The objective of the study was to investigate the role of polymorphonuclear leukocytes (PMNs) in a mouse model of Escherichia coli -induced labor.
Intraperitoneal injection of rabbit antimouse PMN antiserum or control was performed in CD-1 mice 29 hours and 5 hours prior to laparotomy and intrauterine injection of either killed E coli or phosphate-buffered saline on day 14.5 of pregnancy. Preterm delivery was defined as delivery of at least 1 pup within 48 hours. Circulating leukocyte counts were determined manually or by flow cytometry at the time of surgery and 8, 24, and 48 hours afterward. Maternal and fetal tissues were analyzed in a separate group of animals 8 hours after surgery.
Pretreatment with anti-PMN antiserum significantly decreased the numbers of circulating leukocytes and the proportion of neutrophils among all leukocytes by 70–80% at surgery and at least 8 hours thereafter. Neutrophil depletion significantly reduced 2 markers of neutrophil activation in the uterus and placenta (neutrophil elastase and myeloperoxidase activity) and neutrophil infiltration into gestational tissues in bacterially treated animals to baseline (control) levels but did not affect preterm birth rates. The large E coli -induced increases in uterine inflammatory markers (interleukin-1β, tumor necrosis factor, chemokine ligand-5, cyclooxygenase-2) were not affected or were only minimally affected by neutrophil depletion.
Although PMN antiserum reduces both neutrophil number and activity, it does not diminish sensitivity to bacterially induced delivery or meaningfully alter the expression of inflammatory markers in the mouse model. Preterm birth and inflammation in this model are not likely to depend on neutrophil function.
Preterm birth is the most common cause of neonatal morbidity and mortality in the developed world. Up to 50% of cases of preterm labor are associated with acute bacterial colonization of the gestational compartment.
The role of inflammation per se in the genesis of labor in women is not always clear. In multiple animal models, both intrauterine and systemic sterile inflammation can induce preterm birth. In humans, intraamniotic inflammation (ie, the presence of elevated inflammatory cytokines and other markers) is found approximately twice as often as are cultivable bacteria in preterm labor and preterm premature rupture of the membranes, suggesting that sterile inflammation or inflammation resulting from infection in remote compartments may play a role in the pathophysiology of preterm birth.
Polymorphonuclear leukocytes (PMNs), also called granulocytes, include 3 main types of cells: neutrophils, eosinophils, and basophils. These 3 types of PMNs have roles in host responses to different kinds of threats. Neutrophils are the most common type of PMN, representing 50–70% of circulating leukocytes in humans and 10–25% in mice, with the great majority of the remainder being lymphocytes. Neutrophils are both prototypical first responders to inflammatory foci and a source of proinflammatory signals. They are responsible for eliminating pathogens, helping to coordinate the acute inflammatory response, participating in tissue remodeling, and other processes.
Peripheral blood leukocytes become primed as labor approaches. Leukocyte infiltration into gestational tissues has a role in the initiation, maintenance, and resolution of parturition, with major roles ascribed to macrophages and neutrophils. Other functions are ascribed to resident cell types (eg, myometrium, decidua, amnion, chorion) and various other immune cells that reside in or infiltrate these tissues, such as uterine natural killer cells, invariant natural killer T cells, and memory T cells. Such cells may play different roles in infection/inflammation-induced preterm labor than in spontaneous parturition at term.
In a recent study, a massive influx of neutrophils into myometrial tissues occurred in association with endotoxin-induced but not other forms of labor (ie, spontaneous or progesterone withdrawal-induced labor), suggesting the existence of different pathways to parturition. Nonetheless, the question remains as to whether, during infection and inflammation, these leukocytes cause labor, are a consequence of labor, or play an unrelated role.
Given the importance of PMN function in the response to acute bacterial infection, we sought to characterize the role of PMNs in a model of infection-induced preterm labor using intrauterine killed Escherichia coli as the stimulating agent.
