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Immunologic factors in human milk during the first year of lactation
April 1982 The Journal of P E D I A T R I C S
563
Immunologic factors in human milk during the first year of lactation The efJects of the duration of lactation upon lactoferrin, lysozyme, total lgA, SIgA, SIgA antibodies to Escherichia coil somatic antigens and leukocytes in human milk were investigated. Longitudinal and cross-sectional studies were performed with milk collected from women 20 to 35 years of age during the first year o f lactation. Collection and storage conditions and immunologic analyses were controlled to minimize confounding variables. The concentrations o f lactoferrin, total lgA, and leukocytes and the uptake of JH-thymidine by phytohemagglutinin-stimulated lymphocytes fell during the.first several weeks o f lactation; afterward, the levels o f lactoferrin and lgA stabilized, Approximately 90% of total lgA in human milk during the year was SIgA. Secretory lgA antibody titers to E. coil increased in some individuals studied longitudinally suggesting that the enteromammary gland pathway o f SIgA antibody production was active after several weeks of lactation. Moreover, the concentrations of lysozyme, after falling to a nadir of 20 to 30 ~g/ml at 2 to 4 weeks, rose to 200 to 300 ~g/ml by six months and remained elevated. The immunologic system in human milk undergoes remarkable changes which may represent adaptations Jor the recipient inJant.
Armond S. Goldman, Cutberto Garza, Buford L. Nichols, and Randall M. Goldblum, H o u s t o n , T e x a s
HUM AY M1LK is characterized by a host of immunologic components that protect the recipient infant against infections. The effects of these agents upon the developing infant are poorly understood in part because the composition of human milk is subject to change during lactation. In order to investigate the influence of the duration of lactation upon the immunologic components, the effects of processing and storage of human milk were first examined. The type of container ~'2 and the length of storage ~'2 are important. The highest numbers of leukocytes in the fluid phase of mature milk are found after storage for 24 hours at 4~ whereas lysozyme concentrations in mature milk are highest when stored at 37~ In contrast, lysozyme and From the Departments o f Pediatrics of the University of Texas Medical Branch, Galveston, Texas and Baylor College of Medicine, Supported by the National Institutes o f Child Health and Human Development (DHEW No. 1-HD-8-2828) and in part by USDA/SEA, Children's" Nutrition Research Center, Department of Pediatrics, Baylor College o f Medicine and Texas Children's Hospital.
0022-3476/82/040563+05500.50/0 9 1982 The C. V. Mosby Co.
lactoferrin concentrations in mature milk fall after storage at 4~ for 24 hours. Immunologic components are more stable in colostrum than in mature milk? Because of those studies, we were able to standardize collection and storage procedures. The effects of the duration of lactation upon selected components in human milk were examined in this study. Abbreviations used lg: immunoglobulin SIgA: secretory lgA PHA: phytohemagglutinin-P CPM: countsper minute RSV: respiratory syncytial virus
METHODS Research assurance. The research was approved by the Institutional Review Boards; informed consent was obtained from each subject before particiPation. Subject selection and collection procedures. Women, 20 to 35 years of age, who had one or two term pregnancies were studied. Multiple birth pregnancies were excluded. Colostrum donors were recruited from a public maternity Vol. 100, No. 4, pp. 563-567
564
Goldman et al.
The Journal of Pediatrics April 1982
| | ,J 5H-Thymidine Uptake
CPM C,ells/mll(~
r~ ~ 2
Weeks of Lactation
4
6
8
I0
12
Weeks of lactation
Fig. 1. A, A longitudinal study of the numbers of leukocytes, and B, the uptake of 3H-thymidine in lymphocytes. The same subjects were examined during the second through the twelfth week of lactation. The data are presented as the means _+ SD of macrophages-neutrophils (= =) and lymphocytes (o o ) and of stimulated (e, e,) and unstimulatcd (o o ) lymphocytes. Table. N u m b e r s of leukocytes, the degree of uptake of 3H-thymidine by P H A stimulated lymphocytes and the concentrations of lactoferrin, total and secretory IgA, and reciprocal titers of SIgA antibodies to E. coli somatic antigens in milk; a separate group of donors was obtained for each lactation period; cross-sectional d a t a are presented as the mean +_ S D Phase of lactation 4 wk
12 wk
24 wk
36 wk
52 wk
Macrophages-neutrophils (106/ml)
3.6 _+ 2.7
2-3 days
0.06 • 0.12
< 0.01
0,04 + 0.09
< 0.01
< 0.01
Lymphocytes(106/ml)
0.2 + 0.1 2.2 + 2.2
0.02 + 0.03 3.1 • 1.5
< 0.01 *
0.01 _+ 0.02 3.1 _+_3.7
< 0.01 *
< 0.01 *
5.3 + 1.9
1.9 _+ 0.3
0.8 +_ 0.4
1.4 • 0.4
0.9 • 0.2
1.0 _+ 0.2
2.1 _+ 2.3 2.0 _+ 2.5 111 • 150
1.0 • 0.2 1.0 • 0.3 2.3 • 2.4
0.5 • 0.1 0.5 • 0.1 3.8 _+ 3.0
0.6 _+ 0.1 0.5 -+_ 0.1 1.6 _+_3.2
0.9 • 0.2 0.8 -+ 0.2 0.1 + 2.7
1.0 _+_0.5 1.0 • 0.3 8.1 _+ 12.1
3H-thymidine uptak e (cpm • 10 3) Lactoferrin (mg/ml) IgA (mg/ml) Total Secretory SIgA antibodies (reciprocal titers)
I
*Insufficient lymphocytes to culture.
