The Concentrations of Inflammatory Cytokines and Acute-Phase Proteins in the Peripheral Blood and Uterine Washings in Cows with Pyometra
Contents
The development of pyometra in cows depends largely on the state of local immunity of the uterus. The objective of the study was to evaluate the concentration of the following proinflammatory cytokines: tumour necrosis factor (TNF-α) and interleukin-6 (IL-6); anti-inflammatory cytokine: interleukin-10 (IL-10); and acute-phase proteins (APPs): haptoglobin (Hp) and serum amyloid A (SAA), in serum and uterine washings in cows with pyometra and healthy animals. The study was performed on 20 cows divided into two groups based on the results of cytological and ultrasonographic tests: a pyometra and a healthy group (10 cows per group). Experimental material consisted of blood serum and uterine washings. The levels of the following cytokines, TNF-α, IL-6, IL-10 and APPs – Hp and SAA, in the study material were determined by ELISA. The results showed that the values of TNF-α, IL-6, IL-10 as well as SAA and Hp were significantly higher in serum of cows with pyometra compared to controls (p < 0.001). The uterine washings had significantly higher levels of IL-6, IL-10, and Hp in pyometra cows compared to the control (p < 0.001). Our results indicate that it is possible to monitor the course of pyometra in cows based on the evaluation of the concentration of cytokines and Hp in the serum and uterine washings. Simultaneous evaluation of selected indicators of antagonistic interaction can be helpful in determining the current status of local immunity of the uterus. On this basis, it could be possible to properly select an adjunctive therapy in the form of immunomodulating preparations.
Introduction
Pyometra is characterized by the accumulation of purulent material within the uterine lumen in the presence of a persistent corpus luteum and a closed cervix (Sheldon et al. 2006). Accumulation of purulent secretion in the uterine cavity is the result of infection with pathogenic bacteria inducing the inflammatory condition of the endometrium (Noakes et al. 1990; Kaneko and Takagi 2013). Pyometra, similarly to other inflammatory conditions of the uterus, represents a serious problem in dairy cow herds, causing large economic losses, entailing the necessity to cull animals in the herd due to infertility (López-Gatius et al. 1996; Potter et al. 2010; El-Tahawy Ael and Fahmy 2011; Gilbert 2011). Pyometra may occur in cows at various stages of lactation; however, it is most common over 6 weeks post-partum, which is predestined by an early ovulation and formation of the corpus luteum (Noakes et al. 1990; Chapwanya 2008).
Cellular and humoural mechanisms of local non-specific and specific immunity play an essential role in the elimination of uterine infection. The development of bovine uterine inflammation is associated with very complex signalling processes involving the detection of bacterial components by innate immune cells via Toll-like receptors, the production of the tumour necrosis factor-α (TNF-α) and other proinflammatory cytokines (e.g. interleukins), and the mobilization of neutrophils, followed by phagocytosis of invading pathogens within the uterine lumen (Beutler et al. 2003; Herath et al. 2009; Sheldon et al. 2009; Turner et al. 2012). Proinflammatory cytokines (e.g. TNF-α, IL-1β, and IL-6) and chemokines (e.g. IL-8) stimulate neutrophil and monocyte diapedesis and chemoattraction, and promote increased phagocytosis (Butterfield et al. 2006; Singh et al. 2008).
Proinflammatory cytokines are also potent stimulators of the production of acute-phase proteins (APPs), such as haptoglobin, acid glycoprotein, ceruloplasmin or serum amyloid A (Tothova et al. 2008). Their task is to assist in the elimination of infection, e.g. through the modulation of other immune proteins or stimulation of phagocytosis. Of significant importance is also the protective function of APPs from the damaging effects of enzymes formed during inflammatory response that can lead to organ damage. Acute-phase proteins are produced in the liver, and their concentration in the blood serum of cows increases over the first few weeks after birth, in response to uterine infection caused by microorganisms (Sheldon et al. 2001; Tothova et al. 2008). Although there is a possibility that the synthesis of APPs takes place outside the liver, their presence in the cells of the uterine endometrium of cows was not confirmed in vitro (Davies et al. 2008). However, research conducted by Chapwanya et al. (2013) suggests that the production of serum amyloid A (SAA) by uterine endometrium cells in cows is possible.
