Interleukin-8 and Its Receptors in Human Milk from Mothers of Full-Term and Premature Infants

Abstract

In addition to its nutritional benefits, human milk also has bioactive elements. Limited immunological functions of newborns are supported and altered by the immunological elements of mother milk. Chemokines are of importance among these immune factors. Interleukin-8 (IL-8) has been demonstrated in mother's milk, and its receptors, CXC chemokine receptors (CXCR)-1 and CXCR-2, were detected on cells, responsible for immunological reactions and mammary glandular cells. The soluble forms of these receptors are yet to be described in human milk. In this study, it was aimed to assess the IL-8 levels and the concentrations of its receptors in colostrum and mature mother's milk in regard to preterm and term delivery. The results of this study indicated a decline in IL-8 levels with the lactation stage, but no difference was observed between term and preterm mother's milk. Regarding the CXCR-1 and CXCR-2, the concentrations of these receptors were similar in both colostrum and mature milk. Furthermore, there was not any significant difference between term and preterm mother's milk. In conclusion, this is the first study to investigate the concentrations of CXCR-1 and CXCR-2 with the levels of IL-8 in colostrum and mature human milk of term and preterm newborns. The alterations in IL-8 levels were similar in some of the studies reported. CXCR-1 and CXCR-2 levels did not demonstrate any significant difference. Further studies are required to investigate the soluble forms of these receptors and their relation to IL-8 with larger cohort.

Introduction

Human neonate has a limited ability to give rapid and effective response to infections. Both B-cell and T-cell functions and phagocytic defense remain impaired in this immunological insufficiency period.¹ During this developmental delay of the immune system, neonatal immunity is affected and supported by mother's immune elements transferred through breastfeeding.²

Breastfeeding has shown to be the optimal nutrition alternative for newborns. In addition to its nutritional benefits, human milk also has bioactive elements, which have immune modulating, immune stimulating, anti-inflammatory, and antimicrobial effects. These bioactive factors are varied and tend to differ in amounts depending on the stage of lactation.³ Several immunological elements like cytokines and chemokines have been identified in human milk. This has been a new research area and discovering new cytokines and chemokines and obtaining information about their structures and functions have been of great interest in the last two decades.²

Interleukin-1 (IL-1) is probably the first cytokine to be detected in human milk,⁴ followed by the identification of tumor necrosis factor-alpha (TNF-α)⁵ and Interleukin-6 (IL-6)^6,7 in colostrum. Chemokines, which have discrete target cell selectivity on chemotaxis, have been determined in human milk soon after.⁸ Interleukin-8 (IL-8), monocyte chemotactic protein (MCP-1), and RANTES (regulated on activation, normal T cell expressed and secreted) are some of the chemokines found in human milk.^9–11

Chemokines are divided into two subgroups as follows: CXC chemokines and CC chemokines. CXC chemokines in human milk have been implicated in the chemotactic activity especially for neutrophils and provide host defense against bacterial infections. IL-8 is an important CXC chemokine especially for neutrophil chemotaxis and is expressed in macrophages, endothelial, and epithelial cells. Although the existence of IL-8 has been demonstrated in human milk, there are conflicting results about its amount either being higher in colostrum or mature milk and either after preterm or term birth.¹² CXC chemokine receptors (CXCR) are a large family of G protein-linked integral membrane proteins that specifically bind and respond to cytokines of the CXC chemokine family.¹³ Seven CXCR have been described in mammals. CXCR-1 and CXCR-2 are the most studied receptors for IL-8.¹⁴ CXCR-1 and CXCR-2 are structurally similar receptors that recognize CXC chemokines, which contain E-L-R amino acid motif. CXCL-8 (known as interleukin-8) and CXCL-6 can both bind CXCR-1, and the rest of the ELR-positive chemokines bind only CXCR-2.¹⁵

CXCR-1 and CXCR-2 are expressed on neutrophils and other immune cells,¹⁶ as well as mammary stem cells and some epithelial and endothelial cells.^17–19 These receptors were detected in fetal intestinal cells, and IL-8 exposure was related to inflammation in intestines.²⁰ However, considering the fetus to be exposed to IL-8 by amniotic fluid and the newborn by mother's milk, IL-8 was regarded as a physiological factor for intestines.²¹ Some studies have depicted IL-8 levels in human milk, but concentrations of CXCR-1 and CXCR-2 and their evaluation in connection to IL-8 levels have not been assessed in human milk from mothers without any infection or inflammation in breasts.

In this study, it was aimed to investigate the concentrations of IL-8, CXCR-1, and CXCR-2 in human milk and also assess the relationship between the levels of these immunological elements and gestational age or the phase of lactation.

Materials and Methods

Participants

This study was approved by the Gulhane Military Medical Academy Ethics Committee. An informed consent form was obtained from participating mothers.

The mothers of preterm babies, less than 35 week of gestation, and mothers of term babies, between 37 and 42 weeks of gestation who were being followed-up in Gulhane Military Medical Academy Neonatology unit and outpatient clinic between February and August 2015, were recruited.

Mothers with an anamnesis of any systemic diseases and mothers with a physical examination revealing mastitis, malignity, and any other infectious or inflammatory conditions on breasts were excluded from the study. Mothers were questioned about any infections during pregnancy such as urinary tract infections, vaginal discharge with foul smell, fever, and leukocytosis and any cases with a diagnosis of chorioamnionitis were excluded. Other reasons which could be confounders such as preeclampsia, premature rupture of membranes, hypertension, and maternal diabetes were also evaluated.

Mothers donated milk samples twice; first in the first 3 days after birth and second on the 15th day after birth. Sample collection was made using an electric breast pump (Medela, Inc.), and to provide standardization, milk sampling was made after at least 3 hours later than the last feeding. The hindmilk was collected by obtaining the last 5 mL of pumping session to a sterile injector. Samples were kept in −80°C until CXCR-1, CXCR-2, and IL-8 levels were measured.

Laboratory analysis

Milk samples were kept in sterile plastic tubes and were centrifuged at 2,000 g for 15 minutes. The lipid layer at the surface was passed through using a thin needle, and supernatant was obtained and stored at −80°C.

Microplate wells were washed twice with 400 μL of washing buffer solution. 100 μL of standards (we have applied the external standard dilution procedure) was added to standard wells. 50 μL of sample diluent and then 100 μL of samples were added to sample wells. 50 μL of biotin-conjugate was added to each well and the plate, covered with an adhesive film and incubated for 2 hours at room temperature on a microplate shaker at about 400 rpm. Then, the plate was washed thrice with wash buffer. 100 μL of diluted Streptavidin-HRP was added to each well and the plate, covered with an adhesive film and incubated for 1 hour at room temperature on a microplate shaker at about 400 rpm. After incubation, the plate was washed thrice with wash buffer. 100 μL of TMB substrate was added to each well and incubated in the dark at room temperature for 15 min. 100 μL of stop solution was added to each well. The plate was measured with the absorbance at 450 nm with a Synergy HT plate reader (BioTek Instruments, Winooski, VT). From the standard curve, the IL-8 levels in each test sample were quantitated.

CXCR-1 and CXCR-2 levels were determined by the Sandwich ELISA Kit. CXCR-1 and CXCR-2 concentrations were assayed according to the CXCR-1 and CXCR-2 ELISA protocol of manufacturer's instruction (Human CXCR-1 and CXCR-2 ELISA Kit; Elabscience Biotechnology, Hubei, China). Briefly, 100 μL of standards and samples (human breast milk supernatants) were added to 96-microwell plate wells and incubated 90 minutes at 37°C. After incubation, liquid of each well was aspirated and 100 μL Biotinylated Detection Ab was added to each well. The plate was incubated for 1 hour at 37°C. After incubation, each well was washed thrice with 350 μL wash buffer and 100 μL HRP conjugate was added to each well. The plate was incubated at 37°C for 30 minutes and each well was washed five times with 350 μL wash buffer. 90 μL of substrate solution was added to each well and the plate was incubated in the dark at 37°C for 15 minutes. Then, 50 μL of stop solution was added to each well and the plate was measured with the absorbance at 450 nm with a Synergy HT plate reader (BioTek Instruments). From the standard curve, the CXCR-1 and CXCR-2 levels in each test sample were quantitated.

