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Clinical Chemistry 53: 955-962, 2007. First published March 15, 2007; 10.1373/clinchem.2006.077107
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(Clinical Chemistry. 2007;53:955-962.)
© 2007 American Association for Clinical Chemistry, Inc.

Clinical Immunology

Cytokine Profiling in Human Colostrum and Milk by Protein Array

Miloslav Kverka1,a, Jaroslava Burianova1, Raja Lodinova-Zadnikova2, Ingrid Kocourkova2, Jana Cinova1, Ludmila Tuckova1 and Helena Tlaskalova-Hogenova1

1 Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
2 Institute for the Care of Mother and Child, Prague, Czech Republic.

aAddress correspondence to this author at: Fax 420-24172-1143; e-mail: kverka{at}biomed.cas.cz, immuno{at}biomed.cas.cz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Human colostrum and milk contain components that influence development. Our aim was to use a protein array to determine the cytokine profile of human lacteal secretions and changes that occur during the early postpartum period.

Methods: We collected 17 samples of colostrum during the first 2 days postpartum and a 2nd group of 5 sets of 2 to 3 sequential colostrum or milk samples (at 20- to 30-h intervals). We analyzed the samples with array membranes consisting of 42 or 79 antibodies directed against cytokines.

Results: In most samples, we detected the previously described cytokines interleukin-8 (IL-8)/CXCL8, epidermal growth factor (EGF), growth-related oncoprotein (GRO)/CXCL1–3, angiogenin, transforming growth factor ß-2, and monocyte chemotactic protein 1 (MCP-1/CCL2). In addition, we found 32 cytokines that have not been described before in colostrum. Cytokine concentrations differed among mothers, and the spectrum of cytokines changed with time after delivery. A significant decrease occurred in IL-12 and macrophage inflammatory protein-1{delta}/CCL15 and a significant increase in MCP-1/CCL2. The production of angiogenin, vascular endothelial growth factor, GRO/CXCL1–3, EGF, and IL-8/CXCL8 remained high throughout. The concentrations of 2 selected cytokines measured with the array technique and ELISA showed moderate to strong correlation (r = 0.63 for EGF and r = 0.84 for IL-8/CXCL8).

Conclusion: Despite the lack of precise quantification, the protein array might be suitable for cytokine screening. It allows simultaneous detection of a broad spectrum of cytokines (including those not described before) in lacteal secretions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breastfeeding is an important factor helping the newborn adapt to the environment. Microbial colonization of epithelial surfaces after birth is a potential threat to newborns but is also the main contributor to the proper development of the immune system (1)(2). The beneficial effect of breastfeeding may extend well beyond weaning and has been shown to prevent or mitigate several diseases later in life (3). Many of these beneficial effects are the result, at least partially, of secretory IgA, which is the major immunoglobulin isotype in human lacteal secretions, and to other immunologically active components such as antimicrobial factors, cytokines, chemokines, and growth factors. Our knowledge about the variety of these factors is restricted, however, by limitations of the techniques used to study them.

In recent years, methodological approaches for detection of various biological factors have developed rapidly. Bioassays, in which the biological activity of a given component in the sample is tested on a target cell, were replaced by immunoassays (enzymatic immunoassay, RIA) affording generally higher specificity, sensitivity, and reproducibility. Recently, proteomics has been successfully used to detect multiple proteins at once using various multiplex methods available in commercial form (4)(5). Antibody-based protein arrays are a valuable tool, especially in characterizing the spectra of biologically active components in body fluids and cells.

The main objective of our study was to screen human colostrum and milk for cytokines, chemokines, and growth factors and evaluate their changes early after delivery using a proteomic method based on protein array.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
characteristics of the colostrum or milk donors and breast milk sample collection
We collected 31 colostrum (within the first 72 h postpartum) or milk (after 72 h postpartum) samples from 22 mothers within 4 days postpartum. Median (SD) age of the colostrum and milk donors was 29 (4.7) years, and in 12 cases (54.5%) it was their first delivery. The specimens were collected into sterile plastic tubes, immediately frozen at –20 °C, transported to the laboratory, and stored at –20 °C until analysis, which was performed within 8 weeks. To address the question of cytokine degradation during storage, we measured a pool of samples that included both fresh samples and samples frozen at both –20 °C and –70 °C. Freezing the samples did not affect the results of the assay (data not shown). The breast milk fatty layer and cellular elements were removed by 2 centrifugations, at 680g for 10 min at 4 °C, after which the supernatants were removed and then at 10 000g for 30 min at 4 °C. The resulting translucent whey was used for analysis.

protein array
RayBioTM Human Cytokine Array (Raybiotech) was used for detection. Two groups of samples differed according to the protein array used. The 1st group of 17 samples was taken during the first 2 days postpartum and was tested using RayBio Human Cytokine Array V. This array is designed to detect 79 cytokines. The 2nd group, 14 colostrum samples comprising 5 sets of 2 to 3 sequential samples (obtained at 20- to 30-h intervals) from the same mother, was tested using RayBio Human Cytokine Array III, which can detect 42 cytokines.