Galvão et al. (2011) and Ghasemi et al. (2012) showed that the expression of inflammatory cytokine mRNA in uterine tissue was related to the development of bovine clinical or subclinical endometritis. Other studies have also demonstrated significant differences in the serum levels of proinflammatory cytokines between cows with endometritis and healthy individuals (Islam et al. 2013; Kasimanickam et al. 2013). Higher expression of the genes of proinflammatory cytokines in endometrial tissue and/or increase in the levels of these cytokines in the serum is considered to be a sensitive prognostic indicator of the development of endometritis in cows (Galvão et al. 2011; Ghasemi et al. 2012; Islam et al. 2013; Kasimanickam et al. 2013). Recent studies have shown that the assessment of the level of proinflammatory cytokines is possible not only in the uterine tissue and serum, but also in fluid collected from the uterus. Cytokine levels in uterine washings are significantly higher in cows with clinical endometritis than in healthy animals (Kim et al. 2014).
Cited studies do not apply, however, to pyometra. There is no data available in literature regarding the levels of cytokines and APPs during the course of pyometra in cows. It is also unclear whether the analysis of the level of these proteins makes it possible to evaluate the occurrence of such an advanced inflammation condition of the uterus. In addition, it is still unknown whether the assessment of the levels of cytokines and/or APP may be helpful in choosing a method of treatment or starting any adjunctive therapy in the case of pyometra in cows.
The objective of the study was to evaluate the concentration of the following proinflammatory cytokines: tumour necrosis factor (TNF-α) and interleukin-6 (IL-6); anti-inflammatory cytokine: interleukin-10 (IL-10); and acute-phase proteins (APPs): haptoglobin (Hp) and serum amyloid A (SAA), in serum and uterine washings in cows with pyometra and healthy animals in the same post-partum period.
Materials and Methods
Experimental animals
The study was performed on 20 Holstein-Friesian (HF) cows in the second or third lactation. The animals were selected from two herds and kept under similar feeding and environmental conditions. The study initially included the assessment of the overall health status of the animals and a detailed evaluation of the reproductive system by ultrasonography using a Honda 1500 Ultrasound (Honda Electronics, Toyohashi, Japan). The pyometra group consisted of 10 cows with pyometra, diagnosed between day 70 and 90 DPP during routine examination of the cows that did not show signs of oestrus. The proper diagnosis of pyometra was based on a rectal examination (extended size of the uterus), combined with ultrasonography (presence of fluid with a typical echogenicity in the uterus and corpus luteum in the ovary), while vaginoscopy confirmed closed cervix. After collecting specimens, cows with pyometra were properly treated, which consisted of a one-time injection of 25 mg of dinoprost – Dinolytic (Zoetis, Puurs, Belgium), according to the literature data (Kaneko and Takagi 2013). The healthy group consisted of 10 cows in the luteal phase of the ovarian cycle with a normal course of the post-partum period, in which no inflammatory changes in the uterus were discovered during cytological tests (the number of neutrophils did not exceed 5%). Cows in the healthy group were in the same post-partum period as animals from the pyometra group. Cytological tests in both groups were performed on uterine smears collected with intrauterine brushes adapted for cows (Jiangsu Yada Technology, Jiangsu, China), as well as the evaluation of serum progesterone concentration (P4) was performed. Peripheral blood and uterine washings were used for immunological tests. The biological material collected for laboratory tests was sent to the laboratory within an hour.