Statistical analysis

“Statistical Package for the Social Sciences for Windows 21.0” was used for analysis of the data. Frequencies and percentages were used as descriptive statistics for categorical variables. To describe scale variables, mean ± standard deviation and median (min-max) were used. Differences between groups were assessed by Student's t-test in variables with normal distribution and Mann–Whitney U-test for other variables. Chi-Square tests were applied for comparing discrete variables. p < 0.05 was considered as significant.

Results

A total of the mothers of 20 preterm and 20 term babies were included in the study. Female to male ratio of the newborns was 7/13 and 10/10 in preterm and term groups, respectively. Mean gestational age in preterm group was 31.05 ± 3.1 weeks and 38.77 ± 1.17 weeks in term group. Mean gestational weight was 1648.25 ± 553.96 g and 3346.00 ± 449.53 g in preterm and term groups, respectively. The rate of antenatal steroid application was 25% (n = 5), preeclampsia was 10% (n = 2), and premature rupture of membranes was 10% (n = 2) among preterm births. The clinical features are presented in Table 1. The rate of cesarean section (C/S) deliveries in the preterm group was 80%, while it was 40% in the term group. The reason for high C/S delivery in preterm group was preterm labor.

Table 1.

Demographic and Clinical Features

	Preterm group (n = 20)	Term group (n = 20)
Female/male	7/13	10/10
Gestational age (weeks)	31.05 ± 3.10	38.77 ± 1.17
Birth weight (g)	1648.25 ± 553.96	3346.00 ± 449.53
Delivery
Vaginal n (%)	4 (20)	12 (60)
C/S n (%)	16 (80)	8 (40)
Antenatal steroid application n (%)	5 (25)	0
Preeclampsia n (%)	2 (10)	0
Premature rupture of membranes n (%)	2 (10)	0
Maternal hypertension n (%)	1 (5)	2 (10)
Maternal diabetes n (%)	1 (5)	0

C/S, cesarean section.

Mean IL-8 levels tended to decrease by time in both groups. The mean IL-8 levels in colostrum of preterm group were significantly higher than the mature milk of the same group (p < 0.001). In term group, the IL-8 levels presented similar decline and the colostrum consisted of higher IL-8 levels compared to mature milk (p = 0.001). The levels of IL-8 in colostrum and mature milk were similar in preterm and term groups. The comparison of decreasing in IL-8 concentrations between preterm and term groups did not reveal any significant difference (Table 2).

Table 2.

The In-Group and Between-Group Comparison of Alterations in Immunological Markers

	Preterm group (n = 20)			Term group (n = 20)
	Colostrum	Mature milk	p^a	Colostrum	Mature milk	p^a	p^b
CXCR-1 (ng/mL)	1.87 ± 3.57	1.69 ± 2.03	0.211	1.73 ± 1.43	3.11 ± 3.44	0.391	0.365
CXCR-2 (ng/mL)	11.74 ± 11.06	9.96 ± 10.57	0.411	9.52 ± 8.94	15.58 ± 16.83	0.196	0.560
IL-8 (pg/mL)	1092.73 ± 925.75	126.55 ± 161.18	<0.001	1139.33 ± 1206.85	228.23 ± 534.84	0.001	0.689

p-value for in-group comparison of the immunological markers.

p-value for between-group comparison of the alterations in immunological markers.

CXCR, CXC chemokine receptors; IL-8, interleukin-8.

The mean CXCR-1 levels in colostrum were not different from the mature milk in both preterm (p = 0.211) and term (p = 0.391) groups, and the mean CXCR-1 levels in colostrum of preterm group were slightly higher than the term group (p = 0.041). The same difference was not present between mature milk of both groups (Table 3). The comparison of alterations in levels of CXCR-1 in colostrum and mature milk between groups was not significant (Table 2).

Table 3.

The Comparison of Immunological Markers in Colostrum and Mature Milk

	Colostrum			Mature milk
	Preterm group (n = 20)	Term group (n = 20)	p	Preterm group (n = 20)	Term group (n = 20)	p
CXCR-1 (ng/mL)	1.87 ± 3.57	1.73 ± 1.43	0.041	1.69 ± 2.03	3.11 ± 3.44	0.137
CXCR-2 (ng/mL)	11.74 ± 11.06	9.52 ± 8.94	0.874	9.96 ± 10.57	15.58 ± 16.83	0.203
IL-8 (pg/mL)	1092.73 ± 925.75	1139.33 ± 1206.85	0.820	126.55 ± 161.18	228.23 ± 534.84	0.841

The results for CXCR-2 were similar to that of CXCR-1. There was no significant difference between colostrum and mature milk samples in both preterm (p = 0.411) and term (p = 0.560) groups. The CXCR-2 level was similar in colostrum of preterm and term groups and was also similar in mature milk levels of preterm and term groups (Table 3). Between groups, comparisons of changes in CXCR-2 levels from colostrum to mature milk did not present any difference (Table 2).

Prophylactic antenatal steroid administration was present in 25% of preterm newborns. No difference was observed in milk content of CXCR-1 (p = 0.266), CXCR-2 (p = 0.842), and IL-8 (p = 0.156) between mothers who underwent steroid protocol and mothers without antenatal steroid application. Similar to antenatal steroid application, no significant difference was seen between the milk of mothers with preeclampsia, premature rupture of membranes, and without any of these disorders in regard to CXCR-1, CXCR-2, and IL-8.

No correlation between gestational age, birth weight, and immunological markers of CXCR-1, CXCR-2, and IL-8 levels neither in colostrum nor in mature milk was detected. The results are not presented.

Discussion

The immune content of mother's milk changes during different stages of lactation, but not much is known about the alterations in immune ingredient by time. In this study, the changes in the concentrations of IL-8 and its receptors CXCR-1 and CXCR-2 during lactation, by investigating colostrum and mature milk samples, were investigated. In addition, the differences in these immunological elements between milk of mothers after term and preterm delivery were also evaluated.

This is the first study to investigate the IL-8, CXCR-1, and CXCR-2 levels in human milk in regard to being term or preterm and colostrum or mature milk. The results of this study suggest that IL-8 levels were higher in colostrum compared to mature milk regardless of being after term or preterm birth. The decline in IL-8 levels of mother's milk on the 15th day from birth was similar after both term and preterm deliveries. CXCR-1 and CXCR-2 levels in human milk were similar in colostrum and mature milk in both preterm and term groups regardless of the decreased IL-8 levels in mature milk after both term and preterm deliveries.

IL-8 is an important chemotactic pro-inflammatory cytokine.²² It was first determined in milk samples by Basolo et al.¹⁰ and by Palkowetz et al.⁶ and was demonstrated to be secreted from mammary cells and from the cells included in the milk.⁹ IL-8 probably presents its pro-inflammatory functions on neutrophils of milk. In addition, a study by Maheshwari et al.²¹ investigated and reported that IL-8 was not only a pro-inflammatory cytokine but also has a trophic effect on the developing intestines of a newborn.

