Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6 (2), R75-91

Involution of the Mouse Mammary Gland Is Associated With an Immune Cascade and an Acute-Phase Response, Involving LBP, CD14 and STAT3

Affiliations

Involution of the Mouse Mammary Gland Is Associated With an Immune Cascade and an Acute-Phase Response, Involving LBP, CD14 and STAT3

Torsten Stein et al. Breast Cancer Res.

Abstract

Introduction: Involution of the mammary gland is a complex process of controlled apoptosis and tissue remodelling. The aim of the project was to identify genes that are specifically involved in this process.

Methods: We used Affymetrix oligonucleotide microarrays to perform a detailed transcript analysis on the mechanism of controlled involution after withdrawal of the pups at day seven of lactation. Some of the results were confirmed by semi-quantitative reverse transcriptase polymerase chain reaction, Western blotting or immunohistochemistry.

Results: We identified 145 genes that were specifically upregulated during the first 4 days of involution; of these, 49 encoded immunoglobulin genes. A further 12 genes, including those encoding the signal transducer and activator of transcription 3 (STAT3), the lipopolysaccharide receptor (CD14) and lipopolysaccharide-binding protein (LBP), were involved in the acute-phase response, demonstrating that the expression of acute-phase response genes can occur in the mammary gland itself and not only in the liver. Expression of LBP and CD14 was upregulated, at both the RNA and protein level, immediately after pup withdrawal; CD14 was strongly expressed in the luminal epithelial cells. Other genes identified suggested neutrophil activation early in involution, followed by macrophage activation late in the process. Immunohistochemistry and histological staining confirmed the infiltration of the involuting mammary tissue with neutrophils, plasma cells, macrophages and eosinophils.

Conclusion: Oligonucleotide microarrays are a useful tool for identifying genes that are involved in the complex developmental process of mammary gland involution. The genes identified are consistent with an immune cascade, with an early acute-phase response that occurs in the mammary gland itself and resembles a wound healing process.