Sampling of blood and uterine washings
Blood samples (9 ml) were collected from the external jugular vein into clot activator tubes, whereas uterine washings were collected into Vacutest standard tubes (Vacutest Kima srl, Arzergrande, PD, Italy). Blood samples were then centrifuged at 2500 × g for 10 min at 4°C, and the serum was harvested and transferred to 2-ml microcentrifuge tubes and stored at −80°C until analysis.
Fifty millilitre of isotonic phosphate-buffered saline (PBS) was injected into the uterus and washings were collected using a Foley catheter (Jorgen Kruuse, Marslev, Denmark) with a syringe. The uterus was massaged through the rectum for a few minutes, and the saline solution was aspirated by pulling back the syringe plunger to create negative pressure in the catheter. In most cases, approximately 40 ml of fluid was recovered. The uterine fluid was centrifuged at 2500 × g for 10 min at 4°C, and the supernatant was transferred to 2-ml microcentrifuge tubes and stored at −80°C until analysis.
Cytological tests
Imprint cytology specimens were prepared from the smears, which after thorough drying was fixed and stained by means of the Hemacolor method (Merck, Darmstadt, Germany). The preparations were evaluated under a microscope (Olimpus CX 41; Olympus, Tokyo, Japan) at a magnification of 1000×. Cytological evaluation of the preparations was based on the count of neutrophils in the smears. Counting covered 100 cells (epithelial cells and leucocytes) visible in different fields of vision on each preparation, and the percentage of neutrophils was then calculated, according to literature (Kasimanickam et al. 2004, 2005; Sheldon et al. 2006).
Serum progesterone concentration
Serum progesterone concentrations in cows of both groups were measured as single values using a commercial ELISA kit (Biomerieux, Marcy L'Etoile, France). The intra- and interassay coefficients of variation for progesterone were <4.3% and <5.4%, respectively. Absorbance readings and subsequent calculations of final concentrations were performed on a Benchmark Plus spectrophotometric microplate reader with microplate manager Software v. 5.2.1 (Bio-Rad Laboratories, Berkeley, CA, USA).
Measurements of cytokines in uterine washings and blood serum
The concentrations of the tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) in blood serum and uterine flush samples were determined using commercially available kits, that is, bovine enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-6 and IL-10 (USCN Life Science Inc., Houston, TX, USA). The inter- and intra-assay coefficients of variation (CV) for all examined cytokines were <12% and <10%, respectively. All procedures were performed according to the guidelines provided by the manufacturers and methods available in the literature (Kim et al. 2014). Absorbance readings were performed on an automatic microplate reader (ASYS Expert Plus; Biochrom Ltd., Cambridge, UK) at 450 nm.
Measurements of acute-phase proteins in uterine washings and blood serum
Measurements of the level of Serum Amyloid A (SAA) in blood serum and uterine flush were performed using a commercial ELISA kit (Tridelta Development Ltd., Maynooth, Kildare, Ireland). The inter- and intra-assay coefficients of variation for SAA analysis were <12.1% and <7.5%, respectively. The determination of haptoglobin in blood serum and uterine flush was performed using a commercial colorimetric assay kit (Tridelta Development Ltd.). The inter- and intra-assay CVs for Hp analysis were <5.7% and <6.3%, respectively. Procedures were performed according to the manufacturers' instructions and literature methods (Suojala et al. 2008; Tothova et al. 2012). Absorbance readings and the subsequent calculations of final concentrations were performed on an automatic microplate reader (ASYS Expert Plus; Biochrom Ltd.) at 450 nm, 630 nm for Hp, and 630 nm as a reference for SAA. Lyophilized bovine acute-phase serum was used as a standard, and calibration was performed according to the European Union concerted action on standardization of animal APPs (No. QLK5-CT-1999-0153).
Statistical analysis
All values are presented as means ± SEM. Statistical analysis was performed using the statistica software (Statsoft Inc., Tulsa, OK, USA). Data were found to be normally distributed, as demonstrated by Kolmogorov–Smirnov test and Lilliefors correction. Mean values were compared between healthy and pyometra groups using non-paired Student's t-test. p-value <0.05 was considered statistically significant.
