Temporal Changes in Myeloid Cells in the Cervix during Pregnancy and Parturition Brenda C. Timmons, Anna-Marie Fairhurst and Mala S. Mahendroo This information is current as of September 26, 2021. J Immunol 2009; 182:2700-2707; ; doi: 10.4049/jimmunol.0803138 http://www.jimmunol.org/content/182/5/2700 Downloaded from References This article cites 50 articles, 6 of which you can access for free at: http://www.jimmunol.org/content/182/5/2700.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 26, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Temporal Changes in Myeloid Cells in the Cervix during Pregnancy and Parturition1 Brenda C. Timmons,* Anna-Marie Fairhurst,† and Mala S. Mahendroo2* Preterm birth occurs at a rate of 12.7% in the U.S. and is the primary cause of fetal morbidity in the first year of life as well as the cause of later health problems. Elucidation of mechanisms controlling cervical remodeling is critical for development of therapies to reduce the incidence of prematurity. The cervical extracellular matrix must be disorganized during labor to allow birth, followed by a rapid repair postpartum. Leukocytes infiltrate the cervix before and after birth and are proposed to regulate matrix remodeling during cervical ripening via release of proteolytic enzymes. In the current study, flow cytometry and cell sorting were used to determine the role of immune cells in cervical matrix remodeling before, during, and after parturition. Markers of myeloid cell differentiation and activation were assessed to define phenotype and function. Tissue monocytes and eosinophils increased in the cervix before birth in a progesterone-regulated fashion, whereas macrophage numbers were unchanged. Neu- Downloaded from trophils increased in the postpartum period. Increased mRNA expression of Csfr1 and markers of alternatively activated M2 macrophages during labor or shortly postpartum suggest a function of M2 macrophages in postpartum tissue repair. Changes in cervical myeloid cell numbers are not reflected in the peripheral blood. These data along with our previous studies suggest that myeloid-derived cells do not orchestrate processes required for initiation of cervical ripening before birth. Additionally, macro- phages with diverse phenotypes (M1 and M2) are present in the cervix and are most likely involved in the postpartum repair of tissue. The Journal of Immunology, 2009, 182: 2700–2707. http://www.jimmunol.org/ reterm birth is the number one cause of infant morbidity in and loss of tissue integrity (5–7). Upon initiation of uterine con- the first year of life (1, 2). Identification of the specific tractions, the cervix dilates and birth occurs. Immediately follow- P functions of immune cells in the normal parturition pro- ing parturition, a repair process is initiated in which inappropri- cess is critical for understanding the causes of preterm birth. ately assembled matrix molecules must be removed as the cervix Parturition involves the coordination of two major events: uterine regains its rigidity and tensile strength. contractions and remodeling of the cervical extracellular matrix The molecular processes that bring about the dramatic changes (ECM).3 The cervical ECM is comprised primarily of fibrilar col- in the cervical matrix during parturition have been attributed to lagen, proteoglycans, and elastin (3). The cervical collagen fibers activation of the immune response system during ripening (8–14). by guest on September 26, 2021 maintain a structure that provides strength and rigidity to the tissue Histological assessments of cervices from numerous species iden- during pregnancy. During parturition, collagen fiber structure must tify an increased migration of leukocytes into the cervical stroma be extensively altered to allow the cervix to open sufficiently for matrix during ripening. This led to a model in which these cells safe delivery of the fetus. The first phase of remodeling, termed release proteolytic enzymes, which in turn degrade ECM compo- softening, is gradual beginning with increased collagen solubility nents leading to increased tissue compliance. Certainly, mono- and increased tissue compliance (4). Cervical softening overlaps cytes/macrophages have the capacity to generate large amounts of with an accelerated phase termed ripening at the end of pregnancy. proinflammatory