hospital; other donors of milk were from middle to upper socioeconomic populations. O t h e r criteria for selecting donors have been described? '~ Specimens were collected three hours after the last nursing. T h e breast was emptied with each collection by means of a low-pressure electric pump (Egnell, I n c . ) . The collection procedures are presented in d e t a i l in a separate r e p o r U Specimens~ were stored at 4 ~ in polypropylene containers for four hours; immunologic analyses of the samples were conducted immediately thereafter.
Two research designs were used. A longitudinal study was based on milk collected from eight women at 2, 4, 6, 8, 10, and 12 weeks of lactation. A cross-sectional study was performed using milk collected from separate groups at two or three days (10 women), four weeks (11 women), 12 weeks (7 women), 24 weeks (11 women), 36 weeks (4 women), and 52 weeks (5 women). Immunologic analyses. Lysozyme and lactoferrin were quantified by electroimmunodiffusion. L3 Total a n d secretory IgA were measured by a fluorescent i m m u n o s o r b e n t
Volume 100 Number 4
Immunologic factors in human milk
(~)
|
140-
3--
565
120-
2-
Lysozyme 80~g/ml 6O-
1-
40-
Lactoferrin
mg/ml
20-
2' 4'
2' ,l g 8' lb
lb l'z
Weeks of Lactation
Weeks of Lactation
Fig. 2. A longitudinal study of, A, lactoferrin and, B, lysozyme in human milk (means _+ SD). The same subjects were examined during the second through the twelfth week of lactation. The concentration of lactoferrin progressively decreased through the first eight weeks (r = 0.69) (2 vs. 8 weeks; P < 0.02), but not thereafter. In contrast, lysozyme levels steadily increased from the fourth through the twelfth week (r = 0.76) (4 vs 12 weeks; P < 0.0i).
1.25I0
I,O-
RecrprocaF 8S IgA Antibodies
IgA 0.75mg/ml
6~
E. coil
05-
4-
2-
0.25-
)
~
;
;
~;
i)
~
Weeks of Loclotion
4
i
G
i
,b
,'2
Weeks of Loc|otion
Fig. 3. A longitudinal study of A, total and secretory IgA and, B, the reciprocal of SIgA antibody titers to E. coli somatic antigens in human milk, The same subjects were examined during the second through the twelfth week of lactation. Total ( 9 ) and secretory IgA ( o ) data are presented as the mean _+ SD. SIgA antibody titers to E. coli somatic antigens from each subject are represented by different symbols.
technique which discriminates between serum-type and secretory IgA. 1'4 Secretory IgA antibodies to E s c h ~ i c h i a coli somatic antigens were measured by a micromodification of an enzyme-linked immunosorbent assay 5 with anti-human secretory component conjugated to alkaline phosphataseJ Viable leukocytes were enumerated and prepared for functional studies as described previously. ~ Counts of neutrophils and macrophages were combined. Incorporation of 3H-thymidine by lymphocytes stimulated with P H A was measured by a microculture assayJ Statistical analyses. Data from the longitudinal study were analyzed by paired t test; data from the crosssectional study were analyzed by unpaired t test. Correla-
tions between the levels of the components or their functions and duration of lactation were tested by linear regression analysis. RESULTS The volume of eolostrum was low compared to other milk samples (mean, 24 and 71 ml, respectively; P < 0.01). No significant differences were found in the volumes of milk samples collected between the second week through the twelfth month of lactation. The numbers of leukocytes (Table; Fig. I) and the degree of P H A stimulation fell significantly during the first 12 weeks to negligible levels. The concentrations of lactoferrin and total IgA also
5 66
Goldman et al.
