ml and 4 pg/ml, respectively. The dengue immunecomplexes, which were first detected by inhibition latex agglutination on day 2 of fever or 3 days beforeshock or subsidence of fever (titre of 1:8), had a concentration of 30 pg which was equivalent to heat-aggregated human IgG or 39 pg preformed dengue immune complexes. There was a gradual increaseto a maximum value on day 4 or day 5 of fever, or on the day of shock, or the day of subsidence of fever(titre 1: 128) which was equivalent to approximately 310 pg/ml of heat-aggregated human IgG or 150pg/ml of preformed dengue immune complexes. By the Raji cell assay, the amount of immune complexesincreased gradually when stained with conjugated anti-human IgG. The maximum peak was on day 1after shock or after the subsidence of fever which was equivalent to approximately 150pg/ml ofperformeddengue immune complexes. In addition, the amount of circulating immune complexes obtained by usingboth techniques showed a significant increase that varied with the severity of the disease.

Absorption with Raji cells caused a reduction in serum immune complexes and a decrease in reactivitywith the Raji cell immunofluorescence assay, as well as in the inhibitory titre by IgG-coated latex agglu-tination with Clq.

DISCUSSION

In the present study, circulating immune complexes could be detected in the serum of patients havingDHF by both methods. By the inhibition of latex agglutination activity, 84% of ninety-four sera werepositive. By the Raji cell immunofluorescent technique, 75% were positive for immunoglobulin G and58% were positive for dengue antigen. The number ofcirculating immune complexes reached a maximumvalue on day 5 of fever, on the day that patients developed shock or when the fever subsided, anddeclined thereafter.The detection of circulating immune complexes in the serum of patients with DHF was achieved by

using their inhibitory activity on the agglutination of IgG-coated latex particles by Clq. This method isbased on the property of Clq that can precipitate immune complexes (Agnello, Winchester & Kunkel,1970; Agnello et al., 1971). Molecules of Clq can react with immune complexes which are confinedto the restricted zone of antigen excess just beyond the equivalent point (Lurhuma et al., 1976). Inthe present study, the highest inhibitory titre (1: 128) of the immune complexes was found on day 4 or 5after the onset of fever, the day of shock or the day that the fever subsided. This would seem to indicatethat in the early days of the disease, the dengue antigen-antibody complexes were in the stage of antigenexcess and they were found to be small in size. This latex technique for detecting immune complexesappears to be simple, semi-quantitative method which could demonstrate the presence of immunecomplexes in the serum of patients during the early course of DHF.As regards the method of detecting immune complexes, Theofilopoulos et al., (1974) showed that

large size immune complexes could be detected by the Raji cell immunofluorescent method. Ourresults indicated that the large size circulating immune complexes were developed in the later stageof DHF. It would seem reasonable to assume that they were formed in the stage of antigenexcess. Our results also indicate that soluble, complement-containing dengue antigen-antibody com-

plexes react with the complement receptor on the surface of Raji cells. Moreover, removal of thesecomplexes by absorption with Raji cells, reduced or eliminated the reactivity in the Raji cell immuno-fluorescence assay as well as in the latex technique. These absorption studies confirmed the specificityof the reaction between the dengue antigen-antibody complexes in DHF serum and the surface of theRaji cells. However, our studies indicated the presence of dengue antigen-antibody complexes in theserum of patients with DHF by both techniques. This is in agreement with results reported by Thec-filopoulos et al. (1976) who demonstrated circulating immune complexes in 62% of patients withDHF by means of a radioimmunoassay and with a mean value equivalent to 58-83 pg AHG/ml on theday of shock. Substantial evidence has accumulated by Sobel et al. (1975) who have also demonstrated,by using the Clq deviation technique, that the number of immune complexes in sera correlates with theseverity of the disease. By immunofluorescent staining of the surface oflymphocyte suspensions in DHF,

Immune complexes and DHF 53Boonpucknavig et al. (1976b) showed that B lymphocytes increased during the course of the disease.The number of lymphocytes bearing dengue antigen and PlC/a on the surface increased to a maximumvalue on the day of shock or subsidence of the fever (Boonpucknavig et al., 1976b). In addition, serumcomplement levels have been studied and found to be significantly depressed during the stage of shockor at the lowest level of PI C/a on day 6 after the onset of the illness (Bokisch et al., 1973; Russel, 1969).These findings confirm the presence of circulating immune complexes in patients with DHF and supportthe possibility that they may play a role in the immunopathological mechanism of shock in this disease.

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