Materials and Methods
Anti-PMN serum and control injections
A rabbit polyclonal antimouse PMN antiserum and a control rabbit serum were purchased from Accurate Chemical Corp (Westbury, NY; catalog numbers AIA31140 and AIS403). Serum was diluted 1:10 in phosphate-buffered saline (PBS) and administered intraperitoneally as recommended by the supplier in a volume of 0.5 mL (ie, approximately 1.9% of total body water of a typical 40 g pregnant CD-1 mouse).
In pilot experiments modeled after published mouse models, it was determined that 2 injections administered 29 hours and 5 hours prior to surgery resulted in the greatest reduction in circulating PMN counts at the time of surgery. Control animals were treated with either the control serum or vehicle (PBS) in a similar regimen. Control serum was compared with PBS to verify that the control serum did not on its own cause observable changes (see Table 1 and related text). Because no differences were found, results for both were combined.
|Variable||IU injection||Total WBC per μL blood||Neutrophils, %|
|Pretreatment (IP)||Pretreatment (IP)|
|Control serum||PBS||P value||Control serum||PBS||P value|
|At surgery (time 0)||—||6428 ± 1908 |
(n = 10)
|5281 ± 1406 |
(n = 9)
|.20||24 ± 6.7||25 ± 10.1||.8|
|24 h after surgery||PBS||6105 ± 2536||5258 ± 1876||.64||22 ± 7.8||31 ± 15.9||.3|
|E coli||3560 ± 1607||4650 ± 2092||.38||43 ± 13.9||51 ± 18.3||.4|
|48 h after surgery a||PBS||5680 ± 1818||8506 ± 2967||.10||30 ± 2.8||32 ± 5.9||.6|
a Leukocyte counts 48 hours after surgery were available only for mice injected IU with PBS because the majority of animals injected with E coli delivered and underwent necropsy prior to 48 hours. Numbers of animals per IU treatments are 4-6 per group.
Preparation of bacteria
Bacteria were freshly grown, heat killed, concentrated to 2 × 10 9 organisms per milliliter, and frozen in aliquots at –80°C, as previously described. Aliquots were thawed and diluted as needed prior to each experiment.
All procedures involving animals were approved by the NorthShore University HealthSystem Animal Care and Use Committee and conform to the Guide for Care and Use of Laboratory Animals (1996, National Academy of Sciences).
CD-1 female mice (housed in groups separately from males) were identified as being in estrous by the gross appearance of the vaginal epithelium as previously described. Each receptive female was placed individually with a male CD-1 stud in the afternoon and removed the following morning. Presence of a vaginal plug was considered evidence of copulation (morning of plugging was day 0.5 of pregnancy). Intraperitoneal injections of either rabbit antimouse PMN antiserum or the same volume of control solution (either PBS or normal rabbit serum) was performed on the mornings of days 13.5 and 14.5 of pregnancy. Five hours after the second injection, animals were anesthetized with 0.015 mL/g body weight of avertin (2.5% tribromoethyl alcohol and 2.5% tert-amyl alcohol in PBS), as previously described.
At surgery a 1.5 cm midline incision was made in the lower abdomen. Mice underwent an intrauterine injection of either 2 × 10 7 or 6 × 10 7 heat-killed E coli bacteria (an inoculum sufficient to cause preterm delivery in greater than 80% of animals) or an equivalent volume (100 μL) of PBS in the midsection of the right uterine horn at a site between 2 adjacent fetuses. The abdomen was closed in 2 layers, with 4–0 polyglactin sutures at the peritoneum and wound clips at the skin.
Animals recovered in individual cages in the animal facility. Observations were made twice daily for preterm delivery (defined as at least 1 fetus in the cage or lower vagina within 48 hours of surgery). Maternal blood was collected in heparinized capillary tubes from the right retroorbital sinus at the time of surgery (while the animal was anesthetized), from the left retroorbital sinus 24 hours after surgery (under isofluorane anesthesia) and from the right side again 48 hours after surgery upon euthanasia and just prior to necropsy.