Figures

Figure 1
Figure 1
RNA expression profiles of selected control genes. The graphs show the scaled average signals (y-axis) for the milk protein genes casein α, β, γ, δ, κ and butyrophilin (a), as well as the transcription factor genes MSX2 (b) and LMO4 (c), and the pro-apoptotic gene BAX (d) at the different stages of adult mouse mammary gland development (x-axis).
Figure 2
Figure 2
Pearson correlation clustering of involution specific genes. (a) One hundred and three upregulated probe sets identified through SOM clustering were clustered with the Pearson correlation clustering program of GeneSpring (Silicon Genetics). The colours (see colour bar) represent a relative measure for the expression, with 1 representing the median, the red colour upregulated and blue downregulated gene expression. The resulting gene tree was grouped into 10 clusters of similarly expressed genes (shown bracketed at the right) and their average expression patterns are shown on the right as graphs (red lines) with standard deviations (blue lines) (DMT®; Affymetrix). Note that the y-axes represent only arbitrary expression levels and are not scaled identically between graphs. (b) Sixteen downregulated genes were clustered by using the software mentioned above and their average expression (red line) and standard deviations (blue lines) are shown in the graph on the right (DMT®; Affymetrix). Abbreviations: vir, virgin; preg, pregnancy; lac, lactation; inv, involution.
Figure 3
Figure 3
B-lymphocyte response during involution. (a) Pearson correlation clustering of immunoglobulin genes. The colours (see colour bar) represent a relative measure for the expression level, with 1 representing the median. Forty-nine immunoglobulin genes that were upregulated during involution were clustered by using the Pearson correlation clustering function from GeneSpring (Silicon Genetics); their average expression (red line) and standard deviations (blue lines) are shown in the graph on the right (DMT®; Affymetrix). Note that the y-axis represents only an arbitrary level of expression. Abbreviations: vir, virgin; preg, pregnancy; lac, lactation; inv, involution. (b) Cell count of plasma cells and B220+ B lymphocyes present in the mammary tissue during involution. Tissue sections were stained either with methyl green and pyronin or with an antibody against CD45R, and plasma cells and B lymphocytes were counted. Cell counts were taken from 20 high-power fields from each section in triplicate from three individual mice.
Figure 4
Figure 4
Activation of neutrophils, eosinophils and macrophages during involution. (a) RNA expression for GRO1/IL-8/KC and LRG derived from microarray analysis. (b) RNA expression for BMAC, gp39, cathepsin S, MPS1, galectin 3 and CD68 derived from microarray analysis. The x-axes of both graphs describe the different developmental stages: virgin (V10, V12), pregnancy (P1, P2, P3, P8.5, P12.5, P14.5, P17.5), lactation (Lac1, Lac3, Lac7) and involution (Inv1, Inv2, Inv3, Inv4, Inv20). The y-axes describe the averaged scaled signal of the triplicate samples for each time point. The intensity data for LRG have been scaled down 10-fold for display. (c) Semi-quantitative RT–PCR for GRO1, LRG, BMAC and CD68 from a fourth independently collected RNA sample. (d) Identification of macrophages, neutrophils and eosinophils. The figure shows the immunohistochemical staining of a paraffin-embedded tissue section from a mouse mammary gland after 4 days of involution. Macrophages were identified by staining with F4/80 antibody and by their nuclear and cellular shapes. Eosinophils were identified by cross-reaction with the F4/80 antibody, polymorphonuclear structure and eosin staining. Neutrophils were identified through their polymorphonuclear structure, clear cytoplasm, slight eosin staining and non-staining with the F4/80 antibody. (e) Cell counts of neutrophils, eosinophils and macrophages present in the mammary tissue during involution. Tissue sections were stained with an antibody against F4/80 and counterstained with H&E; neutrophils and macrophages were counted. Cell counts were taken from 20 (neutrophils and eosinophils) and 50 (macrophages) high-power fields, respectively, from each section in triplicate from three individual mice.
Figure 5
Figure 5
LBP and CD14 expression in the mouse mammary gland. (a) Western blot for LBP and CD14. Protein extracts from mouse mammary glands from the developmental stages virgin 12 weeks, pregnancy days 1, 2, 3, 8.5, 12.5 and 17.5, lactation days 1, 3 and 7, and involution days 1, 2, 3, 4 and 20 were separated on a denaturing gel; Western blotting was performed with anti-LBP, anti-CD14 and anti-β-actin antibodies. (b) Localisation of CD14 protein by immunohistochemistry. Paraffin-embedded sections from 7 days lactating (i, ii) and 2 days involuting (iii, iv) mouse mammary glands were stained with an anti-CD14-specific antibody. Negative controls contained no primary antibody.
Figure 6
Figure 6
Model for early neutrophil and late macrophage activation. Apoptotic cells are recognised and phagocytosed by their neighbouring epithelial cells or professional and semi-professional cells (curved black arrows). This recognition and/or phagocytosis might involve LBP, CD14, signalling down by means of the Toll-like receptor 4 (TLR4). Activation of this receptor could lead to secretion of the neutrophil-attracting chemokine GRO1/IL-8 (red arrow) and to the attraction of neutrophils to the area of trauma (yellow arrow). This is followed by secretion of the CD14+-monocyte-attracting chemokine BMAC (broken arrow), which could lead to monocyte infiltration on days 3 and 4 of involution (yellow arrow); monocytes would subsequently differentiate into macrophages.

Comment in

  • Evolving Views of Involution
    SR Master et al. Breast Cancer Res 6 (2), 89-92. PMID 14979913. - Review
    The developmental preparation of the mammary gland for milk production that occurs during pregnancy is followed by an equally dramatic process of involution as the gland …

Similar articles

See all similar articles

Cited by 151 PubMed Central articles

See all "Cited by" articles

References

    1. Richert MM, Schwertfeger KL, Ryder JW, Anderson SM. An atlas of mouse mammary gland development. J Mammary Gland Biol Neoplasia. 2000;5:227–241. doi: 10.1023/A:1026499523505. - DOI - PubMed
    1. Masso-Welch PA, Darcy KM, Stangle-Castor NC, Ip MM. A developmental atlas of rat mammary gland histology. J Mammary Gland Biol Neoplasia. 2000;5:165–185. doi: 10.1023/A:1026491221687. - DOI - PubMed
    1. Lund LR, Romer J, Thomasset N, Solberg H, Pyke C, Bissell MJ, Dano K, Werb Z. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways. Development. 1996;122:181–193. - PMC - PubMed
    1. Li M, Liu X, Robinson G, Bar-Paled U, Wagner K-U, Young WS, Hennighausen L, Furth PA. Mammary derived signals activate programmed cell during the involuting mammary gland. Proc Natl Acad Sci USA. 1997;94:3425–3430. doi: 10.1073/pnas.94.7.3425. - DOI - PMC - PubMed
    1. Marti A, Lazar H, Ritter P, Jaggi R. Transcription factor activities and gene expression during mouse mammary gland involution. J Mammary Gland Biol Neoplasia. 1999;4:145–152. doi: 10.1023/A:1018721107061. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback