Inherited deficiency of C8 in a patient with recurrent meningococcal infections: Further evidence for a dysfunctional C8 molecule and nonlinkage to the HLA system

Journal of Clinical Immunology, Vol. 3, No. 1, 1983

Inherited Deficiency of C8 in a Patient with Recurrent Meningococcal Infections: Further Evidence for a Dysfunctional C8 Molecule and Nonlinkage to the HLA System

P. D E N S E N , ~ E. J. BROWN, 2 G. J. O ' N E I L L , 3 F. TEDESCO, 4 R. A. CLARK, 1 M. M. FRANK, 2 D. WEBB, 5 and J. MYERS 6

Accepted: September 1, 1982

An adult male with recurrent meningococcal infections is reported whose serum lacked functional C8 activity but possessed antigenic C8. The addition of 1500 U of purified C8/ml of serum restored hemolytic activity to normal. Four to five times more C8 was required to restore bactericidal activity than to restore hemolytic activity. Bactericidal activity could also be restored by mixing the patient's serum with a second C8-deficient serum that lacked detectable antigenic or functional C8. The patient's serum contained bactericidal antibody for groups A, B, C, and Y meningococci and specific antibody to group Y capsular polysaccharide. There was two to three times more bactericidal antibody activity in the serum than in a pool of normal sera for the infecting strain. Family studies disclosed a sibling who was HLA identical to the patient but whose serum contained normal amounts of total hemolytic and C8 functional activity. KEY WORDS: C8 deficiency; recurrent meningococcaI infection; HLA linkage.

1Evans Memorial Department of Clinical Research and Depart- ment of Medicine, Boston University Medical Center, Boston, Massachusetts 02118.

2Laboratory of Clinical Investigation, National Institute of Aller- gy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205.

3Memorial Sloan-Kettering Cancer Center, New- York, New York 10021. 4Centro Trasfusionale e di Immunologia dei Trapianti, Ospedale Policlinico, Milano, Italy.

5Department of Medicine, U.S. Public Health Service Hospital, Boston, Massachusetts 02135.

6Department of Medicine, Mountainside Hospital, Montclair, New Jersey 07042.

INTRODUCTION

The complement system is an important element in host defense, the inflammatory response, and the production of tissue injury (1). Investigations of patients with complement deficiency states have been particularly valuable in elucidating the role played by the system as a whole as well as by individual components in each of these important responses (1, 2). Clinical and laboratory investigations have established that patients with early complement component deficiencies (through C3) have an increased f requency of autoimmune disorders and, in some instances, of bacterial infection caused by multiple pathogens (2). The latter association is believed to be a direct consequence of impaired antimicrobial defenses, while the former association may reflect genetic linkage of some of the early complement components to the H L A system. Pa- tients with a deficiency of one of the terminal complement components are selectively susceptible to systemic infections caused by Neisser ia but not other bacteria (3). An association between deficiencies of terminal complement components and autoimmune disorders is not as striking as with the early component deficiencies (4). Studies of a possible genetic linkage between deficiencies of the late components and the H L A system have yielded contradictory results but, in general, have not sup- ported linkage (5-9). In this paper, we report the results of the immunologic and genetic evaluation of an individual with recurrent Neisser ia meningi t idis infection whose complement deficiency is due to a dysfunctional C8 molecule.

90 0271-9142/83/0100-0090503.00/0 ~ 1983 Plenum Publishing Corporation

C8 DYSFUNCTION AND MENINGOCOCCAL DISEASE 91

Case Report. This previously healthy, Caucasian male experienced his first episode of meningococcal infection at 16 years of age. The details of that illness were published previously to illustrate the management of disseminated intravascular coagulation (10). Briefly, following a 3-day prodromal illness, the patient developed shock with meningeal signs and a purpuric rash. Laboratory values showed a mild leukocytosis, pronounced thrombo- cytopenia, markedly prolonged prothrombin and partial thromboplastin times, and elevated fibrin degradation products. The cerebrospinal fluid (CSF) contained 42 leukocytes/mm 3, 66% of which were neutrophils, but the glucose and protein were normal. Cultures of the CSF and blood were negative but a nasopharyngeal culture yielded group C N. meningitidis. Therapy with fluid replacement, antibiotics, steroids, and heparin resulted in gradual improvement and the patient was discharged after 27 days, with eventual complete recovery.

