Role of Prolactin in Promotion of Immune Cell Migration into the Mammary Gland

Abstract

Immune cells in the mammary gland play a number of important roles, including protection against infection during lactation and, after passing into milk, modulation of offspring immunity. However, little is known about the mechanism of recruitment of immune cells to the lactating gland in the absence of infection. Given the importance of prolactin to other aspects of lactation, we hypothesized it would also play a role in immune cell recruitment. Prolactin treatment of adult female mice for a period equivalent to pregnancy and the first week of lactation increased immune cell flux through the mammary gland, as reflected in the number of immune cells in mammary gland-draining, but not other lymph nodes. Conditioned medium from luminal mammary epithelial HC11 cell cultures was chemo-attractive to CD4+ and CD8+ T cells, CD4+ and CD8+ memory T cells, B cells, macrophages, monocytes, eosinophils, and neutrophils. Prolactin did not act as a direct chemo-attractant, but through effects on luminal mammary epithelial cells, increased the chemo-attractant properties of conditioned medium. Macrophages and neutrophils constitute the largest proportion of cells in milk from healthy glands. Depletion of CCL2 and CXCL1 from conditioned medium reduced chemo-attraction of monocytes and neutrophils, and prolactin increased expression of these two chemokines in mammary epithelial cells. We conclude that prolactin is an important player in the recruitment of immune cells to the mammary gland both through its activities to increase epithelial cell number as well as production of chemo-attractants on a per cell basis.

Keywords: Chemoattraction; Mammary epithelium; Migration; Milk immune cells; Prolactin.

Fig. 1. Representative dot plots of the gating strategy used for cell types discussed in this manuscript

Gates were determined on the basis of isotype control staining.

Fig. 1. Representative dot plots of the gating strategy used for cell types discussed in this manuscript

Gates were determined on the basis of isotype control staining.

Fig. 2. After 14 days, estrous cycling returns to normal in prolactin-treated animals

Vaginal smears were collected every morning between 8 and 10 am for the last two weeks of a 28-day treatment. Estrous cycle stage was determined on the basis of ratios of cell types present. Data are presented as the average number of days in the 14 day period that mice in each group (n = 6 per group) experienced (A) Diestrus (B) Proestrus (C) Estrus (D) Metestrus. * P < 0.05, ** P < 0.01, and *** P < 0.0001

Fig. 3. Prolactin increased CD4+, CD8+, and CD19+ cell numbers in mammary gland-draining, but not the popliteal lymph nodes

Numbers of indicated cell types were determined for each animal by total cell counts per lymph node and flow cytometric analysis of the markers. n = 6 per group. * P < 0.05, ** P < 0.01, and *** P < 0.0001

Fig. 4. Media conditioned by mammary epithelial cells increased migration of immune cells

Splenocytes were loaded into the top of Transwells™ with either serum-free medium (M) or mammary epithelial cell-conditioned serum-free medium (CM) in the bottoms of the Transwells™. Data are presented as relative migration of immune cells compared to control (set at 1). n = 9 per group. P < 0.05, ** P < 0.01, and *** P < 0.0001

Fig. 4. Media conditioned by mammary epithelial cells increased migration of immune cells

Splenocytes were loaded into the top of Transwells™ with either serum-free medium (M) or mammary epithelial cell-conditioned serum-free medium (CM) in the bottoms of the Transwells™. Data are presented as relative migration of immune cells compared to control (set at 1). n = 9 per group. P < 0.05, ** P < 0.01, and *** P < 0.0001

Fig. 5. Neutrophils that migrated towards media conditioned by mammary epithelial cells expressed more CD11b

Relative mean fluorescence intensity (MFI) of labeled CD11b on neutrophils that migrated to the bottoms of Transwells™ containing either serum-free medium (M) normalized to 1 or mammary epithelial cell conditioned serum-free medium (CM). n = 9 per group. P < 0.05, ** P < 0.01, and *** P < 0.0001

Fig. 6. Prolactin does not act as a direct chemoattractant for immune cells in the time frame used

Splenocytes were loaded into the top of Transwells™ and either serum-free medium (M) or serum-free medium containing 100 ng/mL prolactin (PRL) was placed in the bottoms of the Transwells™. Data are presented as relative migration of immune cells where the control medium (M) is set as 1. n = 9 per group. P < 0.05, ** P < 0.01, and *** P < 0.0001

Fig. 6. Prolactin does not act as a direct chemoattractant for immune cells in the time frame used

Splenocytes were loaded into the top of Transwells™ and either serum-free medium (M) or serum-free medium containing 100 ng/mL prolactin (PRL) was placed in the bottoms of the Transwells™. Data are presented as relative migration of immune cells where the control medium (M) is set as 1. n = 9 per group. P < 0.05, ** P < 0.01, and *** P < 0.0001

Fig. 7. Prolactin treatment of mammary epithelial cells increased migration of most immune cells

Splenocytes were loaded into the top of Transwells™ with either mammary epithelial cell-conditioned serum-free medium (CM) or mammary epithelial cell- conditioned serum-free media from HC11 cells treated with 100 ng/mL prolactin (PRL) in the bottoms of the Transwells™. Data are presented as relative migration of immune cells with the regular conditioned medium set as 1. n = 9 per group. P < 0.05, ** P < 0.01, and *** P < 0.0001

Fig. 7. Prolactin treatment of mammary epithelial cells increased migration of most immune cells

Splenocytes were loaded into the top of Transwells™ with either mammary epithelial cell-conditioned serum-free medium (CM) or mammary epithelial cell- conditioned serum-free media from HC11 cells treated with 100 ng/mL prolactin (PRL) in the bottoms of the Transwells™. Data are presented as relative migration of immune cells with the regular conditioned medium set as 1. n = 9 per group. P < 0.05, ** P < 0.01, and *** P < 0.0001

Fig. 8. Effect of Prolactin on CCL2 and CXCL1 expression and cell number, and effect of CCL2 and CXCL1 depletion on monocyte and neutrophil migration

Relative HC11 mRNA expression of CCL2 (A) and CXCL1 (B) normalized to GAPDH without (DPBS) or with prolactin (100ng/mL) treatment (PRL). (C) Cell numbers, as determined by hemocytometer counts, with the same treatment. Relative migration after depletion of CCL2 (D) and CXCL1 (E). Migration of each cell type in response to serum-free medium is subtracted from each condition Data are presented with control = 1. n = 9 per group. P < 0.05, ** P < 0.01, and *** P < 0.0001