We processed human cytokine array membranes according to the manufacturer’s recommendation. Briefly, the membranes were blocked by incubation with the blocking buffer at room temperature for 30 min and incubated with the sample at room temperature for 90 min. Membranes were washed 3 times with Wash Buffer I and 2 times with Wash Buffer II at room temperature for 5 min per wash and incubated with biotin-conjugated antibodies at room temperature for 90 min. Finally, the membranes were washed and incubated with horseradish peroxidase–conjugated streptavidin at room temperature for 2 h and with detection buffer for 2 min.

We used a luminescence detector (LAS-1000, Fujifilm) for detection, and the data were digitized and subjected to image analysis (AIDA 3.28, Raytest). By subtracting the background staining and normalizing to the positive controls on the same membrane, we obtained relative protein concentrations. We then compared the mean values for each cytokine detected 30, 60, and 90 h postpartum. The manufacturer claims that the imprecision (CV) of the array is <10%. We tested a pool of colostrum samples on 5 arrays. Each of the 5 results was within 11% of the mean of the 5 results.

We measured total milk protein concentration by the bicinchoninic acid protein assay (Pierce).

epidermal growth factor and interleukin-8/cxcl8 elisa
The results of the array were compared with data obtained by ELISA. The concentrations of epidermal growth factor (EGF)1 and interleukin-8 (IL-8)/CXCL8 were analyzed in all 31 samples with commercial ELISA kits from R&D according to the manufacturer’s protocol. All samples and calibrators were analyzed in duplicate, and the mean value was used. The absorbance of the controls was subtracted from the absorbance of the calibrators and samples.

The influence of breast milk components on the measured EGF and IL-8/CXCL8 concentrations was evaluated by adding known amounts of recombinant cytokines to colostrum samples, which were then analyzed. The recovery was tested with a concentration expected to be in the middle of the linear part of the standard curve. The recovery was 93% for EGF and 94% for IL-8/CXCL8. Our results are in agreement with recovery studies performed by other authors who tested broader spectra of cytokines in colostrum and maternal milk by ELISA (6).

statistics
We performed statistical analysis by use of the MedCalc package for Windows version 8.1.1.0 (MedCalc Software). We used Passing and Bablok regression analysis and Bland–Altman plots for method comparisons (7)(8) and the cusum test to evaluate linearity of the paired data. To analyze the association between the 2 methods, we used the Pearson correlation coefficient. The data obtained at various time points were compared by paired Student t-test with post hoc Bonferroni correction.

ethics
The Local Ethics Committee for Human Research at the Institute for Care of Mother and Child approved the study, and all participants gave informed consent.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
spectrum of cytokines
Using an array designed to detect 79 proteins, we found altogether 68 proteins, 32 of which were detected in human colostrum or milk for the 1st time. Three cytokines [EGF, IL-8/CXCL8, and growth-related oncoprotein (GRO)/CXCL1–3] were present in all the tested samples, and 19 were found very often (≥50% samples) (Table 1 ). The median number of cytokines detected in samples was 20 (range 4 to 38) (Fig. 1 ). The variability in cytokine spectrum of individual samples is shown in Fig. 1 .


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  		Table 1. Comprehensive array map (RayBio Human Cytokine Array V) of cytokines present in human colostrum.


Figure 1
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  		Figure 1. Examples documenting the variability of the individual samples.

Sample A shows 4 (minimum), sample B 16, sample C 22, and sample D 38 (maximum) positive cytokines. These arrays are designed to detect 79 cytokines, chemokines, or growth factors. See Table 1Up for cytokine identification.

The cytokine concentration in colostrum or milk taken from an individual mother was quite variable, and changes of the same protein were not consistent over time (Fig. 2 ). The number of detected cytokines did not change significantly with time, although there was an overall decreasing tendency (Fig. 3 ). The mean number of cytokines (SD) we found 20 to 30 h after delivery was 22.80 (8.61), 50 to 60 h after delivery, 18.60 (5.32), and 80 to 90 h after delivery, 15.75 (5.32).


Figure 2
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  		Figure 2. Changes in cytokine relative concentration with time after delivery.

