Quantitation of the biosynthesis of immunoglobulin in peripheral blood lymphocytes of normal and immunodeficient patients

October 1975

The Journal o f P E D I A T R I C S

545

Quantitation of the biosynthesis of immunoglobulin in peripheral blood lymphocytes of normal and immunodeficient patients A solidphase radioimmunoassay was applied to the study of biosynthesis ofimmunoglobufins in lymphocytes cultured from peripheral blood of normal and immunodeficient patients. Total immunoglobulin and IgG were readily detected in lymphocyte culture fluids in studies of all normal individuals (age range 28 weeks, gestation to adulthood). In contrast, synthesis of immunoglobufins was absent or markedly reduced in cultures from patients with humoral immunodeficiencies. Inasmuch as this method requires relatively small amounts of blood, it may be of particular value in the diagnosis of humoral immunodeficiencies in infants and young children.

Stephen H. Polmar, Ph.D., M.D.,* and Patricia A. Chase, B.S., Cleveland, O h i o

THE DIAGNOSIS of humoral immunodeficiency in infancy is often difficult to establish because of the presence of maternal IgG and the normally low levels of the infant's own serum immunoglobulins. In addition, administration of exogenous immunoglobulins (immune serum globulin, whole blood, or plasma) to children suspected of being immunodeficient, prior to adequate immunologic evaluation, may obscure or delay correct diagnosis. Since the ability to synthesize immun0gobulins develops relatively early in fetal life? studies of immunoglobulin synthesis in a readily accessible lymphoid tissue might facilitate early detection of immunoglobulin deficiency states. With the use of a sensitive solid phase From the Department of Pediatrics, Case Western Reserve University School of Medicine, and the Division of Pediatric Immunology, Rainbow Babies and Childrens Hospita. L Supported in part by Grants AM-08305 and HL06009from the National Institutes of Health and by a grant from the Cleveland Cystic Fibrosis Foundation and the Health Fund of Cleveland. Presented at the annual meeting of the American Pediatric Society-Society for Pediatric Research, May, 1974. *Reprint address: Rainbow Babies and Childrens Hospital, 2103 Adelbert Rd., Cleveland, Ohio 44106.

radioimmunoassay technique, synthesis of immunoglobulin was readily detected and quantitated in cultures of lymphocytes from peripheral blood of normal infants, children, and adults. In contrast, immunoglobulin biosynthesis in lymphocytes was found to be absent or markedly diminished in patients with humoral immunodeficiencies. This method has the advantages of requiring relatively small amounts of blood and being capable of discriminating between normal and immunodeficient individuals over a broad age range. Abbreviations used Ig: immunoglobulin BAC: bromacetyl cellulose

MATERIALS AND M E T H O D S Patients and control subjects. Samples of heparinized venous blood were obtained from three patients with agammaglobufinemia (late onset) and two patients with a familial dysgammaglobulinemia. Thirteen patients with unrelated illnesses (ranging in age from birth to 12 years) and five adult laboratory and hospital personnel served as normal control subjects. Three newborn infants with gestational ages of 28, 30, and 36 weeks, respectively, were studied within 24 hours of birth. Studies were performed using 2 to 3 ml of blood from young infants, 5 ml from

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546

Polmar and Chase

The Journal of Pediatrics October 1975

Table 1. Immunodeficient patients Serum immunoglobulins Patient

IgG IgA Igm IgE* (mg/ (rag~ (mg/ (U/ dl) dl) dl) ml)

Age (yr)

Sex

S.S.

4

F

6

0

19

4

J.P.

6

F

280t

0

0

4

J.C.

12

M

92t

0

4

3

E.M.

12

M

290

0

63

3

C.M.

30

F

126

0

49

3

Diagnosis Late-onset agarnmaglobulinemia Late-onset agammaglobulinemia Late-onset agammaglobulinemia Dysgammaglobulinemia Dysgammaglobulinemia

*IgE levelsin the 3 to 4 U/ml range are at the lowerlimitof detectionfor the radioimmunoassay. ]Patient was receivinggammaglobulininjections.