He developed his second episode of meningococcal infection 5 years later, at age 21 years, during a stint in the coast guard. Over the course of a day, he developed gastrointestinal symptoms, meningeal signs, and a petechial, purpuric rash. Although alert on admission, he rapidly became comatose and his course was complicated by grand real seizures. The peripheral blood contained 25,300 leukocytes and 86,000 platelets/mm 3, and a mild abnormality of clotting function was evident. The CSF contained 131 leukocytes/mm 3 (32% neutrophils); the glucose and protein were 68 and 52 mg/dl, respectively. Cultures of the blood and CSF grew group Y N. meningitidis. Treatment was initiated with antibiotics, dilantin, and mannitol. Repeat lumbar puncture on hospital day 3 demonstrated a CSF formula more typical of bacterial meningitis with a marked leukocytosis, moderately elevated protein, and de- pressed glucose. Despite antibiotic therapy, culture of the CSF remained positive. CSF obtained on hospital day 7 was sterile and demonstrated a marked improvement in all laboratory parameters. Skull films and examination for a CSF leak were negative. Serum immunoglobulins were normal. The total hemolytic complement was < 10 hemolytic units (normal, 99-126). The patient was discharged after 2 weeks of therapy and 1 week of observation off antibiotic therapy.

One week following discharge, he again developed fever, vomiting, and meningeal signs. There was no rash but an acutely enlarged, tender anterior cervical lymph node was noted. Studies of clotting

function were normal. The CSF contained 39 leukocytes/mm 3, 89% of which were lymphocytes, and the glucose and protein were 58 and 48 mg/dt, respectively. Cultures and gram stain of the CSF were negative for organisms but blood cultures grew N. meningitidis. The isolate was not sero- grouped. The patient defervesced promptly with antibiotic therapy. At the time of discharge, additional therapeutic measures included bivalent-meningococcal vaccine, fresh-frozen plasma to restore complement function, and prophylactic minocy- cline and rifampicin, which were continued for 4 weeks.

Two months following the discontinuation of prophylactic antibiotics, he presented to another hospital with fever, myalgias, vomiting, diarrhea, headache, meningismus, and rash and was thought to have aseptic meningitis. On transfer the next day, he was lethargic, febrile, and hypotensive and had a purpuric truncal rash. Petechiae were present on mucosal membranes and he again had tender right cervical and supraclavicular adenopathy. Lab- oratory studies demonstrated a leukocytosis of 40,000/ram 3 with a marked left shift. The platelet count was normal but the prothrombin and partial thromboplastin times were moderately prolonged. The CSF contained 3 cells/ram 3, of which 2 were neutrophils; the glucose and protein were normal. Cultures of the CSF and blood were negative. Dramatic clinical improvement occurred within 24 hr of starting penicillin, which was then continued for 2 weeks. The patient again received meningococcal vaccine and was discharged on antibiotic prophylaxis. One year later prophylaxis was dis- continued, and the patient has remained healthy during the ensuing 2 years.

MATERIALS AND METHODS

Sera. Blood was collected from the patient and members of his family, allowed to clot at room temperature for t hr, and centrifuged at 1500g for 15 rain at 4°C. The serum was removed and stored in small aliquots at -70°C. In some studies, a pool of normal human sera (PHS) from I0 men and 10 women who were not taking antibiotics and who denied any history of neisserial infection was used as a control.

Meningococci. The identity of the group Y meningococcus isolated from the patient during his second meningococcal infection was reconfirmed by sugar fermentation, positive oxidase test, typical

Journal o f Clinical l?mrnunology, Vol. 3, No. 1, 1983

92 DENSEN, BROWN, O'NEILL, TEDESCO, CLARK, FRANK, WEBB, AND MYERS

appearance on gram stain, and serogrouping using anticapsular antibody specific for group Y meningococci. This strain was used in most studies employing bacteria. Where indicated, other clinical neisserial isolates were utilized. Neisseria were grown to log phase in proteose peptone broth containing 0.82% glucose and washed twice in 0.01 M potassium phosphate buffer, pH 7.0, containing 0.1% bovine serum albumin prior to use.

Neutrophils. Human polymorphonuclear neutrophils (PMN) were purified from venous blood by dextran sedimentation and hypotonic lysis of the contaminating erythrocytes. This method routinely yields leukocyte preparations that are 85-90% neutrophils.

