Circulating Immune Complexes and Complement Sequence Activation in Infectious Mononucleosis

JACK R. WANDS, M.D. JOSEPH L. PERROTTO, M.D. KURT J. ISSELBACHER,M.D. Boston, hksechusetts

The role of immune complex formation was investtgated in a patient with infectious mononucleosis compikated by an urtkariai rash. Circulating cryoprotein immune complexes were identified during the urtkariai phase of the illness, and disappeared during recovery. These compiexes were composed of immunogtobuitns G (IgG), M (IgM) and A (IgA), complement components C3, C4 and C5, Epstein-Barr (EB) virus capsid antibody and particles resembling EB virus. The igG subtypes identified in the immune complexes were the complement fixing IgG-1, igG-2 and igG-3. The C3 activator of the properdin complex was detected in serum obtained during that acute phase but not after recovery. Thus, the transient appearance of circulating compiement-fixtng immune complexes was associated in this patient with activation of both classic and aiternate complement pathways. The findings suggest that these complexes may be involved in the rash associated with infecttous mononucleosis. Infectious mononucleosis is occasionally accompanied by rash and arthralgias [ 11. These clinical features resemble a “serum sickness-like” syndrome. Previous studies have suggested that circulating immune complexes are important in the pathogenesis of another “serum sickness-like” syndrome associated with acute viral hepatitis [2, 31. In an attempt to isolate and characterize circulating immune complexes, we performed a serial study on a patient with infectious mononucleosis whose course was complicated by an extensive urticarial rash. The role of the classic and afternate complement pathways and their activation by immune complexes in the pathogenesis of the rash associated with infectious mononucleosis was studied. MATERIAL

From the Departments of Medicine, Harvard Medicsi School and the GsstrointestinalUnit, MassachusettsGeneral Hospital, Boston, Massachusetts 02114. This work was supportedin part by grants from the American Cancer Society (M-146) and from the National Institutes of Health (CA-12389). Requests for reprints should be addressed to Dr. J. R. Wands, Gastrointestinal Unit, Massachusetts General Hospital, EMSton, Msesechusetts 02114. Manuscript accepted June 2, 1975.

AND METHODS

Case Report. The patient, a 24 year old man, presented with a one week history of pharyngitis, fever, chills, anorexia and dark urine. Physical examination revealed an extensive urticariil rash over the arms, legs and trunk, bilateral axillary and cervical adenopathy, exudative pharyngitis and hepatosplenomegaly. Pertinent laboratory data included a hematocrit value of 40 per cent, and a white blood cell count of 5,600/mm3 with 60 per cent lymphocytes and 10 per cent atypical lymphocytes. The results of liver function tests and Epstein-Barr (EB) virus antibody titers are shown in Table I. The heterophil titer was 1:1,792 which absorbed completely with beef red cells but not with guinea pig kidney. Hepatftis-B surface anti-

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TABLE I

Characterization of the Cryoprotein Immune Complexes in Infectious Mononucleosis Clinical

SGOT (IU)

(::, 100 ml)

TP”

c3

c4

C5

IgG

IgM

IgA

EBV Antibody Titert

1

256

9.4

7.4

f

+

+

+

+

+

1:50

1:160

>1:400

>I:5120

5 9 18 53

ND 130 ND 29

ND 7.2 ND 0.4

5.2

0

0

0

+

+

+

ND

ND

ND

3.4 2.2 0

0 0 0

0 0 0

0 0 0

+ 0 0

+ + 0

+ 0 0

ND ND 0

ND ND 0

ND ND 1:400

ND ND ND I:160

Dav of Study Pharyngitis+ Rash

Whole Serum

CryoproteinComplex Beef RBC Titer

EBV Antibody Titer

Beef RBC Titer

--*

IgG Subtypes in the Cryoprotein Immune Complexes

NOTE:

IgG-l*

IgG-2

IgG-3

lgG-4

1.60 (1)

0.10

0.108

0

ND = not determined.

lCryoprecipitable protein concentration (mg/ml) was calculated as the amount of protein measured in the cryoprecipitate resolubilized in 0.5 ml of the same BSAcontaining buffer minus the amount of protein measured in 0.5 ml of the same WA-containing no cryoprecipitate was detectable. ?lFA (Kindly measured by Dr. Paul M. Feorino, Viral Oncology Branch, CDC, Atlanta, Georgia). SConcentration

expressed as mg/ml (number

in parenthesis represents day of study).

gen (HB,Ag) and toxoplasmosis antibody were undetectable by radioimmunoassay and complement fixation. The cytomegalic virus antibody titer was 1:16, by complement fixation. The patient made an uneventful recovery; the rash disappeared within one week, and the hepatosplenomegaly and adenopathy regressed at one month.