Thin lines represent the trends in relative protein concentration in individual mothers. The thick line represents the mean values. *Significant differences between the groups of samples taken at the same time.


Figure 3
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  		Figure 3. Cytokine profile 30 h (A), 60 h (B), and 80 h (C) after delivery in one of the sample sets, demonstrating the changes in cytokine spectrum.

The rightmost array (D) represents a negative control array. These arrays are designed to detect 42 cytokines, chemokines, or growth factors. See Table 2 for cytokine identification.

We found 32 proteins that have not been described in human colostrum or milk before. Some of these were present in more than 3 samples: growth factors (fibroblast growth factor 4, placental growth factor), chemotactic factors [B lymphocyte chemoattractant/CXCL13, macrophage inflammatory protein (MIP)-1ß/CCL4, MIP-1{delta}/CCL15, MIP-3{alpha}/CCL20, pulmonary and activation-related chemokine (PARC)/CCL18, leukemia inhibitory factor, oncostatin M], and antiinflammatory factors [tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, macrophage-derived chemokine (MDC)/CCL22].

The protein content in 14 samples was in the range of 23.4 to 203 g/L, with a mean (SE) of 70.67 (16.30) g/L. We did not find any statistically significant difference between the protein content in milk at 20 to 30 h [135.48 (27.10) g/L], 50 to 60 h [38.68 (6.28) g/L], and 80 to 90 h postpartum [29.70 (1.69) g/L].

comparison of elisa with protein array
We statistically evaluated and compared the data obtained for IL-8/CXCL8 and EGF using ELISA and protein array. The correlation between the array and ELISA was moderate to strong (r = 0.63, P <0.001 for EGF and r = 0.84, P <0.001 for IL-8/CXCL8). The Passing and Bablok regression analysis (Fig. 4 A and B) and Bland–Altman plot (Fig. 4 C and D) did not suggest any constant or proportional difference between the 2 methods. The cusum test did not show a significant deviation from linearity for either of the cytokines (P >0.10 for EGF and P >0.05 for IL-8/CXCL8).


Figure 4
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  		Figure 4. Passing and Bablok regression scatter plot for EGF (A) and IL-8/CXCL8 (C).

Solid line, regression line (y = –7.7398 + 1.0913x for EGF and y = 10.1347 + 1.1076x for IL-8); dashed lines, 95% confidence interval for the regression line; dotted line, line of identity. Bland–Altman difference plot for EGF (B) and IL-8 (D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the approaches to characterize the proteomic profile in biological samples is protein array. The cytokine array we used in our experiments belongs to a multiplex immunoassay that allows detection of many proteins simultaneously. Nowadays, more multiplex techniques based on antigen–antibody binding are available. Because it uses the largest number of cytokines, the protein array is most suitable to screen for biological factors and their changes.

The cytokine arrays give information about relative changes in cytokine concentrations, but they do not provide specific quantitative information as does an ELISA. The sensitivity for each protein of interest varies because of differences in antibody affinity. However, correlation with ELISA, at least for the 2 cytokines we tested (EGF and IL-8/CXCL8), is moderate to strong. Correlation between these 2 methods for IL-8/CXCL8 detection was also reported in human plasma (9). Interestingly, the concentrations of EGF and IL-8/CXCL8 in colostrum are >1000 times higher than concentrations in plasma (10).

The advantages of this approach are that the number of proteins detected on an array is high and steadily increasing (moreover, custom arrays are offered); the manufacturer (Raybiotech) tests the sensitivity and specificity for each protein of interest and claims that no cross-reactivities have been found for the antibodies on the array; and according to the user’s manual, the detection range may be at least 100-fold greater (IL-2) with the protein array than with ELISA. We used this advantageous technique to measure the content of cytokines in biologically important body fluids—human colostrum and milk. The quality of measurement is influenced by many factors including preparation, processing, and standardization of samples. Moreover, interindividual, diurnal, and other variations may affect the evaluation of the biologically active components of mucosal and exocrine gland secretions (11). The number of cytokines detected in individual samples of colostrum and milk shows considerable interindividual variability. The problem of normalization of the cytokine content to some constant in mucosal fluids is not yet solved and standardized (12).

It seems that cytokines are secreted in the mammary gland mostly by the epithelium and resident leukocytes, but a minor part derives from the serum (13). The production of chemotactic factors and specific adhesion molecules in the mammary gland allows the cells of mucosal origin to enter the mammary gland from the circulation and increase the production of immunologically active proteins in situ. Human colostrum and milk contain high concentrations (106 to 107 for colostrum, 105 to 106 for milk) of numerous cells capable of producing various cytokines even in the infant’s gut (12). Besides nutritional components, antibodies, and major antibacterial proteins (e.g., lysozyme, lactoferrin, defensins), a wide array of immunomodulatory factors such as cytokines, chemokines, and growth factors are present (3). These factors may influence not only the suckling’s gut, but the whole organism as well, as the complex proteins may be absorbed into the newborn’s bloodstream through the permeable intestinal barrier and thus help to adapt the newborn’s immune system to the environment (14)(15). Moreover, all the above factors are similarly important for protecting the maternal mammary gland (13).