Table 11. Comparison of biosynthesis of immunoglobulins in lymphocytes cultured from immunodeficient patients and control subjects Patients

dpm* Ig

%Igt

I dpmIgG 1%IgG

0.03 90 0.05 S.S. 58 0.18 161 0.12 J.P. 345 0.05 73 0.07 J.C. 50 0.82 9 0.0l E.M. 619 0.40 218 0.37 C.M. 316 Control subjects 4.03 1,581 1.47 Mean 3,731 Range 873-15,846 0.77-10.40 301-7,505 0.20-5.43

were measured by solid phase radioimmunoassay? '~ Specifc anti-IgG and anti-Fab were prepared by elution from insoluble immunoadsorbents and covalently linked to bromacetyl cellulose? Specificity of the insoluble antibodies was established, in part, by binding studies with radioiodinated human IgG, IgA, IgM, and albumin, and with rabbit y-globulin. In addition, analysis of molecular weights on sodium dodecyl sulfate acrylmide gels of newly synthesized proteins eluted from BAC-antibodies confirmed that BAC-anti-IgG bound only IgG while BAC-anti-Fab bound IgG, IgA, and IgM, but not other proteins. Quantitation of IgG synthesis was performed as follows: an aliquot of dialyzed cultured fluid was incubated with BAC-anti-IgG and a second aliquot was incubated with BAC-anti-IgG which had been inhibited with excess unlabeled IgG. After centrifugation and washing both BAC preparations were counted in a liquid scintillation counter. The difference between the uninhibited and inhibited BAC-anti-IgG count rates was a measure of the labeled IgG in the culture fluid. Counts were corrected for BAC-antibody binding efficiency and counting efficiency. IgG synthesis was expressed as d p m / 106 cells and IgG, as a percentage of the total secreted protein. The same method was used to measure total immunoglobulins employing the BAC-anti-Fab reagent. The standard deviation of repeat determinations was less than _+5%. Other immunologic studies. Quantitation of serum IgG, IgA, and IgM was performed by single radial diffusion. Serum IgE was measured by radioimmunoassay? T- and B-lymphocyte subpopulations were enumerated by rosette techniques using sheep erythrocytes. 7 RESULTS

*dpm/106 cells. tImmunoglobulinas a percentageof newly synthesizedsecretedprotein. children up to 5 years of age, and 10 to 25 ml from older children and adults. Studies were carried out with i n formed consent according to a protocol approved by the University Hospitals Human Experimentation Committee.

Lymphocyte culture and quantitation of lymphocyte immunoglobulin biosynthesis. Lymphocytes were isolated from heparinized peripheral blood using Ficoll-Hypaque gradient centrifugation. 2 Preparations contained greater than 90% lymphocytes. These cells were cultured at a concentration of 1 • 106 cells/ml in leucine-deficient minimal essential medium (Eagle) containing 10% heatinactivated fetal calf serum and 5 t~C of 3H-leucine (specific activity 15C/mmole) in 5% CO~ at 37 ~ C for 24 to 72 hours. Supernatant culture fluids were removed by centrifugation and dialyzed, and the labeled immunoglobulins

Immunologic studies on immunodeficient patients. The serum immunoglobulin levels of the five immunodeficient patients studied are presented in Table I. The three agammaglobulinemic patients had marked reduction or absence of all classes of immunoglobulins studied. The two dysgammaglobulinemic patients had low normal values of IgM, decreased values of IgG, and absence of IgA and IgE. The proportions of T- and B-lymphocyte subpopulations were within the normal range for all patients except Patient J. C. who had a diminished percentage of B-cells (11% versus 25% for age-matched control subjects). Lymphocyte immunoglobulin biosynthetic studies. Synthesis of immunoglobulin was detectable by 24 hours in control lymphocyte cultures and reached a peak at 48 hours in most cultures. A small amount of additional synthesis was detected in some cultures up to 72 hours; generally, however, after 48 hours there was a slight

Volume 87 Number 4

Lymphocyte immunoglobulin biosynthesis

547

~_ !0(1)

m~176 15-

cz

(1)

dp

8

LD (1) c.o

Ee o_

._u--) 09 (1.) ._C: 4 .+--. c

CO

oo ap4 c-+,.--

c>-, 00

CO

~2

H

9 2

c]

.4----

c] IA

, /~ ,A 0

i

~

+

4

5

102b

s'o 40

Age (yrs.)

0

i

~

b 4 s Io 2b so 4'o Age (yrs.)

Fig. 1. Total immunoglobulin synthesis (expressed in dpm/10 ~ ceils) in 48-hour lymphocyte cultures in relation to age. 9 = Normal; A = humoral immunodeficient patient.

Fig. 2. Total immunoglobulin synthesis (expressed as a percentage of the total secreted, dpm Ig/dpm total protein x 100) in 48hour lymphocyte cultures in relation to age. 9 = Normal; A = humoral irnmunodeficient patient.

decrease in the percentage of immunoglobulin in the newly synthesized secreted protein. Immunoglobulin synthesis was readily detectable in lymphocyte cultures from the newborn infants studied. Both the amount of newly synthesized immunoglobulin and the percentage of immunoglobulin in the secreted proteins increased rapidly in the first few months of life (Figs. 1 and 2). Synthesis of IgG followed a similar pattern and accounted for 34% (mean) of total immunoglobulin synthesis. The three agammaglobulinemic patients had distinct diminution of lymphocyte immunoglobulin and of IgG synthesis (Fig. 1 and Table II). The immunoglobulin biosynthetic defect is demonstrated more strikingly by comparison of the immunoglobulin and IgG percentages of secreted proteins with those of the normal control subjects (Fig. 2 and Table II). The rate and percentage of synthesis in the immunodeficient patients were less than those observed in the premature infants studied. The dysgammaglobulinemic patients synthesized more immunoglobulin than did the agammaglobulinemic ones but much less than the normal control subjects.

munoassay. By means of these methods, synthesis of immunoglobulins could be readily detected in newborn premature infants and in all older normal control infants and children. In contrast, biosynthesis of immunoglobulin was markedly diminished or absent in the immunodeficient patients studied. The immunoglobulin biosynthetic defect in the immunodeficient patients is most striking when the percentages of secreted protein that are immunoglobulin are compared to those of the normal control subjects. Our findings are similar to those previously reported by others, a, 9 Unlike these reports, however, young infants could be included in our studies because of the relatively small amounts of blood required for quantitation of immunoglobulin synthesis by the techniques employed. Quantitation of immunoglobulin biosynthesis in short-term lymphocyte cultures would, therefore, appear to be of value in the detection of humoral immunodeficiency, particularly in infancy when serum immunoglobulin concentrations are normally low and most of the serum immunoglobulin is of maternal origin. Studies of immunoglobulin biosynthesis may also be of assistance in the evaluation of patients to whom exogenous immunogtobulins have been administered prior to appropriate evaluation of immune status. Patients with non-X-linked agammaglobulinemias and dysgammaglobulinemias as well as some X-linked agammaglobulinemic individuals have circulating B-lymphocytes, where-

DISCUSSION Quantitative studies of synthesis of immunoglobulins in peripheral blood lymphocytes have been performed on normal infants, children, and adults, as well as on immunodeficient patients using a sensitive solid phase radioim-