The Journal of Pediatrics April 1982
I LysozymE p.g/nll
2 D.
1Mo.
3 Mo. 6 M0. 9 M0. Duration of Lactation
12M0,
Fig. 4. A cross-sectional study of lysozyme in human milk. Separate groups of subjects were examined at each phase of lactation. Data are presented as the mean + SD. Lysozymelevels rose (four weeks vs 25 weeks; P <0.001). decreased during the first 12 weeks but not thereafter (Table; Figs. 2 and 3). Secretory IgA comprised about 90% of the IgA in milk throughout the first ydar of lactation (Table; Fig. 3). Secretory IgA antibody titers to E. coli somatic antigens were highest__ in colostrum (Table). Although there were no significant differences in mean titers of SIgA antibodies to E. coli between the fourth and twenty-fourth week in the cross-sectional study (Table), SIgA antibody titers rose significantly in several individuals examined longitudinally (Fig. 3); the increments were not due to increases in total SIgA levels. The mean concentration of lysozyme in colostrum was 87 ~g/ml (Fig. 4). A nadir of 24 gg/ml was reached at two to four weeks of lactation (Figs. 2 and 4). The mean concentration of lysozyme then rose progressively over the next five months to 245 #g/ml and remained unchanged through the twelfth month of lactation. DISCUSSION Despite a number of important studies6-t6 there is comparatively little information concerning the immunologic components of human milk during the first year of lactation. We confirmed that the numbers of leukocytes and the degree of mitogenic stimulation of lymphocytes sharply declined during the first two or three months of lactation to essentially undetectable levels and that the concentrations of lactoferrin and IgA declined during the first three months of lactation. It has been inferred, based on past studies, that the quantities of these proteins decrease throughout lactation, but our data show that the concentrations of lactoferrin and IgA were mamtamed from three through 12 months. Furthermore, the quantity of the proteins secreted during these phases of lactation may be greater than these estimates because of the
increase in the daily volume of milk as lactation progresses. It has been assumed that lgA in milk measured by anti-c~ chain antisera is principally SIgA throughout lactation, but there are few data on that point. By quantifying total and secretory IgA independently with an immunosorbent technique, we found that approximately 90% of total IgA in milk collected throughout the first year of lactation is SIgA. There is a great deal of information regarding the spectrum of SIgA antibodies in human milk, 17 but there are few longitudinal data concerning specific antibodies during lactation, or the mechanism of the production of these antibodies. Based upon oral immunization experiments with E. coli (083) in the last trimester of pregnancy, Goldblum et aP 8 predicted that SIgA antibodies to enteric pathogens in human milk would arise through an enteromammary gland pathway, Carlsson and her co-workers ~2 reported rises in SIgA antibodies to E. coli in the milk of four out of 12 subjects during the first several weeks of lactation, but it was not ascertained whether these changes were simply a reflection of total SIgA levels. We now find that SIgA antibodies in human milk to E. coli somatic antigens rose in six of the eight individuals examined longitudinally during the first three months of lactation when total SIgA concentrations were diminishing. Recently, Fishaut et al ~ reported that SIgA antibodies to respiratory syncytial virus in human milk appear in the aftermath of RSV infection in the lower respiratory tract. Thus, our study and that of Fishaut suggest that immunologic pathways linking the gut and respiratory tract to the mammary gland may operate throughout lactation to provide antibodies which protect the recipient infant against enteric and respiratory tract pathogens from the mother: With few exceptions,9 lysozyme levels in human milk are reported to remain relatively stable throughout lactation?L 13,16In our investigation the concentrations of lysozyme fell from 87 ~g/ml in colostrum to a nadir of 24 ~g/ml at two to four weeks and then rose to 245 #g/ml at six months. This high level persisted through the remainder of the first year of lactation. Our preliminary data indicate that these levels are maintained throughout the second year of lactation also. The quantities of lysozyme in human milk may be greater than these estimates. As the concentration of lysozyme rises after the first month of lactation, the volume of milk production increases. Therefore, the total amount of lysozyme secreted in milk at six months would be approximately 15 times greater than the amount secreted at one month. The genesis of these high levels of milk lysozyme and their effects upon the recipient infant have not been ascertained.