Isolation and Characterization of Immune Compfaxes. The physical-chemical property of cryoprecipitation was used to isolate circulating immune complexes. Cryoproteins were isolated by allowing 50 ml of venous blood obtained from the patient and two normal laboratory control subjects to clot at 37’C for 45 minutes and then centrifuged at 5,000 g at 37’C for 15 minutes. This serum was incubated at 4OC for 3 to 7 days to allow formation of the ctyoproteins. The cryoproteins were centrifuged for 1 hour at 4’ at 1,000 g, washed three times, resuspended at 37OC in 10 ml of 1 per cent bovine serum albumin (BSA) in phosphate-buffered saline solution, pH 7.6, with 0.01 per cent sodium azide. These cryoprecipitates were placed at 37’C overnight to resolubilize the true cryoproteins. After centrifugation at 2,000 g for 15 minutes at 37OC, the supernates were placed at 4’C for an additional three day period to allow reprecipitation. The cryoproteins were washed three times. and finally redisSolved in 0.5 ml of 1 per cent BSA in phosphate-buffered saline solution with 0.01 per cent sodium azide. These purified cryoproteins showed no reaction in double agar gel diffusion (Ouchterlony) at 37’C against antihuman serum albumin, a-kmacrcglobulln, and a-1-antltrypsin demonstrating the absence of any detectable nonspecific serum contamination. The samples were then analyzed for immunoglobullns G (IgG), M (IgM) and A (IgA) by double diffusion In agar gel at 37OC. Complement components C3, C4 and C5 were assayed by counterimmunoelectrophoresis according to the method of Gocke and Howe [4]. IgG subtypes were measured by radial diffusion in agar gel (monospecific antise-

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rums kindly provided by Dr. Peter Schur, Robert Breck Brigham Hospital). Total protein in the cryoprecipitates was determined by the folin phenol reagent method of Lowry et al. [ 51. Hepatitis-B surface antigen and antibody were measured by hemagglutination inhibition and hemagglutination [6]. Antibody to EB virus cap& antigen was determined by Dr. Feorino (Viral Oncology Branch, CDC., Atlanta, Georgia) using the indirect fluorescent antibody (EBV)-IFA procedure as described by Henle and Henle [ 73. The cryoprotein immune complexes were also examined by electron microscopy using negatlve stainlng. Im mune complexes were solubillzed in normal saline solution by placing them at 37OC for 60 minutes. A drop was then placed on collodioncoated copper grids for 1 minute. A drop of 1.5 per cent solution of phosphotungstic a&I pH 6.0 was added to the grid for 1 minute and the excess material removed with filter paper. The grids were subsequently studied with a Philips 200 electron microscope using 60 KV. Serum Complement Studlas. All serum samples were frozen immediately at -7O’C until they were tested. Actii vator of C3 proactivator of the alternate pathway, in whole serum was determined by the immunoelectrophoretic technic of Scheidegger [6]. Fresh normal serum (NHS) and zymosan activated NHS served, respectively, as neg ative and positive controls. After subjecting control and sample serums to electrophcresis, troughs were filled with anti-C3 activator (Behrlngwerke). Appearance of the C3 activator with gamma mobility, similar to that seen in zymosan-actlvated serum, indicated activation of C3 proactivator.

RESULTS Characterization plex.

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Cryoprotein

of the Cryoproteln Immune Comimmune complexes

consisting of

CRYOPROTEHS

immunoglobulins IgG, IgM and IgA, complement components C3, C4 and C5, and specific EB virus antibody were detected during the urticarial rash phase of the patient’s illness (Table I). These circulating immune complexes disappeared with resolution of the rash. lmmunoglobulins and complement components were undetectable in similarly handled cold precipitated serum obtained from two healthy control subjects. Additionally, the total protein concentration of immune complexes was highest during the rash phase and decreased as symptoms resolved. The subtypes of IgG in the circulating immune complexes were quantitatively measured by radial diffusion using specific antiserums against IgG1, IgG-2, IgG-3 and IgG-4. Table I shows that the subtypes of IgG in the immune complexes obtained from this patient were the complement fixing IgG 1, IgG2 and lgG3. These immune complexes were then examined by electron microscopy. Numerous particles measuring approximately 115 nm in diameter were identified. One such particle is shown in Figure 1. The particles had a center nucleoid and an outer coat consistent with previous description of EB viral particles. Alternate comComplement Sequence Activation. plement pathway activation was demonstrated by the identification of the C3 activator fragment of C3 proactivator in serum obtained during the acute phase. Figure 2 depicts the C3 activator with gamma electrophoretic mobility in serum obtained during the acute phase when the urticarial rash was present. We could not demonstrate alternate pathway activation in serum obtained during recovery. COMMENTS Our sequential study in a patient with infectious mononucleosis demonstrates that circulating cryoprotein complexes were present during the early phase of his illness. The physical characteristic of cryoprecipitation allowed us to isolate, purify and characterize the components in circulating immune complexes. More importantly, the presence, composition and concentration of these circulating immune complexes correlated with the clinical findings of an extensive urticarial rash which suggests an etiologic relationship. These circulating immune complexes were composed of all three classes of immunoglobulin, complement components C3, C4 and C5, EB virus antibody and probably EB virus. It is unlikely that the EB virus capsid antibody titers present in the cryoprotein immune complex represent a nonspecific contamination since, on day 53 (recovery), high titers of EB virus capsid antibody were detectable in the serum but were not detectable in cold precipitated serum. The lack of nonspecific serum contamination was