Our results about IL-8 levels were supportive of the previous studies stating higher IL-8 levels in colostrums.^21,23 However, we did not observe any difference in IL-8 levels between term and preterm colostrum milk samples, which was against our expectation of preterm colostrum to consist of higher immunological support. Previous studies also stated conflicting results. Michie et al.¹² reported lower levels of IL-8 in preterm milk compared to term milk, while Maheshwari et al. indicated the IL-8 levels to be higher in preterm colostrum than term colostrums.²¹

Some cytokines have been detected with their receptors in human milk. For instance, TNF-α has been shown to be included in human milk with its soluble receptor. IL-6 receptor was detected to be synthesized by mammary epithelium and was secreted to human milk.^10,24 The receptors for IL-8, CXCR-1, and CXCR-2, which are expressed mostly on neutrophils, have been also demonstrated on epithelial, endothelial, and mammary cells as well. The expression of these receptors on neutrophils is well known and studied, but their expressions on other cell types are not well investigated.^25,26 The presence of IL-8 in human milk can be in charge of stimulating CXCR expressing cells of mother's milk. Besides, similar to TNF-α, in human milk, IL-8 might partially be with the soluble forms of these receptors, which are not well known. Soluble CXCR-2 was studied in a few studies,²⁷ but no study has been conducted on the soluble form of CXCR-1, except for one investigating the fragments of the receptor after cleavage.²⁸

In our study, CXCR levels were studied in human milk for the first time in the literature. CXCR-1 were slightly higher in preterm group compared to term group in colostrum. However, this was not observed in mature milk. Regarding CXCR-2, the amount in preterm and term groups was similar in both colostrum and mature milk. This could be due to CXCR-1 being expressed on various cell types more than CXCR-2, especially on inflammatory cells, which are in higher amounts in colostrum. Nevertheless, it is not easy to infer its relation to prematurity. CXCR-2 expression has been detected in different cell types as well. However, the cellular diversity of expression is not larger than CXCR-1. This may explain the similar levels of CXCR-2 in colostrum and mature milk in both preterm and term groups. Besides, IL-8 and the receptors were measured using ELISA Sandwich method and the ligand to be bound to its soluble receptors might have affected the detection. Moreover, CXCLR1 and CXCLR2 tend to form homo and heterodimers. This could also affect accurate measurement of the molecules.²⁹ Only the detection of these receptors in human milk after preterm and term delivery was aimed in this study, so additional tests such as Western blotting for differentiation of bound and unbound ligands and dimers of the receptors are of importance and can be evaluated in a novel study.

The concentrations of cytokines in human milk can be affected by various factors such as mastitis, electrolyte concentrations of milk, nutritional and allergic status, and ethnicity of mother.^23,30,31 In addition, drug usage of mothers should also be considered in assessing cytokine levels of mother's milk. In this context, milk samples of mothers who underwent antenatal steroid application in preterm group were compared to the milk samples of mothers without antenatal steroid application. The results did not indicate any significant difference regarding IL-8, CXCR-1, and CXCR-2 levels between the subgroups of mothers with antenatal steroid application and without steroid application among the preterm group in both colostrum and mature milk. The number of mothers with antenatal steroid application in preterm group was 5, which was low to assess the effect of the drug on milk content. Significantly higher C/S delivery rate in preterm group compared to term group was not considered as a confounding factor as the reason for C/S delivery was preterm labor in preterm group and mothers with any cases of infections were not included in the study.

Finally, this is the first study to investigate the concentrations of CXCR-1 and CXCR-2 with the levels of IL-8 in colostrum and mature human milk of term and preterm newborns regarding some clinical features. It can be stated that IL-8 level is higher in colostrum after both term and preterm deliveries and its level in colostrum is similar in mother's milk of term and preterm newborns. CXCR-1 and CXCR-2 levels do not tend to differ in colostrum and mature milk after preterm and term births. Each study group consisted of 20 mothers, which may affect the proper assessment of the levels and relationship between these immunological markers. Larger groups are required to conduct studies to make better assessment of these immune markers in human milk, and further investigations about the soluble forms of these receptors should be conducted to understand IL-8 and its interactions in human milk.

Footnotes

Disclosure Statement

No competing financial interests exist.

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