Additional blood samples were collected by cardiac puncture in a separate group of animals 8 hours after surgery immediately upon euthanasia, at which time necropsies were performed and tissues collected for analysis (see the following text). At necropsy the number of fetuses delivered or remaining in utero and the survival status of these retained fetuses (as determined by cardiac or vascular pulsations in the fetal bodies and membranes) were recorded.
For tissue harvests 8 hours after surgery, the inoculated uterine horn was incised longitudinally along the antimesenteric border. Gestational tissues (uteri, fetal membranes, fetuses, and placentas) were collected, washed in ice-cold PBS, and either flash frozen in liquid nitrogen and stored at –80°C or fixed in 10% buffered formalin for later analysis.
Total white blood cell (WBC) and leukocyte differential counts were performed manually in a hemacytometer after lysis of red blood cells and nuclear staining using the Leuko-TIC kit (Bioanalytic GmbH, Freiburg, Germany) according to the instructions of the manufacturer.
Flow cytometry was conducted on 100 μL whole blood equivalents after lysing red blood cells, centrifugation, and resuspension in PBS. Cells were blocked with rat antimouse CD16/CD32 (Mouse BD Fc Block buffer; BD Pharmingen, San Diego, CA) for 10 minutes and then stained for CD45 fluorescein isothiocyanate, rat antimouse CD11b-PE, and rat antimouse Ly6G PerCP-Cy5.5 (BD Pharmingen) for 45 minutes at room temperature. Appropriate isotype control antibodies were used to exclude false-positive signals. CD45+CD11b+Ly6G+ cells were considered neutrophils. Flow cytometry was performed with BD FACSCalibur and FlowJo software (BD Biosciences).
Assays of neutrophil function
Myeloperoxidase (MPO) activity (mu per gram of tissue) was determined using the MPO fluorometric kit (Enzo Life Sciences, Plymouth Meeting, PA) according to the manufacturer’s instructions. Neutrophil elastase (nanograms per gram of tissue) was measured using a specific enzyme-linked immunosorbent assay kit (LSBio Mouse HNE/Neutrophil Elastase, catalog number LS-F11364; LifeSpan BioSciences, Seattle, WA). A set of standards was run concurrently with the samples for each of these assays.
Tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sections (4–5 μm thick) were assessed for neutrophil infiltration.
Real-time polymerase chain reaction
Total ribonucleic acid (RNA) was extracted from homogenized tissues with TRIzol reagent (Invitrogen, Carlsbad, CA). Two micrograms of total RNA were used as a template for complementary deoxyribonucleic acid (cDNA) synthesis using a qScript cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD). TaqMan polymerase chain reaction ( PCR) primers and probes were purchased from Applied Biosystems (Foster City, CA): interleukin (IL)-1β Mm00434228; chemokine ligand-5 (CCL5) (regulated upon activation, normal T cell expressed, and secreted) Mm01302428; tumor necrosis factor (TNF) Mm00443258; cyclooxygenase-2 (COX-2) Mm00478374; connexin 43 (GJA1) Mm01179639; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (×20) Mm4452339E.
Duplex reactions were performed in a 10 μL mixture containing 1 μL cDNA, with 1 primer pair amplifying the gene of interest and the other an internal reference (GAPDH). Thermocycler parameters were 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Semiquantitative analysis of gene expression was done using the comparative cycle threshold (ΔΔCT) method, normalizing the expression of the gene of interest to GAPDH. PCR assays were performed in triplicate.
Categorical data were analyzed by a Fisher exact test. Continuous variables were analyzed by a Student t test or a Mann-Whitney U test (for nonnormally distributed data) or an analysis of variation or Kruskal-Wallis with Dunn’s post hoc testing (for comparison of more than 2 groups).
Depletion of PMNs does not alter bacterially induced preterm delivery
There were 4 treatment groups: (1) preoperative intraperitoneal (IP) injection of control solution followed by intrauterine (IU) PBS (IP Ctrl/IU PBS); (2) preoperative IP PMN antiserum followed by IU PBS (IP α-PMN/IU PBS); (3) preoperative IP control injections followed by IU killed E coli (IP Ctrl/IU E coli ); and (4) preoperative IP PMN antiserum followed by IU killed E coli (IP α-PMN/IU E coli ).
Compared with the IP injection with PBS, the injection with control serum produced no changes in circulating leukocyte counts, regardless of whether E coli were injected IU ( Table 1 ). Therefore, PBS and control serum results were combined for subsequent analyses.
Intraperitoneal injection of anti-PMN antiserum 29 and 5 hours prior to surgery resulted in large (70–80%) reductions in both the total number of circulating leukocytes and the proportion of these leukocytes that were neutrophils at the time of surgery ( Table 2 , time 0), with the remainder almost exclusively lymphocytes.
|Variable||IU injection||Total WBC/μL blood||Neutrophils, %|
|IP anti-PMN antiserum||IP Control||P value||IP anti-PMN antiserum||IP Control||P value|
|At surgery (time 0) a||—||1628 ± 1053||5884 ± 1745||< .0001||6%||24%||< .0001|
|8 h after surgery b||PBS||—||—||—||3%||48%||< .001|
|E coli||—||—||—||6%||53%||< .0001|
|24 h after surgery a||PBS||4694 ± 1376||5788 ± 2208||.4||16%||25%||.1|
|E coli||1183 ± 199||4105 ± 1850||.002||23%||47%||.007|
|P value||< .001||.1||.3||.005|
|48 h after surgery a , c||Control||4325 ± 1219||6936 ± 2678||.1||32%||31%||.7|
c Leukocyte counts 48 hours after surgery available only for mice injected IU with PBS because the majority of animals injected with E coli delivered and underwent necropsy prior to 48 hours. Blood at surgery and 24/48 hours after surgery was obtained via retroorbital sinus puncture. Blood at 8 hours was obtained via cardiac puncture upon euthanasia. Intraperitoneal controls (PBS or control serum) were combined. Numbers of animals per IU treatments were: controls, 4; E coli , 5-9 per group.
Given the fact that in the mouse, unlike the human, the predominating circulating leukocyte is the lymphocyte, this observation indicates that in addition to a specific anti-PMN effect, the antiserum also depletes lymphocytes. This nonspecific lymphocyte depletion, presumably mediated through Gr-1 antigens, has been reported previously for this antiserum.
Performance of general anesthesia, laparotomy, and IU injection produced a significant increase in neutrophil proportion (from 24% up to about 50%) 8 hours after surgery in animals not pretreated with antiserum, regardless of whether the intrauterine injections contained bacteria or only PBS. Pretreatment with antiserum completely eliminated this 8 hour increase in neutrophil proportion ( Figure 1 and Table 1 ). The suppression of both circulating total leukocyte count and neutrophil proportion persisted through 24 hours after surgery in animals treated with IU E coli but not in animals treated with IU PBS.
To correlate between circulating PMN numbers and function within gestational tissues, an activity assay for MPO and an enzyme-linked immunosorbent assay for neutrophil elastase (markers of neutrophil activation) were conducted in uteri and placentas ( Figure 2 ). MPO activity was increased by IU E coli treatment by 500% in uterus and 50% in placentas (the latter not a significant increase), responses that were eliminated by pretreatment with PMN antiserum. A similar effect was seen on the levels of neutrophil elastase, which were significantly elevated by bacterial exposure in both uteri and placentas.
Thus, anti-PMN antiserum induced large and significant drops in both circulating neutrophil counts and neutrophil activity within tissues to levels at or below baseline during the critical time between the performance of the surgical procedure and delivery 9–13 hours later. Despite these reductions, there were no differences in either the occurrence or timing of E coli -induced preterm delivery or in the number and survival status of fetuses retained in utero ( Figure 3 ).