Assays of Complement Function. Total hemolytic complement activity in serum was measured by the method of Mayer (11) and expressed as the reciprocal of the serum dilution sufficient to lyse 50% of the sensitized sheep erythrocytes (Z = 0.69) used in the assay. In some experiments, deficient serum was reconstituted with partially purified human C8 (Cordis Laboratories, Inc., Miami, FL) prior to the determination of total hemolytic activity. Assays of total hemolytic complement were performed in Veronal-buffered saline (t2).

The titer of functional C8 in serum was measured by a previously described method (12). Briefly, sheep erythrocytes sensitized through C7 (EAClgp4-7hu; Cordis Laboratories, Inc.) are incubated with serial twofold dilutions of the serum being tested in the presence of excess C9 (Cordis Laboratories, Inc.). Functional C8 activity is expressed as the reciprocal of the serum dilution sufficient to provide one hemolytic site per celt (Z = 1.0). Hemolytic C8 assays were carried out in low- ionic strength (Ix = 0.065) dextrose-veronal-buffered saline (12).

The functional activity of other complement components in the patient's serum was determined by previously described methods (12). The concentration of factor B in serum was determined by radial immunodiffusion.

HLA Typing and HLA-Linked Marker Studies. Family members were genotyped for HLA-A, -B, and -C locus antigens by the standard two-stage microcytotoxicity typing technique using a large battery of well-characterized antisera. HLA-DR typing was carried out using B- cells prepared by filtering peripheral blood lymphocytes through nylon-wool columns (13) in a modified two-stage microcytotoxicity test with VIII International Work-

shop reagents. HLA-D typing was performed in a one-way system using 5 x 10 4 responding and 5 x 10 4 stimulating cells. The stimulating cells were irradiated with 2000 rads as previously described (14). Typing for the HLA-linked complement markers, C2, C4, and BF, and the red-cell enzyme, GLO, was carried out by previously described methods (15-18).

Serum Bactericidal Assay. Either 2 x 106 or 2 x 10 7 meningococcal colony-forming units were inocu- lated, respectively, into 10 or 30% human serum in either Hanks balanced salt solution (GIBCO Labo- ratories, Grand Island, NY) or 0.01 M potassium phosphate buffer containing 0.1% bovine serum albumin in a final volume of 0.25 ml. The reaction mixtures were tumbled (12-15 rpm) at 37°C and survival of organisms was quantitated after 60 min by plating aliquots on gonococcal agar base (Difco Laboratories, Detroit, MI) containing 1% isovitalex supplement. Survival is expressed as a percentage of the inoculum at time zero.

Opsonophagocytic Assay. Human neutrophils (2 X 106) were added to 10% human serum in 0.01 M phosphate buffer containing 2 x 10 6 meningococcal colony-forming units in a total volume of 0.25 ml and the mixture was tumbled (12-15 rpm) at 37°C for 60 min. Survival, assessed by dilution plating on agar, was expressed as a percentage of the inocu- lure at time zero.

Assays for Meningococcal Antibody. Antibody titers to groups A, B, C, and Y meningococci were kindly determined by Dr. Carl E. Frasch (Bureau of Biologics, FDA, Bethesda, MD) using a previously described microbactericidal assay (19). In separate experiments, the titer of bactericidal antibody activity for group Y meningococci in the patient's serum was compared to that in PHS. In these experiments, serial dilutions of heated serum (56°C, 30 rain) serving as the antibody source were mixed with 5% hypogammaglobulinemic human serum serving as a complement source. The hypogammaglobulinemic serum by itself was devoid of bactericidal activity for the group Y meningococcus at this concentration. These serum mixtures were then employed in the low-inoculum bactericidal assay described previously. These assays detect total bactericidal antibody activity in serum but not antigen-specific activity. The amount of anticapsular antibody to group Y meningococci present in the patient's serum was kindly determined by Dr. J. MacLeod Griffiss (Channing Laboratory, Boston, MA) using a previously described technique (20) which mea-



Table I, Immunoglobulin Concentration in Patient Serum and Pooled Fractions of Sera o

Ig concentration (mg/dl)

Ig source IgG IgM IgA

PHS 1450 52 100 IgG pool 450 <5 <4.2 IgM pool <18 64 <4.2

C8D 620 190 50 IgG pool 1750 <5 <4.2 IgM pool <18 96 2.3

-Delipidated sera were fractionated by sequential molecular sieve and ion exchange chromatography. Appropriate fractions were pooled and concentrated and the immunoglobulin concentration was determined by radial immunodiffusion. Ig, immunoglobulin; PHS, pooled human serum; C8D, serum deficient in the eighth complement component.

sures the ability of the test serum to precipitate radioactively labeled group Y capsular polysaccharide of a known specific activity. The class of antibody responsible for bactericidal activity was assessed by purifying IgG and IgM from PHS and the patient's serum by a combination of molecular- sieve and ion-exchange chromatography as previously described (21). The purity of these preparations is shown in Table I. An amount of immunoglobulin equivalent to 30% of its concentration in PHS was added to 30% hypogammaglobulinemic serum in 0.01 M phosphate buffer containing 0.1% bovine serum albumin. This reconstituted hypogammaglobulinemic serum was then utilized in the high-inoculum bactericidal assay described above.

Neutrophil Function Studies. Previously described techniques were utilized to assess oxygen consumption (22) and chemotaxis (23) in response to a standard stimulus.

R E S U L T S

Studies o f the Patient's Complement System. The patient's serum lacked hemolytic activity in both the total hemolytic assay and the C3-C9 assay, which provides a measure of terminal component activity. Further studies (Table II) demonstrated that the patient lacked functional C8 activity.

Hemolytic activity in the patient's serum could be restored to normal by the addition of 1500 U of partially purified (Cordis) C8/ml of serum. In con- trast, in excess of 2000 U of (Cordis) C8/ml of serum was required to restore complement-dependent bactericidal activity in the patient's serum to a level comparable to that in PHS for his own infecting meningococcal isolate. The precise amount of Cor-

dis C8 required to restore serum bactericidal activity completely could not be ascertained due to inhibition by other constituents contained in the relatively large amounts of the C8 preparation required in the assay. The addition of 6666-8333 U of highly purified C8 (activity adjusted to that in the Cordis preparation by the ability to restore total hemolytic activity in C8-deficient serum to normal) restored bactericidal activity in the deficient serum to normal.

The possibility that the patient's serum might contain a functionally nonreactive but immuno- chemically reactive C8 molecule was examined using rabbit anti-human C8 (Fig. 1). The patient's serum reacted with the antibody to give a line of partial identity with C8 in normal serum. No immunoreactive C8 was detected in the serum from a previously reported patient with C8 deficiency examined concurrently using the same antiserum as well as three additional antisera with C8 specificity. These results suggested that the biochemical basis for these two deficiencies might be different. Conse- quently, equal volumes of the two C8-deficient sera were combined and the mixture was examined for bactericidal and hemolytic activity. The results of these studies (Table III) demonstrate that, although neither deficient serum alone possesses significant activity, the mixture of the two has potent bactericidal and substantial hemolytic activity.

Studies of the Complement System and Genetic Markers in the Patient's Family. The results of investigations of the complement system, HLA genotype, and genetic markers for the sixth chromosome in the patient and his family are presented in Tables IV and V. In spite of the fact that the mother and father are obligate heterozygotes for C8

Table II. Complement Levels in C8-Deficient Serum-

U/ml

Component assay Patient Normal

Total hemolytic complement (CHso) 0 58-103 C3-C9 0 20-40 C3 69,000 50,000-100,000 C5 137,000 100,000-300,000 C6 18,000 20,000-70,000 C7 450,000 200,000-500,000 C8 0 1.9,000-71,000 C9 24,570 10,000-20,000

Factor B 17.4 mg/dl 13-22 mg/dt

~Functional complement assays were performed as described under Materials and Methods. Factor B was quantitated by radial immunodiffusion.


94 DENSEN~ BROWN, O'NEILL, TEDESCO, CLARK, FRANK~ WEBB~ AND MYERS

Fig. 1. Examination of sera deficient in the eighth component of complement (CSD) for immunoreactive C8 by double diffusion in agarose. The central well contains rabbit antihuman C8. Peripheral wells 1 and 2 contain pooled normal human serum; wells 3 and 6, C8D~ (subject of the present report); and wells 4 and 5, C8Dz [subject of a previous report (33)].

deficiency, there is no clear correlation among the heterozygous state, functional C8 activity, and total hemolytic activity. The data in these two tables also indicate that the patient has an HLA-identical brother. Despite identity at the HLA loci and other loci on the sixth chromosome, the patient and his brother clearly differ with respect to C8 activity. These studies provide strong evidence for the lack of linkage of the C8 locus with HLA loci.

