Antibody deficiency with normal immunoglobulins in a child with hypoplastic anemia

CLINICAL

IMMUNOLOGY

Antibody

AND

IMMUNOPATHOLOGY

Deficiency

with Normal lmmunoglobulins with Hypoplastic Anemia

ALAN P. KNUTSEN**? Department

of Pediatrics, Hematology-Oncology,

36, 330-337 (1985)

Divisions

AND DENNIS

in a Child

M. O’CONNOR*+

of *Allergy and iClinical St. Louis University Medical St. Louis, Missouri 63104

Immunology and $Department Center, 146.5 S. Grand,

of

We report a young child with hypoplastic anemia and antibody deficiency with normal serum immunoglobulin levels. Studies of antibody responses to bacteriophage +X 174 revealed an amnestic response but was predominantly limited to IgM. A similar response was observed following booster tetanus toxoid immunization. Analysis of in vitro pokeweed mitogen-induced immunoglobulin synthesis by the patient’s B cells suggested an intrinsic B-cell defect. 0 1985 Academic Press. Inc.

INTRODUCTION Congenital hypoplastic anemia (CHA)’ is a rare red cell aplasia presenting in early infancy characterized by a marked paucity of erythroid precursors beyond the level of proerythroblasts (1, 2). There is persistence of fetal hemoglobin (HbF), macrocytosis, and “i” antigen as well as other red cell enzymes characteristic of fetal-newborn erythrocytes. In addition, a short, webbed neck similar to that seen in Turner’s syndrome and abnormalities of the thumb are frequently observed. Associated humoral immunodeficiency is a rare association, having been reported only twice (3, 4). Though the etiology of CHA is unknown, an intrinsic erythroid progenitor cell defect is the most likely cause (5-8). In this report, we describe a child with CHA, transient hypoplastic neutropenia, and an intrinsic B-cell immunodeficiency, characterized by normal serum immunoglobulins and deficient antibody function. CASE REPORT M.T., a 4-year-old black female, was referred to St. Louis University Medical Center at 6l/2 months of age for evaluation of a macrocytic anemia and pneumonia. Laboratory studies (Table 1) revealed a low hemoglobin (3.7 gm/dl), a low reticulocyte count (O.S%), elevated MCV (9.3 Fm3), weakly positive indirect Coombs test, and normal serum folate (4.8 mg/ml), vitamin B,, (838 pg/ml) concentrations, and serum erythropoietin (38 mu/ml). The patient had normal numbers of neutrophils, lymphocytes, and platelets. Chromosome analysis was performed for ’ Abbreviations used: AET-SRBC, aminoethylisothiouronium bromide-treated sheep red blood cells; CHA, chronic hypoplastic anemia; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; HbF, fetal hemoglobin; Ig, immunoglobulin; IVIG, intravenous gammaglobulin; MCV, mean cellular volume; MNC, mononuclear cells; PBS, phosphate-buffered saline; PMN, polymorphonuclear cells; PWM, pokeweed mitogen; WBC, white blood cells. 330 0090-1229/85 $1.50 Copyright All rights

8 1985 by Academic Press, Inc. of reproduction m any form reserved.

ANTIBODY

PERIPHERAL

BLOOD

331

DEFICIENCY

TABLE 1 CELLS IN A PATIENT WITH HY~~PLASTIC HUMORAL IMMUNODEFICLENCY

ANEMIA

AND

Age (months)

Hb,

gm/dl

MCV, Frn’ WBC/mm3 PMN/mm3 Lymphocytes/mm’ Reticulocyte, % HbF, % Erythropoietin, mu/ml