Volume 100 Number 4
O u r studies provide evidence of a complex immunologic system in h u m a n milk which is unique for the early, mid, and late phases of lactation, and suggest t h a t the levels of immunologic constituents in h u m a n milk are regulated, although the nature of the controls are undetermined. T h e impact of these changes upon the infant is also poorly understood. Longitudinal studies of infants fed h u m a n milk should include evaluations of the immunologic system of the recipient a n d the fate of these m a t e r n a l factors in vivo. If the changes in the composition of h u m a n milk during lactation are optimal adaptations for the recipient infant, it follows t h a t stored h u m a n milk for feeding should be selected from a phase of lactation which approximates the postnatal age of the child. Dr. E. O. Smith provided the statistical analyses. The technical assistance of M. A. Armendariz, D. Nannen, M. Fehl, M. Douglas, and K. Andree; the nursing assistance of E. Cherry, C. Hines, S. Post, and J. Hopkinson; and the secretarial help of T. O'Neal were greatly appreciated. REFERENCES
1. Goldblum RM, Garza C, Johnson CA, Nichols 13]~, and Goldman AS: Human milk banking. I. Effects of container upon immunologic factors in mature milk, Nutr Res (in press). 2. Goldblum RM, Garza C, Johnson CA, Nichols BL, and Goldman AS: The stability of immunologic factors in colostrum, Acta Paediatr Scand (in press). 3. Laurell CB: Electrophoretic and electro-immunochemical analyses of proteins, Scand J Clin Lab Invest 29:(Suppl 124):1, 1972. 4. Goldblum RM, Powell GK, and Van Sickle G: Secretory IgA in the serum of infants with obstructive jaundice, J PEDIATR 97:33, 1980, 5. Engvoll E, and Perlmann P: Enzyme-linked immunosorbent assay, ELISA. Ill. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen coated tubes, J lmmunol 109:129, 1972. 6. Amman A J, and Stiehm ER: Immune globulin levels in colostrum and breast milk and serum from formula-and. breast-fed newborns, Proc Soc Exp Biol Med 122:1098, 1966.
Immunologic factors in human m i l k
567
7. Hanson LA, Borssen R, Holmgren J, Jodal V, Johansson BG, and Kaijser B: Secretory IgA, in Hagan B, and Stiehm R R, editors: Immunologic incompetence, Chicago, 1971, Year Book Medical Publishers, Inc. p 39 8. Mata L J, and Wyatt RG: Host resistance to infection, Am J Clin Nutr 24:976, 1971. 9. Rao PU, and Belanady B: Protein fractions in human milk. Part II. Isolation and characterization of basic protein from human milk and the lytic activity of milk samples, Indian J Biochem Biophys, 10:87, 1973. 10. Hyslop NE, Kern KC, and Walker WA: Lysozyme in human colostrum and breast milk, in Osserman E F, editor: Proceedings of lnternaional Symposium on Lysozyme, New York, 1974, Academic Press, Inc., p 449. 11. Peitersen B, Bohn L, and Anderson H: Quantitative determination of immunoglobulins, lysozyme, and certain electrolytes in breast milk during the entire period of lactation, during a 24-hour period, and in milk from the individual mammary gland, Acta Paediatr Scand 64:709, 1975. 12. Carlsson B, Gothefors L, Ahlstedt S, Hanson LA, and Winberg J: Studies of Escherichia coli O antigens specific antibodies in human milk, maternal serum and cord blood, Acta Paediatr Scand 65i216, 1976. 13. Reddy V, Bhaskaram C, Rahuramulu M, and Jagadeesan V: Antimicrobial factors in human milk, Acta Paediatr Scand 66:229, 1977. 14. Ogra SS, and Ogra PL: Immunologic aspects of human colostrum and milk. I. Distribution characteristics and concentrations of immunoglobulins at different times after the onset of lactation, J PEDIATR 92:546, 1978. 15. Ogra SS, and Ogra PL: Immunologic aspects of human colostrum and milk. II. Characteristics of lymphocyte reactivity and distribution of E-rosette forming cells at different times after the onset of lactation, J PEDIATR 92:550, t978. 16. McClelland DBL, McGrath J, and Samson RR: Antimicrobial factors in human milk. Studies of concentration and transfer to the infant during the early stages of lactation, Acta Paediatr Scand 67 (Suppl 271): 1, 1978. 17. Smith CW, and Goldman AS: Host resistance factors in human milk, J PEDIATR 82:1082, 1973. 18. Goldblum RM, Ahlstedt S, Carlsson B, Hanson LA,, Jodal U, Lidin-Janson G, and Sohl-Akerlund A: Antibody-forming cells in human colostrum after oral immunization, Nature 257: 797, 1975. 19. Fishaut M, Murphy D, Neifert M, Mclntosh K, and Ogra PL: Bronchomammary axis in the immune response to respiratory syncythial virus, J Pediatr 99:186, 1981.
563
Immunologic factors in human milk during the first year of lactation The efJects of the duration of lactation upon lactoferrin, lysozyme, total lgA, SIgA, SIgA antibodies to Escherichia coil somatic antigens and leukocytes in human milk were investigated. Longitudinal and cross-sectional studies were performed with milk collected from women 20 to 35 years of age during the first year o f lactation. Collection and storage conditions and immunologic analyses were controlled to minimize confounding variables. The concentrations o f lactoferrin, total lgA, and leukocytes and the uptake of JH-thymidine by phytohemagglutinin-stimulated lymphocytes fell during the.first several weeks o f lactation; afterward, the levels o f lactoferrin and lgA stabilized, Approximately 90% of total lgA in human milk during the year was SIgA. Secretory lgA antibody titers to E. coil increased in some individuals studied longitudinally suggesting that the enteromammary gland pathway o f SIgA antibody production was active after several weeks of lactation. Moreover, the concentrations of lysozyme, after falling to a nadir of 20 to 30 ~g/ml at 2 to 4 weeks, rose to 200 to 300 ~g/ml by six months and remained elevated. The immunologic system in human milk undergoes remarkable changes which may represent adaptations Jor the recipient inJant.
Armond S. Goldman, Cutberto Garza, Buford L. Nichols, and Randall M. Goldblum, H o u s t o n , T e x a s
HUM AY M1LK is characterized by a host of immunologic components that protect the recipient infant against infections. The effects of these agents upon the developing infant are poorly understood in part because the composition of human milk is subject to change during lactation. In order to investigate the influence of the duration of lactation upon the immunologic components, the effects of processing and storage of human milk were first examined. The type of container ~'2 and the length of storage ~'2 are important. The highest numbers of leukocytes in the fluid phase of mature milk are found after storage for 24 hours at 4~ whereas lysozyme concentrations in mature milk are highest when stored at 37~ In contrast, lysozyme and From the Departments o f Pediatrics of the University of Texas Medical Branch, Galveston, Texas and Baylor College of Medicine, Supported by the National Institutes o f Child Health and Human Development (DHEW No. 1-HD-8-2828) and in part by USDA/SEA, Children's" Nutrition Research Center, Department of Pediatrics, Baylor College o f Medicine and Texas Children's Hospital.
0022-3476/82/040563+05500.50/0 9 1982 The C. V. Mosby Co.
lactoferrin concentrations in mature milk fall after storage at 4~ for 24 hours. Immunologic components are more stable in colostrum than in mature milk? Because of those studies, we were able to standardize collection and storage procedures. The effects of the duration of lactation upon selected components in human milk were examined in this study. Abbreviations used lg: immunoglobulin SIgA: secretory lgA PHA: phytohemagglutinin-P CPM: countsper minute RSV: respiratory syncytial virus
METHODS Research assurance. The research was approved by the Institutional Review Boards; informed consent was obtained from each subject before particiPation. Subject selection and collection procedures. Women, 20 to 35 years of age, who had one or two term pregnancies were studied. Multiple birth pregnancies were excluded. Colostrum donors were recruited from a public maternity Vol. 100, No. 4, pp. 563-567
564
Goldman et al.
The Journal of Pediatrics April 1982
| | ,J 5H-Thymidine Uptake
CPM C,ells/mll(~
r~ ~ 2
Weeks of Lactation
4
6
8
I0
12
Weeks of lactation
Fig. 1. A, A longitudinal study of the numbers of leukocytes, and B, the uptake of 3H-thymidine in lymphocytes. The same subjects were examined during the second through the twelfth week of lactation. The data are presented as the means _+ SD of macrophages-neutrophils (= =) and lymphocytes (o o ) and of stimulated (e, e,) and unstimulatcd (o o ) lymphocytes. Table. N u m b e r s of leukocytes, the degree of uptake of 3H-thymidine by P H A stimulated lymphocytes and the concentrations of lactoferrin, total and secretory IgA, and reciprocal titers of SIgA antibodies to E. coli somatic antigens in milk; a separate group of donors was obtained for each lactation period; cross-sectional d a t a are presented as the mean +_ S D Phase of lactation 4 wk
12 wk
24 wk
36 wk
52 wk
Macrophages-neutrophils (106/ml)
3.6 _+ 2.7
2-3 days
0.06 • 0.12
< 0.01
0,04 + 0.09
< 0.01
< 0.01
Lymphocytes(106/ml)
0.2 + 0.1 2.2 + 2.2
0.02 + 0.03 3.1 • 1.5
< 0.01 *
0.01 _+ 0.02 3.1 _+_3.7
< 0.01 *
< 0.01 *
5.3 + 1.9
1.9 _+ 0.3
0.8 +_ 0.4
1.4 • 0.4
0.9 • 0.2
1.0 _+ 0.2
2.1 _+ 2.3 2.0 _+ 2.5 111 • 150
1.0 • 0.2 1.0 • 0.3 2.3 • 2.4
0.5 • 0.1 0.5 • 0.1 3.8 _+ 3.0
0.6 _+ 0.1 0.5 -+_ 0.1 1.6 _+_3.2
0.9 • 0.2 0.8 -+ 0.2 0.1 + 2.7
1.0 _+_0.5 1.0 • 0.3 8.1 _+ 12.1
3H-thymidine uptak e (cpm • 10 3) Lactoferrin (mg/ml) IgA (mg/ml) Total Secretory SIgA antibodies (reciprocal titers)
I
*Insufficient lymphocytes to culture.