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ASSOCIATED WITH INFECTIOUS MONONUCLEOSIS-WANDS

ET AL.

mune complex revealing a viral particle with a dense outer core and apparent central nucleoid. Magnification X 2 13,000, reduced by 23 per cent.

also demonstrated by the absence of serum albumin and other serum proteins in the cryoprotein immune complexes on counterelectrophoreses. The electron microscopic appearance of the particles seen in the isolated immune complexes is consistent with complete EB virus [ 91. Furthermore, the demonstration of specific EB virus capsid antibody in the immune complexes supports our morphologic observation that we were viewing EB viral particles. Finally, we have used the cryoprecipitation technic to demonstrate antigenic components in circulating immune complexes in other disease states in which immune complex formation appears to play a role in the pathogenesis [2, 101. Preformed antigen-antibody immune complexes activate both the alternate and classic complement

Figure 2. A, C3PA activation by demonstrating the C3 activator fragment with gamma ektrophoreric mobility in serum obtained from a patient with infectious mononuckosis and urticarial rash. El, C3 activator fragment was undetectable after recovery (day 53).

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pathways in vitro [ 11, 121. We have demonstrated that circulating immune complexes in infectious mononucleosis may activate both the classic and alternate (properdin) complement pathways since C4, an early component of the complement sequence, was found in the cryoprotein immune complex, and the C3 activator fragment of the properdin complex was demonstrable in the serum during the acute phase of the patient’s illness when circulating immune complexes were present. We could not demonstrate alternate complement pathway activation during

ET AL.

recovery when immune complexes were undetectable. Thus, our study suggests that EB virus may be present in the blood with the subsequent formation of circulating immune complexes composed of IgG (complement fixing subtypes IgEl, IgG-2 and IgG-3) IgM, IgA, C3, C4, C5 and EB virus capsid antibody. These immune complexes activate both the classic and alternate complement pathways and appear important in the pathogenesis of the occasionally observed urticarial rash in infectious mononucleosis.

REFERENCES 1. Glade PR: Infectious Mononucleosis: Proceedings of a Symposium, Philadelphia, J.B. Lippincott & Co., 1973, p 5. 2. Wands JR. Mann E, Alpert E, et al.: The pathogenesis of arthritis associated with acute HB.Agposltive hepatitis. Complement activation and characterization of circulating immune complexes. J Clin Invest 55: 930, 1975 3. Alpert E, lsselbacher KJ, Schur PH: The pathogenesis of arthritis associated with hepatitis. N Engl J bled 285: 185, 1971. 4. Gocke DJ, Howe C: Rapid detection of Australia antigen by counterimmunoelectrophoresis. J lmmunol 140: 1031, 1970. 5. Lowry OH, Rosebrough NJ, Farr AL, Randall, RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 287, 1970. 6. Vyas GN, Shulman NR: Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170: 332.1970.

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7. Henle G, Henle W: lmmunofluorescence in cells derived from Burkii’s lymphoma. J Bacterial 91: 1248, 1966. 8. Scheldegger JJ: Une micro-m&hod0 de I’immuno-&rctrophorese. Int Arch Allergy 7: 103, 1955 9. Epstein MA, Achong BG: The EB virus, Burkitt’s Lymphoma (6urkitt DP, Wright DH, eds), Edinburgh and London, E. & S. Livingstone, 1970, p 231. 10. Wands JR, LaMont JT, Mann E, lsselbacher KJ: The pathogenesis of arthritis associated with the jejunal-ileal bypass procedure for morbM obesity: Complement activation and characterization of circulating immune complexes. Clin Res 22: 696, 1974. 11. Ruddy S, Gigli I, Austin KF: The complement system of man. N Engl J Med 287: 489; 545; 592; 642; 1972. 12. Sandburg AL, Gijtze 0, MDller-Eberhard HJ, Osler AO: Complement utilization by guinea pig y-l and y-2 immunoglobulins through the C3 activator system. J lmmunol 107: 920‘1971.

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Circulating immune complexes and complement sequence activation in infectious mononucleosis.

Circulating Immune Complexes and Complement Sequence Activation in Infectious Mononucleosis JACK R. WANDS, M.D. JOSEPH L. PERROTTO, M.D. KURT J. ISSE...
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