Residency and activation of myeloid cells during remodeling of the prepartum murine cervix

Abstract

Remodeling of the cervix is a critical early component of parturition and resembles an inflammatory process. Infiltration and activation of myeloid immune cells along with production of proinflammatory mediators and proteolytic enzymes are hypothesized to regulate cervical remodeling as pregnancy nears term. The present study standardized an approach to assess resident populations of immune cells and phenotypic markers of functional activities related to the mechanism of extracellular matrix degradation in the cervix in preparation for birth. Analysis of cells from the dispersed cervix of mice that were nonpregnant or pregnant (Days 15 and 18 postbreeding) by multicolor flow cytometry indicated increased total cell numbers with pregnancy as well as increased numbers of macrophages, the predominant myeloid cell, by Day 18, the day before birth. The number of activated macrophages involved in matrix metalloproteinase induction (CD147) and signaling for matrix adhesion (CD169) significantly increased by the day before birth. Expression of the adhesion markers CD54 and CD11b by macrophages decreased in the cervix by Day 18 versus that on Day 15 or in nonpregnant mice. The census of cells that expressed the migration marker CD62L was unaffected by pregnancy. The data suggest that remodeling of the cervix at term in mice is associated with recruitment and selective activation of macrophages that promote extracellular matrix degradation. Indices of immigration and activities by macrophages may thus serve as markers for local immune cell activity that is critical for ripening of the cervix in the final common mechanism for parturition at term.

FIG. 1

Preparation of tissue for flow cytometry of resident immune cells. Systemic perfusion reduces monocytes from preparations of dispersed cervix. A) Flow cytometry of living monocytes (7AAD⁻CD45⁺Neu7/4⁺ GR-1⁻) in dispersed cervix from NP mice that were perfused (green dots) or not perfused (red dots). B and C) The percentage of monocytes per total live cells in the dispersed cervix from individual mice that were NP or pregnant (D18). Data in A are enumerated in B for the same pair of NP individuals (perfused and not perfused). The NP and D18 mice were simultaneously processed on the same day and replicated in an additional pairing of mice. D) Study design to evaluate effects of tissue processing on Mφ viability. Samples I and II compared collagenase versus mesh dispersal of spleen. Sample III was seeded with splenocytes with a known number of Mφ, and sample IV assessed the effect of collagenase dispersal on census of living Mφ in the cervix from an NP mouse.

FIG. 2

Flow cytometry gating strategy to identify leukocyte populations within spleen and cervix. Dispersed cells were stained to detect living myeloid cells based on coexpression of CD45⁺, F4/80⁺, and GR-1 and gated as follows: light-scatter for intact cells (I), CD45⁺ leukocyte gate (II), 7AAD^– gate to identify living cells (III), F4/80⁺ gate to identify myeloid cells (IV), and GR-1^– to eliminate granulocytes and identify Mφ (V). The gates established in A were applied to dispersed cervical cells (B) to identify resident Mφ in cervix from each NP mouse. This same approach was used to establish gates to identify Mφ in the spleen from D15 and D18 mice (data not shown) and applied to identify resident Mφ in dispersed cells from the cervix of D15 mice (C) and D18 mice (D).

FIG. 3

Mφ increase in the cervix before birth. The number of total living cells (7AAD^–; A), leukocytes (CD45⁺; B), and Mφ (CD45⁺7AAD^–F4/80⁺GR-1^–; C) in the cervix as identified by flow cytometry and gated as described in Figure 2 is shown. Statistical significance (P < 0.05) for D18 (n = 5) versus groups of mice that were ^aNP (n = 6) or ^bD15 (n = 5) is also shown. D18 is pregnant mice on Day 18 postbreeding (n = 5). D) Representative plots of gated living Mφ and Neu in dispersed cervices from NP as well as D15 and D18 mice. E) Mφ stained dark brown by immunohistochemistry (arrows) and counterstained with hematoxylin in sections of cervix from NP and pregnant mice. Bar = 25 μm.

FIG. 4

Mφ in the prepartum cervix increase expression of certain activation markers. Dispersed cells from perfused cervices were stained for flow cytometry, and a standardized gating protocol was used to identify F4/80⁺GR-1^– Mφ. A) Representative histograms of CD11b expression by F4/80⁺GR-1⁻ Mφ in the cervix of from mice that were NP, D15, or D18. B) Percentages of CD11b^+hi Mφ/group (n = 3–5; Student t-test). C–F) Median fluorescence intensity of the various activation markers on F4/80⁺GR-1⁻ Mφ/group (n = 4–6; Mann-Whitney test). For D18, statistical significance (P < 0.05) versus ^aNP or ^bD15 is shown.