Table III. Reconstitution of Complement-Dependent Serum Bactericidal and Hemolytic Activity in Two C8-Deficient Sera.

CHso Condition % survival ~ (U/ml) c

30% PHS <0.04 68.5 15% PHS 5.2 - - 15% C8D1 >161 0 15% C8D2 84.5 0 15% C8D1 +

15% C8D~ 2.0 75

aResults represent the means of two separate experiments performed in duplicate. PHS, pooled human serum; C8D, serum deficient in the eighth component of complement; C8D~, subject of the present report; CSD~, subject of a previous report (33).

bLog-phase group Y meningococci (2 × 107 colony-forming units) isolated from patient C8D~ were incubated for 1 hr under the stated conditions. Survival is expressed as a percentage of the initial inoculum.

"Total hemolytic complement.

Studies of Antimeningococcal Antibody. There was no serum available from the patient at an early stage of his illness, so all studies of antimeningococcal antibody were performed using serum obtained from the patient after his recovery. This convales- cent serum had serogroup-specific antibody at titers of >--1/512 for group A, 1/128 for group B, 1/256 for group C, and 1/64 ~br group Y meningococci. The serum had a capacity to bind 58 ng of group Y capsular polysaccharide/25 txl. Bactericidal antibody specific for the infecting meningococcal isolate was measured in PHS and in the patient's serum (Fig. 2). There was 2-2.5 times as much bactericidal antibody in the patient's serum as in PHS. The immunoglobulin class responsible for bactericidal activity was examined by reconstitut- ing hypogammaglobulinemic serum with IgG or IgM purified from the two sera (Table VI). The results of these studies clearly demonstrate that IgG is primarily responsible for the killing of the group Y meningococcus in C8-deficient serum, but they fail to identify the class of immunoglobulin responsible for bactericidal activity in normal serum. From these data, we cannot exclude the presence of some activity in the IgM pools since bacterial growth in IgM-supplemented hypogammaglobulinemic serum was inhibited relative to that in hypo-



Table IV. Totat Hemolytic Complement, Functional C8, HLA Typing, and Genetic Markers

CHso C8 (19,000- (58-103) ~ 7t ,000) ~ HLA genotype Bf Glo C4 C2 Chido Rodger

Patient b 0 0 A2; Bt5, (w6); Cw4; D R - SS 1-1 FS C + +

Aw33: B14, (w6); tA4+7

Father 58 41,500 A2; B15 (w6); Cw4; D R - SS 2-I FS C + +

A28; Bw35, (w6); Cw4; DRw4

Mother 56 21,250 A2; Bw39(16), (w6); DRw4 SS 2-1 FS C + +

Aw33; B14, (w6); IA4+7

Brother 47 24,000 A2; BI5, (w6); Cw4: D R - SS 2-1 FS C + +

A2; Bw39(16), (w6); DRw4

Brother b 54 71,000 A2; BI5, (w6); Cw4; D R - SS 1-1 FS C + +

AW33; B14, (w6); IA4+7

Brother 47 43,500 A28; Bw35, (w6); Cw4; DRw4 SS 2-2 FS C + +

A2; Bw39(16), (w6); DRw4

aNormal range in parentheses. bHLA identical.

gammaglobulinemic serum alone. A possible explanation for this result is that the bactericidal locus on group Y meningococcal lipopolysaccharide, to which antibody in normal serum is commonly di- rected, is antigenicaily cross-reactive with the agarose beads used for the chromatographic separation of immunoglobulins from serum (24). In this cir- cumstance, the chromatographic gel functions as an affinity column to remove cross-reactive (bactericidal) antibody. That the antibody is not functionally altered during the separation procedure is demonstrated by the finding that purified IgM can kill a serum-sensitive gonococcus as previously reported (25) and that IgG purified from C8-deficient serum can kill the infecting meningococcal strain.

Opsonophagocytic Studies. As a consequence of the functional absence of C8 in the patient's serum,

opsonophagocytic bactericidal activity can be dis- tinguished from complement-dependent serum bactericidal activity. The patient's serum readily sup- ported opsonization and killing of the infecting strain of meningococcus by neutrophils (1.7 -+ 0.5% survival in C8-deficient serum plus neutrophils vs 154 -+ 42% survival in C8-deficient serum alone). Analogous studies employing PHS as the source of meningococcal opsonins proved impossible due to an inability to distinguish phagocytic killing from serum bactericidal activity even when tested over a range of serum concentrations.