Using multiplex analysis, this study confirmed the presence of various cytokines described previously; EGF, GRO/CXCL1–3, and IL-8/CXCL8 were detected in all samples of lacteal secretions. There is little evidence concerning the in vivo activities of human milk cytokines in sucklings; however, their immunomodulatory effects in vitro and in animal models have been proven repeatedly. In an animal model of human celiac disease, we have shown that EGF, in concentrations similar to those in milk, exerts a protective effect when orally administered to newborn rats (16).

In connection with supposed biological activities, it would be interesting to speculate on the total intake of these factors. Based on the known daily volume of milk ingested by breast-fed infants, we can calculate the intake of those cytokines detected in human colostrum, by use of a method that gives quantitative results (17). EGF concentration in colostrum is ~222 µg/L (7.3 µg/day) on the 1st day, 228 µg/L (9.8 µg/day) on the 2nd day, and 268 µg/L (28.7 µg/day) on the 3rd day. For other cytokines, we can use sensitivity values for the array given by the manufacturer: e.g., sensitivity for angiogenin is 10 ng/L; because it was detected in the sample, it could be expected that the suckling ingests >330 pg on the 1st day, >430 pg on the 2nd day, and >1070 pg on the 3rd day.

Interestingly, by use of the protein array, we found several immunologically active proteins not yet described in human colostrum or milk (search performed using PubMed database,http://www.ncbi.nlm.nih.gov/PubMed, accessed July 24, 2006). These proteins (cytokines) differ in their main biological activities; they exert chemoattractant, growth-promoting, and antiinflammatory activities, and some of them display multiple functional properties.

Cytokines with chemoattracting activity found in colostrum or milk for the 1st time included PARC/CCL18, MIP-3{alpha}/CCL20 (mainly for lymphocytes and immature dendritic cells), MIP-1ß/CCL4 (mainly for NK cells and macrophages), and B lymphocyte chemoattractant/CXCL13 (mainly for naive B lymphocytes). Their activity, promoting the attraction of various types of cells, could be advantageous for the proper development and priming of the intestinal lymphocytes, which may protect the newborn against several diseases immediately after birth and later in life (18).

Some of the growth factors found in colostrum and milk for the 1st time, such as leukemia inhibitory factor and fibroblast growth factors, may contribute to developing gut structure and function including epithelial barrier maturation; others, such as placenta growth factor, may mainly influence angiogenesis. Interestingly, we found several neuronal growth factors (brain-derived neurotrophic factor, glial cell line–derived neurotrophic factor, neurotropin-3, and neurotropin-4), which may be important for development of the newborn’s enteric nervous system and possibly also for the proper development of the central nervous system (19).

Along with several cytokines with antiinflammatory activity described earlier, we found the following antiinflammatory factors for the first time: TIMP-1, TIMP-2, and MDC. These factors may help in establishing mucosal homeostasis in both the mammary gland and the suckling’s gut (13)(20)(21).

Because of the increasing interest in the detection of multiple biomarkers, there is a need to identify them in a time-saving and efficient way. The protein array offers parallel identification of individual protein biomarkers, which makes it very suitable for use in basic and clinical research as well as in clinical practice.


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  		Table 2. Comprehensive array map (RayBio Human Cytokine Array III) of cytokines for identification of individual cytokines in Fig. 3Up .


   Acknowledgments
 
Grant/funding support: The study was supported by grants 310/03/H147 and 303/06/0974 of the Czech Science Foundation; S500200572 of the Academy of Sciences of Czech Republic; and Institutional Research Concept Grant AV0Z50200510.

Financial disclosures: None declared.

Acknowledgements: We are grateful to Dr. Brauner, Institute of Physiology, Academy of Sciences of the Czech Republic, who helped us with the use of the chemiluminescence detector LAS-1000.


   Footnotes
 
1 Nonstandard abbreviations: EGF, epidermal growth factor; IL-8, interleukin-8; GRO, growth-related oncoprotein; BLC, B lymphocyte chemoattractant; MIP, macrophage inflammatory protein; TIMP, tissue inhibitor of metalloproteinase; and MDC, macrophage-derived chemokine.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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