7

8

13

3.1 93 8700

6.3 88 6600 264 5412 1.0

8.1 88 9400 5264 1974

4611

2262 0.8

0.1

20 5.5 105 15,100

9.815 3,020 2.0

13.5

17.5

24 12.2

44 10.6

104

104

7300 2555 3869

7500 2625 3450 0.8 7.4

1.4

38

suspected Turner’s syndrome, on the basis of dysmorphic features consisting of short, webbed neck, hypoplastic and wide-spaced nipples, depressed nasal bridge and mild pectus excavatum. The patient’s karyotype was 46XX with normal banding. The patient received packed red blood cell transfusions and intravenous ampicillin. Respiratory synctial virus was isolated from a nasopharyngeal culture, and antibiotics were discontinued. At 8 months of age, the child was hospitalized with an Escherichia coli urinary tract infection. At this time she had developed neutropenia, 264 cells/mm’, concomitant with the persistent anemia. A bone marrow aspirate was done which showed markedly decreased erythroid and myeloid precursors. A diagnosis of CHA was made, and the patient received blood transfusions every 4 to 6 weeks. The neutropenia spontaneously resolved after 2 months. At 11 months of age, prednisone therapy, 2 mg/kg, was initiated for CHA. Thereafter, the patient began having significant bacterial infections. She was hospitalized for pneumonia at 12 months and then for a Haemophilus injluenzae osteomyelitis of the right hip at I3 months. The prednisone was discontinued thereafter because of an absence of a clinical response. The patient was readmitted at 18 months of age for a H. influenzae type b (blood isolate) pneumonia. Over the next 4 months, she had several episodes of otitis media, sinusitis, and pneumonia. At 23 months of age, intravenous gammaglobulin (Gammimmune; Cutter) was begun. In the past 2 years, the patient has had only two episodes of otitis media. Additionally, the anemia has resolved since beginning IVIG, though the macrocytosis and an elevated HbF persist. METHODS In vitro immunoglobulin synthesis was studied. In vitro pokeweed mitogen (PWM)-stimulated B-cell synthesis of immunoglobulin was performed as previously described (9). Briefly, mononuclear cells (MNC) were isolated from heparinized venous blood by Ficoll-Hypaque density centrifugation, and subpopulations enriched for T cells and for B cells were obtained by rosette depletion using AET-treated sheep erythrocytes and a second Ficoll-Hypaque centrifu-

332

KNUTSEN

AND O'CONNOR

gation step. Unfractionated MNC, 2 x IO6 cells, were placed in duplicate l-ml cultures in RPM1 1640 medium containing 10% fetal calf serum with and without PWM and held at 37°C in a humidified 5% CO, atmosphere for 10 days. Cocultures contained lo6 MNC and T cells or lo6 T cells and 0.5 x IO6 B cells from each donor. Culture supernatant IgM and IgG concentrations were measured by double antibody radioimmunoassay, using rabbit anti-human immunoglobulin heavy chain antisera (CappeIi Laboratories) as the first antibodies and a donkey antirabbit IgG (developed in this laboratory) as the second antibody for all assays. Data were transformed logarithmically for statistical analysis. RESULTS OF IMMUNOLOGICAL

STUDIES

Quantitative serum immunoglobulins (Table 2), initially obtained when the patient was 7 months old, revealed low normal IgG and IgA levels and an elevated IgM. This pattern persisted until she was 19 months when the serum IgG (860 mg/dl) and IgA (105 mg/dl) rose and the serum IgM remained elevated (200 mg/ dl). Serum IgG,, IgG,, and IgG, concentrations were also normal at 19 months (kindly measured by Dr. Moon Nahm, using a radioimmunoassay). The serum IgE was <5 IU/ml. Isohemagglutinins, anti-A and anti-B, were low (1:2) at 10 months and undetectable at 19 months. After the patient’s third diphtheria-pertussis-tetanus (DPT) immunization, the diphtheria antibody titer was I:81 (normal > 1:2000) and the tetanus antibody titer was 1: 19,603 (normal > 1: 10,000). Two weeks after a booster DT immunization, both adiphtheria and atetanus antibody titers rose as determined by a tanned-cell hemagglutination assay (I 0) to > 1:53 1,441. However, after 2-mercaptoethanol reduction, the titers to diphtheria and tetanus were reduced to I:729 and 1:6561, respectively, suggesting the majority of the antibody responses to be IgM. Likewise, IgG-specific antitetanus antibody, measured by an ELISA assay (1 I), revealed only a rise from 0.21 to 0.30 U/ml, also suggesting that the major increase in antibody was in the IgM isotype following the DT booster. The patient’s antibody responses following immunization with bacteriophage $X 174 (Fig. 1) was also evaluated (measured by Dr. Hans Ochs. using a phage neutralization method (12)). After the primary immunization at 19 months, the patient developed a normal antibody titer, but it quickly declined to a subnormal titer. Following the secondary immunization, an amnestic response was observed, but it was quantitatively deficient compared to normal control values with only 34% being IgG (Fig. lb). The patient had a normal percentage of B cells with 8% smIgM and 4% smIgD as determined by immunofluorescent studies. Analysis of T lymphocyte phenotypes revealed 79.7% OKT3+ (normal 74 ? 6) and 78.9% OKTl I ’ cells (normal 80 ? 5). OKT4+ (T helper) cells were 45.3% (normal 25 + 6). Blastogeneic responses of the patient’s isolated mononuclear cells to the mitogens phytohemagglutinin, conconavalin A, and pokeweed mitogen, and to the antigens Candida and tetanus toxoid were normal. To evaluate T-cell helper and suppressor activity and B-cell function, lymphocyte subpopulations from the patient and control were cocultured in vitro in the T-cell-dependent PWM-stimulated B-cell synthesis of immunoglobulin. The patient’s unfractionated MNC, compared to a normal adult control MNC, synthe-

360 16 320 <5

7

0.21

370 22 75 <5 I:2 I:2 81 19,603

10

0.30

531,441 531,441

425 16 250

13

0

0

0

860 105 200

19 269-913 8-54 27-80

loo0 170 56

>2-fold

>1:16b >I:8 >2,000 > 10,000

7-12

47 363- 1005 14-85 37-190

13-24

Normal values for age”

518-1447 23-137 43-198

36-60

u Normal age-related Ig values reported by the clinical laboratory. b Normal titer for >18 months. L Diphtheria and tetanus antibody titers are expressed as reciprocals of dilution performed by tanned RBC hemagglutination. Results are responses observed 2 weeks after the third and fourth immunizations, respectively. d Specific IgG antibody to tetanus toxoid by microtiter ELISA technique. Antibody response in 5 children was greater than twofold rise following tetanus toxoid immunization with mean 2.38 +- 0.13 (log,,).

IgG, mg/dl IgA, mg/dl IgM, mg/dl IgE. W/ml oA CXB aDiphtheria’ oTetan& aTetanusd (IgG-ELISA) U/ml AS0

Age (months):

Clinical values

TABLE 2 HUMORAL IMMUNE PROFILE

334

KNUTSEN

a

AND

O’CONNOR

b

Weeks

Weeks

FIG. 1. Antibody responses of patient (solid lines) after primary (a) and secondary (b) immunizations with bacteriophage 4X 174 compared to normal subjects (shaded areas). Although an amnestic antibody response was observed following the secondary immunization, only 34% was of the IgG isotype (Type IV response).

sized little IgM (102 vs 3150 rig/ml) or IgG (120 vs 1800 rig/ml) as seen in Fig. 2. Furthermore, when the patient’s B cells were cocultured with control T lymphocytes, providing normal T help, there was still markedly decreased IgM (30 ng/ ml) and IgG (325 &ml) secretion by the patient’s B cells. To examine for increased T-suppressor activity, the patient’s T cells were cocultured with control MNC cells. In this study, the patient’s T cells compared to an analogous coculture with control T cells enhanced IgM synthesis of control B cells (2290 vs 1370 ng/ ml) and suppressed IgG synthesis from 1100 to 460 rig/ml. DISCUSSION

The patient described in this report has a humoral immune defect characterized by specific antibody deficiency in the presence of normal serum immunoglobulins;

MNC

M&c

&

MNC

ML

i&l

FIG. 2. In vitro PWM-stimulated B-cell synthesis of IgM and IgG. Patient’s MNC (P) synthesized very little Ig compared to control’s MNC (C), and cocultures of patient and control MNC (Co) resulted in 20 and 38% suppression of expected IgM and IgG synthesis, respectively. The patient’s T cells (Tp) enhanced IgM and suppressed IgG synthesis (58%) by control MNC (MNCc), compared to similar culture with control T cells (Tc). The patient’s B cells (Bp) did not increase Ig synthesis when cocultured with normal T help (Tc + Bp).

ANTIBODY

DEFICIENCY

335

a hypoplastic anemia associated with dysmorphic features; and a transient neutropenia. In the spectrum of immunodeficiencies, this type of humoral defect is now well recognized (8, 13- 16). These patients have repeated episodes of bacterial infections, similar to patients with hypogammaglobulinemia. Though serum immunoglobulin levels are normal or near normal in these patients, there is a deficiency of antibody formation to specific antigens. Naturally occurring antibodies, such as to streptococcus antigen (ASO) and to isoagglutinins were absent in our patient and in other reported patients (8, 13-15). Some patients, however, may produce antibodies to some antigens, such as to AS0 and E. coli as reported by Rothbach et al. (14). It is of note, that the antibody to E. coli in that patient was determined to be mainly IgM. Likewise, antibody responses to immunizations with tetanus (8, 14, 15), typhoid (13, 14), pneumococcal polysaccharides (8, 14), and bacteriophage +X 174 (8, 16) are usually abnormal as well. Our patient, after repeated DT immunizations appeared to produce normal antibody titers to these antigens; however, further evaluation revealed diminished IgG antibody titers. This predominant IgM antibody response is consistent with the persistent IgM antibody response seen in the bacteriophage system in our patient and in others (8-16). Furthermore, in one patient reported by Ochs et al. (12) quantitatively normal antibody titers were seen following a third bacteriophage immunization. In vitro immunoglobulin synthesis studies in our patient suggested an intrinsic B-cell defect of IgM and IgG secretion associated with increased T suppressor cells for IgG synthesis. The patient’s MNC synthesized very little IgM and IgG compared to a normal control. Since the defect may be lack of T help, excessive T-suppressor or deficient B-cell activity, cocultures of T- and B-cell populations were performed. The patient’s B cells secreted little IgM or IgG when cocultured with allogeneic T cells from a normal control, suggesting an intrinsic B-cell defect of Ig synthesis or release. When the patient’s T cells were cocultured with control MNC, enhancement of IgM and suppression of IgG synthesis was observed. In similar studies, Saxon et al. (15) reported on two patients with specific antibody deficiency and normal serum immunoglobulins. They observed increased T-suppressor-cell activity for polyclonal and specific antitetanus IgG and IgM synthesis. Dissimilar to our patient, though, their patients’ B cells were able to synthesize normal quantities of immunoglobulins when cocultured with normal T help, suggesting abnormal T-cell suppression accounting for the antibody deficiency. However, in one patient, specific antitetanus IgG and IgM responses were absent, suggestive of an intrinsic B-cell defect. Undoubtedly, the functional defects of antibody synthesis in these patients is complex and may be at multiple sites, as it is in patients with common variable immunodeficiency. Our patient had a hypoplastic anemia characterized by macrocytosis, elevated fetal hemoglobin, and decreased erythroid precursors seen in the bone marrow aspirate. She also had dysmorphic features suggestive of Turner’s syndrome but with a normal chromosomal karyotype. These features are reminiscent of congenital hypoplastic anemia as described by Diamond et al. (1, 2). Neutropenia, found transiently in our patient, is rarely seen in this disorder. Hypoplastic neutropenia, though, has been well documented to occur in several hypogamma-

336

KNUTSEN

AND O’CONNOR

globulinemic disorders (17, 18). Our patient’s anemia also did not respond to corticosteroid treatment which it does in approximately 85% of patients with CHA (2). She did have a spontaneous recovery of anemia with persistence of an elevated HbF and macrocytosis. features observed in patients with CHA (2). Though cellular immune defects, such as nucleoside phosphorylase deficiency and thymoma have been associated with red cell aplasia, humoral immune deficits have rarely been reported. Brookfield and Singh (3) reported a child with CHA and hypogammaglobulinemia, and Feldman et ul. (4) reported a girl very similar to ours with recurrent infections, CHA, transient episodes of neutropenia, Turner’slike phenotype, and antibody deficiency with normal serum immunoglobulins. An intrinsic B-cell defect of PWM-stimulated immunoglobulin synthesis was found in our patient, which was not examined in the others. The etiology of CHA remains unknown, though an intrinsic defect of an erythroid progenitor cell seems to be most likely. Immune studies of patients with CHA have demonstrated normal T-helper and no excessive T-suppressor activity of erythroid burst- and colony-forming units on normal subjects’ progenitor cells (5-7). Unfortunately, similar studies in our patient were not obtained, especially since some increased T-cell suppression of IgG was seen. In summary, this patient seems to have abnormalities of three hematopoietic cell lines, namely hypoplastic anemia, hypoplastic transient neutropenia, and abnormal antibody responses. Though the defect remains unknown, significant imbalance of T-cell regulation was unlikely in this subject.

ACKNOWLEDGMENTS The authors are grateful to Ms. Kathleen M. Mueller for technical assistance, to Ms. Beverly Denison for secretarial skills, and to Dr. Herbert Krantman for review of the manuscript.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11.

Diamond, L. K., Allen, D.. and Magil, E B., Amer. J. Dis. Child. 102, 149. 1961. Diamond, L. D.. Wang, W. C., and Alter, B. P., Adv. Pediurr. 22, 349. 1976. Brookfield, E. G., and Singh, P., J. Pediatr. 85, 529, 1974. Feldman, K. W., Ochs, H. D., Price, T. H., Wedgwood, R. J., J. Pediutr. 88, 979, 1976. Nathan, D. G., Hillman, D. G., Chess, L., Alter, B. P, Clarke, B. J., and Housman, D. E.. N. Engl. J. Med. 298, 1049, 1978. Ershler, W. B.. Ross, J., Finlay, G. L., and Shahidi, N. T., N. Engl. J. Med. 302, 1321, 1980. Freedman, M. N., and Saunders, E. F., Blood 51, 1125. 1978. Finlay, J. L., Shahidi, N. T., Horowitz, S., Borcherding, W., and Hong, R.. J. C/in. Impest. 70, 619, 1982. Knutsen, A. P., and Buckley, R. H., In “Primary lmmunodeficiencies: INSERM Symposium No. 16” (M. Seligmann and W. H. Hitzig, Eds.), Vol. 13, pp. 13-22. Elsevier/North Holland Biomedical Press, 1980. Knutsen, A. I?, Merten, D. F.. and Buckley, R. H.. J. Pediatr. 98, 420. 1981. Kendall, D., lonescu-Matui, I., and Dreesman, G. R.. J. Immunol. Methods 56, 329, 1983.

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12. Ochs, H. D., Davis, S. D., and Wedgwood, R. J.. J. C/in. Invest. 50, 2559, 1971. 13. Blecher, T. E., Soothill. J. F., Voyce, M. A., and Walker, W. H. C., C/in. Exp. Immunol. 3, 47, 1968. 14. Rothbach, C., Nagel. J., Rabin, B., and Fireman, P, J. Pediatr. 94, 250, 1979. 15. Saxon, A., Kobayashi, R. H.. Stevens, R. H., Singer, A. D.. Stiehm, E. R., and Siegel, S. C., C/in. Immunol. Immunoputhol. 17, 235, 1980. 16. Ochs, H. D., and Wedgwood, R. J., In “Immunologic Disorders in Infants and Children” (E. R. Stiehm and V. A. Fulginiti, Eds.), p. 250. Saunders, Philadelphia, 1980. 17. Gitlin, D., In “Cellular and Humoral Aspects of the Hypersensitive States” (H. S. Lawrence, Ed.), pp. 394-395, 403-404. Hoeber. New York, 1959. 18. Good, R. A., In “Progress in Allergy” (P. Kallos and B. H. Waksman, Eds.). Vol. VI, pp. 251252. Karger, Basel. 1962. Received October 10, 1984; accepted with revision April 3, 1985