Neutrophil Function Studies. Neutrophils isolated from the C8-deficient patient demonstrated normal oxygen consumption upon stimulation by phor- bol myristate acetate (17.0 -+ 3.0 ~I O2/106 PMN/hr vs the normal 17.3 -+ 2.2 txl O2/106 PMN/hr). The

Table V. Mixed Lymphocyte Culture (Thymidine Incorporation; cpm)

Stimulating cells

Responder Normal Pool Pool cells Patient Father Mother Brother Brother control I a 2"

Patient b 584 10,858 11,022 470 8,796 15,248 19,696 36,018 Father 17,489 764 27,988 9,224 17,948 30,340 31,472 37,600 Mother 21,546 29,862 3,866 17,002 28,496 35,088 41,902 38,030 Brother b 490 12,530 I6,550 640 8,764 22,104 20,552 27,676 Brother 21,024 19,482 22,822 15,188 1,676 27,528 30,864 35,262 Normal

control 36,856 32,264 43,262 27,902 28,106 524 43,928 42,850

"Pool 1 and Pool 2 are pools of lymphocytes from a large number of normal donors. bHLA identical.

Journal o f Clinical Immunology, Vol. 3, No. 1, 1983

96 DENSEN, BROWN, O'NEILL, TEDESCO, CLARK, FRANK, WEBB, AND MYERS

,2c y 100 t /

/

PHS 0 C8D

I 0 I 1 i I I ~ [ I i ~ I i ~ I l I I 2 40 100 200 400 1000

SERUM CONCENTRATION (reciprocal dilution)

Fig. 2. Bactericidal antibody for group Y meningococci in C8-deficient serum (CSD) and pooled normal human serum (PHS). Log-phase meningococci (2 × 106 colony-forming units) were incubated in a mixture of 5% hypogammaglobulinemic serum and serial dilutions of the test serum. Survival is expressed as a percentage of the initial inoculum. Results are the means -+ SE for three separate experiments performed in duplicate.

80 %

SURVIVAL 60

chemotactic response of the patient's neutrophils to zymosan-activated normal serum (1926 -+ 105 cpm) was normal or increased compared with that of neutrophils from a normal donor. There was no evidence of a chemotactic inhibitor in the C8- deficient serum.

DISCUSSION

The association between recurrent systemic infection caused by Neisseria gonorrhoeae or Neis- seria meningitidis and an inherited deficiency of one of the terminal complement components C5-C8 has recently received emphasis in the literature (3). This patient's clinical history prompted us to evalu- ate the possibility of, and ultimately to document, a deficiency of C8 as an explanation for his recurrent episodes of meningococcal infection. Of particular interest was the observation that despite the complete absence of functional C8 activity, his serum contained immunoreactive C8. This material pro-

duced a line of partial identity with normal C8 in an Ouchterlony gel and presumably reflects the presence of a dysfunctional C8 molecule. The C8 molecule is unusual among complement proteins in that it is composed of three polypeptide chains (26, 27), two of which (a and ~/) are convalently linked and noncovalently associated with the third ([3) chain (27). The t3 chain mediates binding of the C8 mole- cute to the C5-C7 complex (28, 29). Deficiency of C8 due to a dysfunctional molecule has been reported (30-32), and the nature of this defect has been delineated recently by Tschopp and associates, who established that three such individuals lacked the C8 13 subunit (32).

Our data, in conjunction with the reported literature (30-36), suggest that there are at least two different types of C8 deficiency, one in which there is a dysfunctional C8 molecule detected antigenical- ly and one in which no immunoreactive material can be detected by conventional means. The latter type of C8 deficiency has been reported to represent the



Table VI. Immunoglobulin Class Responsible for Bactericidal Activity -

%survival

Condition Meningococcus ~ Gonococcus ~

AGS 237 71.8 +IgGpHs 242 60.6 + IgMFHs 123 0.1 +IgGcso 8.2 ND + IgMcsD 92 ND

~qmmunogtobulins were separated from serum by chromatography and used to reconstitute hypogammagtobulinemic serum (AGS) equivalent to 10% normal serum. Log-phase (2 x 10 6 colony-forming units) meningococci or gonococci were added to the mixture and survival was measured at t hr. Survival is expressed as a percentage of the initial inoculum. PHS, pooled human serum; C8D, serum deficient in the eighth complement component; ND, not determined.