54 8

Polmar and Chase

as most patients with X-linked agammaglobulinemia lack these cells. 1~ Enumeration of B-lymphocytes using immunofluorescent techniques to detect surface immunoglobulins or rosette techniques to detect C3 receptors would identify most patients with X-linked agammaglobulinemia but would fail to detect the majority of patients with other forms of humoral immunodeficiency. Measurement of immunoglobulin synthesis by lymphocytes is an assay of function and clearly distinguishes between humoral immunodeficient patients and normal subjects. The relationship between synthesis of immunoglobulins by lymphocytes and serum immunoglobulin concentration is not well defined. Certainly lymphocytes from agammaglobulinemic and dysgammaglobulinemic patients synthesize less immunoglobulin than those of normal individuals. However, since immunoglobulin synthesis by peripheral blood lymphocytes probably contributes very little to total body inlmunoglobulin pools, one would not expect perfect correlation. The origin of the immunoglobulins produced by lymphoid cell cultures, such as those used in these studies, is not entirely dear. Wu and associates 1" have shown that unstimulated lymphoid cell cultures contain less than one cell with intracytoplasmic immunoglobulin per thousand cells. When cultures are stimulated with pokeweed mitogen there is an increase in immunoglobulin synthesis as well as an increase in cells with intracytoplasmic immunoglobulin, suggesting that these cells are largely responsible for the increased immunoglobulin synthesis? ~ Cells with intracytoplasmic immunoglobulin have the histologic appearance of plasma cells or lymphoblasts. We have studied peripheral blood lymphoid cell cultures from a patient with chronic glomerulonephritis in which as much as 43% of the protein secreted was immunoglobulin. Although immunofluorescent studies were not performed, neither plasma cells nor lymphoblasts were present in this child's peripheral blood. Alternatively, the immunoglobulin produced in our lymphocyte cultures may come from B-cells (presumably by elution from the cell membrane). If this is the case then clearly the B-cells of non-X-linked agammaglobulinemic and dysgammaglobulinemic patients, although indistinguishable from normal B-cells by immunofluorescence or C3 receptor studies, are clearly defective in the production of membrane immunoglobulin even in the resting state. At this time data concerning the cells of origin of the secreted immunoglobulin are inconclusive and studies of immunoglobulin biosynthesis on fractionated lymphoid cell populations are needed. In this report, studies were carried out on a relatively small number of normal infants and children. Clearly, a much larger number of such normal control subjects of

The Journal of Pediatrics October 1975

different ages must be studied in order to establish the normal range of values for immunoglobulin synthesis in lymphocytes. Inasmuch as patients with immunodeficiency disorders suffer both acute and chronic infection, the effect of such infections on lymphocyte immunoglobulin synthesis in normal individuals must also be determined. We have shown that quantitative studies of immunoglobulin synthesis can be carried out using small amounts of blood, thus making it possible to study young infants. Unfortunately, we have not as yet studied any patients under the age of 4 years with humoral immunodeficiency. Serial studies comparing synthesis of immunoglobulin in lymphocytes of young children with congenital hypogammaglobulinemia and of those with transient hypogammaglobulinemia of infancy to the values in normal children of the same age would be most informative. The authors wish to express their appreciation to Mr. Paul Shoenfeld and Ms. Erica Wetzler for their technical assistance, to Dr. Dorr Dearborn for his suggestions and criticism of the manuscript, and to Ms. Kathleen Hollosy for her assistance in preparation of the manuscript. REFERENCES

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munoglobulin (Ig) and in vitro production of Ig by lymphoid cells from patients with primary immunodeficiencies, Scand J Immunol 2:9, 1973. !0. Grey HM, Rabellino E, and Pirofsky B: Immunoglobulins on the surface of lymphocytes. IV. Distribution in hypogammaglobulinemia, cellular immune deficiency and chronic lymphatic leukemia, J Clin Invest 50:2368, 1971. 11. Cooper MD, Lawton AR, and Bockman DE: Agammaglobulinemia with B lymphocytes: specific defect in plasma-cell differentiation, Lancet 2:791, 1971. 12. Gajl-Peczalska KJ, Park BH, Biggar WD, and Good RA: B

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and T lymphocytes in primary immunodeficiency disease in man, J Clin Invest 52:919, 1973. 13. Geha RS, Rosen FS, and Merler E: Identification and characterization of subpopulations of lymphocytes in human peripheral blood after fractionation on discontinuous gradients of albumin: the cellular defect in X-linked agammaglobulinemia, J Clin Invest 52:1726, 1973. 14. Wu LYF, Lawton AR, and Cooper MD: Differentiation capacity of cultured B lymphocytcs from immunodeficient patients, J Clin Invest 52:3180, 1973.