mediators, particularly TNF-␣ (15). In addition, In the mouse, ripening is preceded by a decline in progesterone they generate reactive oxygen species and enzymes that have the synthesis and increase in progesterone metabolism and is charac- capacity to degrade the structure and integrity of tissue. Neutro- terized by further matrix disorganization, altered proteoglycan phils also may be stimulated to release reactive oxygen species and composition, and increased synthesis of hyaluronan, a space-filling proteolytic enzymes (16, 17). Therefore, myeloid lineage cells glycosaminoglycan that promotes increased tissue viscoelasticity could be important candidates orchestrating the structural changes occurring during pregnancy and labor. Moreover, determining the specific role that leukocytes play at each stage of cervical remod- *Department of Obstetrics and Gynecology, and †Department of Immunology, Uni- versity of Texas Southwestern Medical Center, Dallas, TX 75390 eling before and after birth may be important for understanding Received for publication September 25, 2008. Accepted for publication December mechanisms leading to preterm birth. 26, 2008. Human studies have demonstrated that inflammatory cells mi- The costs of publication of this article were defrayed in part by the payment of page grate into the cervix and secrete proinflammatory cytokines at charges. This article must therefore be hereby marked advertisement in accordance term. Nonetheless, it remains unclear as to whether leukocytes are with 18 U.S.C. Section 1734 solely to indicate this fact. required for ripening before birth or during postpartum (PP) repair 1 This work is supported by National Institutes of Health R01 HD043154 (to M.S.M.). of the cervix given variations in timing of tissue collection as well 2 Address correspondence and reprint requests to Dr. Mala S. Mahendroo, De- as numerous studies that question the necessity of functional neu- partment of Obstetrics and Gynecology, University of Texas Southwestern Med- ical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9032. E-mail ad- trophils and the timing of macrophage activation relative to birth dress: [email protected] (12, 18–24). We have previously reported no change in macro- 3 Abbreviations used in this paper: ECM, extracellular matrix; Arg1, arginase 1; IL- phage distribution during ripening as compared with earlier in 1ra, IL-1 receptor antagonist; IL-13ra1, IL-13 receptor alpha 1; NIL, not in labor; PP, pregnancy, although other studies suggest increases in macrophage postpartum; WT, wild type; IL, in labor. numbers during ripening (11, 24). The requirement for macro- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 phage-derived proteases in cervical ripening is unclear given the www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803138 The Journal of Immunology 2701 FIGURE 1. Cervical tissue mono- cytes, but not neutrophils, increase before parturition. Cervical suspen- sions were stained with anti-CD45, anti-Gr1, and anti-Neu 7/4. A, Leuko- cytes were gated on CD45 and further analyzed on the Neu 7/4 vs Gr1 dot plot. Monocytes were identified as Neu 7/42ϩ and Gr1 low to intermedi- ate. Neutrophils were defined as Neu 7/4ϩ, Gr12ϩ. These populations were sorted, cytospun onto slides, and stained to visualize cell morphology. Downloaded from Bar of measure represents 20 ␮m. B, Neutrophils are present before cervi- cal ripening, but do not increase sig- nificantly until PP. Monocytes signif- icantly increase by late on gestation day 18 and remain high through PP. http://www.jimmunol.org/ Data represent mean Ϯ SEM of 6–10 p Յ 0.05 ,ء .cervices, as indicated compared with day 15. IL, Indicates in labor samples. by guest on September 26, 2021 activity of collagenase, a proteolytic enzyme made by leukocytes, Materials and Methods is not increased in the term cervix of numerous species (25, 26). Mice Moreover, exogenous collagenase treatment of rat cervices does Ϫ Ϫ Steroid 5␣-reductase type 1-deficient mice (Srd5a1 / ) were generated not replicate the natural remodeling process (27). and genotyped, as described previously (28). Timed pregnant NIH Swiss To better define the role of myeloid cells at each phase of cer- (Harlan) and HSD: ICR (CD-1) (Harlan) were obtained from The Jackson vical remodeling before, during, and after birth, we have