~Results are the means of two separate experiments. cResults are the means of one experiment performed in duplicate.

complete absence of the C8 molecule (33-36). Nei- ther type of C8-deficient serum possesses complement-dependent serum bactericidal or hemolytic activity by itself. However, a combination of equal volumes of these two C8-deficient sera restored normal bactericidal and hemolytic activity to the mixture. These experiments suggest the possibility that the two sera are deficient in different portions of the C8 molecule and that functional C8 activity is reconstituted when the deficient sera are combined. Precedent for this possibility has been established by the demonstration that hemolytic C8 activity can be reconstituted in vitro by mixing either the three purified polypeptide chains or the ~-~ and [3 sub- units together at equimolar ratios (27). Further studies defining the biochemical basis for the C8 defect in these two C8-deficient sera are in progress and preliminary results (37) from these experiments support the hypothesis that the two C8-deficient sera lack different portions of the C8 molecule.

Bactericidal activity and hemolysis are believed to be analogous expressions of complement activity differing only in the nature of the target cell. Thus, the finding that the amount of C8 required to restore bactericidal activity to the deficient serum was greater than that required to reconstitute hemolytic activity was unexpected. These results could reflect technical differences Such as the use of optimal concentrations of antibody to sensitize erythrocytes but not bacteria or the ratio of target cells to serum in the two assays. Alternatively, less complement might be required to produce a lytic site in the lipid

bilayer of an erythrocyte than to produce a bactericidal hit in the multilayer cell wail of a bacterium.

The family studies disclosed a sibling who was HLA identical to the patient but who had normal total hemolytic complement and functional C8 activity; therefore, the genetic locus for C8 is not HLA linked. Thus, our results support those of other investigators who have noted that C8 (6, 9), like other terminal complement components (7, 8), does not exhibit linkage with the HLA system.

The patient's clinical course is notable in several respects. First, he survived four separate episodes of systemic meningococcal infection despite the high mortality associated with this disease (38). Second, the severity of his illness appeared to decrease with successive episodes, suggesting that the immune response might ameliorate the complement defect, perhaps by enhancing the host defense mechanisms. The third episode of infection probably represented a relapse, as judged by its temporal relation to the previous episode and to the discontinuation of antibiotics. Similar episodes of relapse, although uncommon, have been reported in other complement-deficient patients (39, 40), suggesting that antibiotics should be continued for a prolonged period in these patients. The use of bivalent A-C meningococcal vaccine failed to prevent a fourth episode of infection but, since an organism was never isolated during this infection, one cannot conclude that vaccination was without value. Nev- ertheless, many of the reported cases of meningococcal infections in complement-deficient patients have been caused by group Y strains (40), against which this vaccine would not offer protection. This association between group Y meningococcal infection and deficiencies of terminal complement components may reflect the older age of the patients reported in the literature since group Y disease is predominantly a disease of older individuals (41).

The titers of antibody to groups A, B, C, and Y meningococci in our patient's serum were sufficient to be considered protective in normal circum- stances. One complement-deficient patient reported in the literature (42) failed to respond to group Y meningococcal infection with the production of specific capsular antibody. She did respond with specific antibody production when immunized with group Y capsular polysaccharide. Our patient had substantial levels of specific antibody to the group Y capsular polysaccharide; thus, it appears that his ability to respond with a specific immune response is preserved. The fact that the bactericidal antibody

Journal o f Clinical Immunology, Vol. 3, No. 1, 1983

98 DENSEN, BROWN, O'NEILL, TEDESCO, CLARK~ FRANK, WEBB, AND MYERS

present in his serum was of the IgG class, whereas the bactericidal antibody in the pool of normal serum was probably IgM, likely reflects the low prevalence of specific antibody to group Y meningococci in the population.

ACKNOWLEDGMENTS

We are i ndeb t ed to Dr. G. F. Brooks , San F r a n -

cisco, for his g e n e r o u s gift o f the s econd C8-

deficient serum. This work was suppor t ed in par t by

Gran t AI16476 f rom the Na t iona l Ins t i tu tes of

Heal th .

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Documents

Inherited deficiency of C8 in a patient with recurrent meningococcal infections: Further evidence for a dysfunctional C8 molecule and nonlinkage to the HLA system