hospital; other donors of milk were from middle to upper socioeconomic populations. O t h e r criteria for selecting donors have been described? '~ Specimens were collected three hours after the last nursing. T h e breast was emptied with each collection by means of a low-pressure electric pump (Egnell, I n c . ) . The collection procedures are presented in d e t a i l in a separate r e p o r U Specimens~ were stored at 4 ~ in polypropylene containers for four hours; immunologic analyses of the samples were conducted immediately thereafter.
Two research designs were used. A longitudinal study was based on milk collected from eight women at 2, 4, 6, 8, 10, and 12 weeks of lactation. A cross-sectional study was performed using milk collected from separate groups at two or three days (10 women), four weeks (11 women), 12 weeks (7 women), 24 weeks (11 women), 36 weeks (4 women), and 52 weeks (5 women). Immunologic analyses. Lysozyme and lactoferrin were quantified by electroimmunodiffusion. L3 Total a n d secretory IgA were measured by a fluorescent i m m u n o s o r b e n t
Volume 100 Number 4
Immunologic factors in human milk
(~)
|
140-
3--
565
120-
2-
Lysozyme 80~g/ml 6O-
1-
40-
Lactoferrin
mg/ml
20-
2' 4'
2' ,l g 8' lb
lb l'z
Weeks of Lactation
Weeks of Lactation
Fig. 2. A longitudinal study of, A, lactoferrin and, B, lysozyme in human milk (means _+ SD). The same subjects were examined during the second through the twelfth week of lactation. The concentration of lactoferrin progressively decreased through the first eight weeks (r = 0.69) (2 vs. 8 weeks; P < 0.02), but not thereafter. In contrast, lysozyme levels steadily increased from the fourth through the twelfth week (r = 0.76) (4 vs 12 weeks; P < 0.0i).
1.25I0
I,O-
RecrprocaF 8S IgA Antibodies
IgA 0.75mg/ml
6~
E. coil
05-
4-
2-
0.25-
)
~
;
;
~;
i)
~
Weeks of Loclotion
4
i
G
i
,b
,'2
Weeks of Loc|otion
Fig. 3. A longitudinal study of A, total and secretory IgA and, B, the reciprocal of SIgA antibody titers to E. coli somatic antigens in human milk, The same subjects were examined during the second through the twelfth week of lactation. Total ( 9 ) and secretory IgA ( o ) data are presented as the mean _+ SD. SIgA antibody titers to E. coli somatic antigens from each subject are represented by different symbols.
technique which discriminates between serum-type and secretory IgA. 1'4 Secretory IgA antibodies to E s c h ~ i c h i a coli somatic antigens were measured by a micromodification of an enzyme-linked immunosorbent assay 5 with anti-human secretory component conjugated to alkaline phosphataseJ Viable leukocytes were enumerated and prepared for functional studies as described previously. ~ Counts of neutrophils and macrophages were combined. Incorporation of 3H-thymidine by lymphocytes stimulated with P H A was measured by a microculture assayJ Statistical analyses. Data from the longitudinal study were analyzed by paired t test; data from the crosssectional study were analyzed by unpaired t test. Correla-
tions between the levels of the components or their functions and duration of lactation were tested by linear regression analysis. RESULTS The volume of eolostrum was low compared to other milk samples (mean, 24 and 71 ml, respectively; P < 0.01). No significant differences were found in the volumes of milk samples collected between the second week through the twelfth month of lactation. The numbers of leukocytes (Table; Fig. I) and the degree of P H A stimulation fell significantly during the first 12 weeks to negligible levels. The concentrations of lactoferrin and total IgA also
5 66
Goldman et al.
The Journal of Pediatrics April 1982
I LysozymE p.g/nll
2 D.
1Mo.
3 Mo. 6 M0. 9 M0. Duration of Lactation
12M0,
Fig. 4. A cross-sectional study of lysozyme in human milk. Separate groups of subjects were examined at each phase of lactation. Data are presented as the mean + SD. Lysozymelevels rose (four weeks vs 25 weeks; P <0.001). decreased during the first 12 weeks but not thereafter (Table; Figs. 2 and 3). Secretory IgA comprised about 90% of the IgA in milk throughout the first ydar of lactation (Table; Fig. 3). Secretory IgA antibody titers to E. coli somatic antigens were highest__ in colostrum (Table). Although there were no significant differences in mean titers of SIgA antibodies to E. coli between the fourth and twenty-fourth week in the cross-sectional study (Table), SIgA antibody titers rose significantly in several individuals examined longitudinally (Fig. 3); the increments were not due to increases in total SIgA levels. The mean concentration of lysozyme in colostrum was 87 ~g/ml (Fig. 4). A nadir of 24 gg/ml was reached at two to four weeks of lactation (Figs. 2 and 4). The mean concentration of lysozyme then rose progressively over the next five months to 245 #g/ml and remained unchanged through the twelfth month of lactation. DISCUSSION Despite a number of important studies6-t6 there is comparatively little information concerning the immunologic components of human milk during the first year of lactation. We confirmed that the numbers of leukocytes and the degree of mitogenic stimulation of lymphocytes sharply declined during the first two or three months of lactation to essentially undetectable levels and that the concentrations of lactoferrin and IgA declined during the first three months of lactation. It has been inferred, based on past studies, that the quantities of these proteins decrease throughout lactation, but our data show that the concentrations of lactoferrin and IgA were mamtamed from three through 12 months. Furthermore, the quantity of the proteins secreted during these phases of lactation may be greater than these estimates because of the
increase in the daily volume of milk as lactation progresses. It has been assumed that lgA in milk measured by anti-c~ chain antisera is principally SIgA throughout lactation, but there are few data on that point. By quantifying total and secretory IgA independently with an immunosorbent technique, we found that approximately 90% of total IgA in milk collected throughout the first year of lactation is SIgA. There is a great deal of information regarding the spectrum of SIgA antibodies in human milk, 17 but there are few longitudinal data concerning specific antibodies during lactation, or the mechanism of the production of these antibodies. Based upon oral immunization experiments with E. coli (083) in the last trimester of pregnancy, Goldblum et aP 8 predicted that SIgA antibodies to enteric pathogens in human milk would arise through an enteromammary gland pathway, Carlsson and her co-workers ~2 reported rises in SIgA antibodies to E. coli in the milk of four out of 12 subjects during the first several weeks of lactation, but it was not ascertained whether these changes were simply a reflection of total SIgA levels. We now find that SIgA antibodies in human milk to E. coli somatic antigens rose in six of the eight individuals examined longitudinally during the first three months of lactation when total SIgA concentrations were diminishing. Recently, Fishaut et al ~ reported that SIgA antibodies to respiratory syncytial virus in human milk appear in the aftermath of RSV infection in the lower respiratory tract. Thus, our study and that of Fishaut suggest that immunologic pathways linking the gut and respiratory tract to the mammary gland may operate throughout lactation to provide antibodies which protect the recipient infant against enteric and respiratory tract pathogens from the mother: With few exceptions,9 lysozyme levels in human milk are reported to remain relatively stable throughout lactation?L 13,16In our investigation the concentrations of lysozyme fell from 87 ~g/ml in colostrum to a nadir of 24 ~g/ml at two to four weeks and then rose to 245 #g/ml at six months. This high level persisted through the remainder of the first year of lactation. Our preliminary data indicate that these levels are maintained throughout the second year of lactation also. The quantities of lysozyme in human milk may be greater than these estimates. As the concentration of lysozyme rises after the first month of lactation, the volume of milk production increases. Therefore, the total amount of lysozyme secreted in milk at six months would be approximately 15 times greater than the amount secreted at one month. The genesis of these high levels of milk lysozyme and their effects upon the recipient infant have not been ascertained.
Volume 100 Number 4
O u r studies provide evidence of a complex immunologic system in h u m a n milk which is unique for the early, mid, and late phases of lactation, and suggest t h a t the levels of immunologic constituents in h u m a n milk are regulated, although the nature of the controls are undetermined. T h e impact of these changes upon the infant is also poorly understood. Longitudinal studies of infants fed h u m a n milk should include evaluations of the immunologic system of the recipient a n d the fate of these m a t e r n a l factors in vivo. If the changes in the composition of h u m a n milk during lactation are optimal adaptations for the recipient infant, it follows t h a t stored h u m a n milk for feeding should be selected from a phase of lactation which approximates the postnatal age of the child. Dr. E. O. Smith provided the statistical analyses. The technical assistance of M. A. Armendariz, D. Nannen, M. Fehl, M. Douglas, and K. Andree; the nursing assistance of E. Cherry, C. Hines, S. Post, and J. Hopkinson; and the secretarial help of T. O'Neal were greatly appreciated. REFERENCES
1. Goldblum RM, Garza C, Johnson CA, Nichols 13]~, and Goldman AS: Human milk banking. I. Effects of container upon immunologic factors in mature milk, Nutr Res (in press). 2. Goldblum RM, Garza C, Johnson CA, Nichols BL, and Goldman AS: The stability of immunologic factors in colostrum, Acta Paediatr Scand (in press). 3. Laurell CB: Electrophoretic and electro-immunochemical analyses of proteins, Scand J Clin Lab Invest 29:(Suppl 124):1, 1972. 4. Goldblum RM, Powell GK, and Van Sickle G: Secretory IgA in the serum of infants with obstructive jaundice, J PEDIATR 97:33, 1980, 5. Engvoll E, and Perlmann P: Enzyme-linked immunosorbent assay, ELISA. Ill. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen coated tubes, J lmmunol 109:129, 1972. 6. Amman A J, and Stiehm ER: Immune globulin levels in colostrum and breast milk and serum from formula-and. breast-fed newborns, Proc Soc Exp Biol Med 122:1098, 1966.
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7. Hanson LA, Borssen R, Holmgren J, Jodal V, Johansson BG, and Kaijser B: Secretory IgA, in Hagan B, and Stiehm R R, editors: Immunologic incompetence, Chicago, 1971, Year Book Medical Publishers, Inc. p 39 8. Mata L J, and Wyatt RG: Host resistance to infection, Am J Clin Nutr 24:976, 1971. 9. Rao PU, and Belanady B: Protein fractions in human milk. Part II. Isolation and characterization of basic protein from human milk and the lytic activity of milk samples, Indian J Biochem Biophys, 10:87, 1973. 10. Hyslop NE, Kern KC, and Walker WA: Lysozyme in human colostrum and breast milk, in Osserman E F, editor: Proceedings of lnternaional Symposium on Lysozyme, New York, 1974, Academic Press, Inc., p 449. 11. Peitersen B, Bohn L, and Anderson H: Quantitative determination of immunoglobulins, lysozyme, and certain electrolytes in breast milk during the entire period of lactation, during a 24-hour period, and in milk from the individual mammary gland, Acta Paediatr Scand 64:709, 1975. 12. Carlsson B, Gothefors L, Ahlstedt S, Hanson LA, and Winberg J: Studies of Escherichia coli O antigens specific antibodies in human milk, maternal serum and cord blood, Acta Paediatr Scand 65i216, 1976. 13. Reddy V, Bhaskaram C, Rahuramulu M, and Jagadeesan V: Antimicrobial factors in human milk, Acta Paediatr Scand 66:229, 1977. 14. Ogra SS, and Ogra PL: Immunologic aspects of human colostrum and milk. I. Distribution characteristics and concentrations of immunoglobulins at different times after the onset of lactation, J PEDIATR 92:546, 1978. 15. Ogra SS, and Ogra PL: Immunologic aspects of human colostrum and milk. II. Characteristics of lymphocyte reactivity and distribution of E-rosette forming cells at different times after the onset of lactation, J PEDIATR 92:550, t978. 16. McClelland DBL, McGrath J, and Samson RR: Antimicrobial factors in human milk. Studies of concentration and transfer to the infant during the early stages of lactation, Acta Paediatr Scand 67 (Suppl 271): 1, 1978. 17. Smith CW, and Goldman AS: Host resistance factors in human milk, J PEDIATR 82:1082, 1973. 18. Goldblum RM, Ahlstedt S, Carlsson B, Hanson LA,, Jodal U, Lidin-Janson G, and Sohl-Akerlund A: Antibody-forming cells in human colostrum after oral immunization, Nature 257: 797, 1975. 19. Fishaut M, Murphy D, Neifert M, Mclntosh K, and Ogra PL: Bronchomammary axis in the immune response to respiratory syncythial virus, J Pediatr 99:186, 1981.