Results
Results of cytological tests
Cytology in the pyometra group showed that the mean neutrophil count was 77.15 ± 5.49% and was significantly higher (p < 0.001) compared to the cytological test performed in healthy cows (3.1 ± 1.58%). The detailed results of cytological tests are presented in the first article (Brodzki et al. 2014c).
Results of progesterone tests
Progesterone levels in the blood serum of cows with pyometra ranged from 7.3 to 9.4 ng/ml, while in healthy cows from 4.4 to 6.9 ng/ml (Brodzki et al. 2014c).
Results of immunoassays
The results of the level of proinflammatory cytokines: tumour necrosis factor (TNF-α) and interleukin-6 (IL-6); anti-inflammatory cytokine: interleukin-10 (IL-10; and acute-phase proteins: serum amyloid A (SAA) and haptoglobin (Hp) in the blood serum of cows with pyometra and healthy individuals are presented in Table 1. The data show that the levels of TNF-α, IL-6, IL-10 as well as acute-phase proteins (SAA and Hp) were significantly higher in serum of cows with pyometra compared to healthy cows (p < 0.001). The uterine washings had significantly higher levels of IL-6, IL-10 and Hp in cows with pyometra compared to the healthy group (p < 0.001). The level of IL-6 (p < 0.002) as well as Hp and SAA (p < 0.001) was significantly higher in cows with pyometra, while the level of IL-10 was lower (p < 0.001) in blood serum compared to the uterine washings. In healthy cows, the level of IL-6, IL-10, Hp (p < 0.001) and SAA (p = 0.01) was significantly higher in the serum than in uterine washings.
| Group (n = 10 cows per group) | Evaluated parameters | ||||
|---|---|---|---|---|---|
| TNF-α (pg/ml) | IL-6 (pg/ml) | IL-10 (pg/ml) | SAA (μg/ml) | Hp (mg/ml) | |
| Blood serum | |||||
| Pyometra | 341.77 ± 54.63** | 357,32 ± 68.66**,## | 126,88 ± 26.68**,## | 62,5 ± 9.01**,## | 314,8 ± 110.89**,## |
| Healthy | 271.58 ± 46.01 | 275.4 ± 67.88## | 53.59 ± 3.19## | 35.4 ± 6.90# | 60.53 ± 11.88## |
| Uterine washings | |||||
| Pyometra | 349.43 ± 65.29** | 264.98 ± 31.81** | 219.49 ± 29.98** | 29.3 ± 3.6 | 48.8 ± 5.03** |
| Healthy | 251.11 ± 52.09 | 172.2 ± 43.37 | 14.37 ± 1.45 | 27.5 ± 6.62 | 22.29 ± 4.62 |
- Values are expressed as mean ± SEM, **Statistical significance at p ≤ 0.001 compared with the control. Significant differences between blood serum and uterine washings in the two groups at ##p ≤ 0.001 and #p ≤ 0.01.
Discussion
The aim of the study was to evaluate the concentration of proinflammatory cytokines: tumour necrosis factor (TNF-α) and interleukin-6 (IL-6); anti-inflammatory cytokine: interleukin-10 (IL-10); and acute-phase proteins: haptoglobin (Hp) and serum amyloid A (SAA), in the serum and uterine washings of cows with subclinical endometritis and healthy animals. This is the first study of this type conducted on cows with pyometra. The research is a continuation of previously published studies by the authors concerning phagocytic activity of granulocytes and monocytes in peripheral blood and uterine washings in cows with pyometra (Brodzki et al. 2014c). Presented in our previous studies, higher levels of inflammatory cytokines TNF-α, IL-6 and IL-10, and acute-phase proteins, Hp and SAA, in the serum of cows with pyometra may be associated with the occurrence of severely advanced forms of uterus inflammation. Increased activity of immunocompetent cells, stimulated mainly in the uterus and in peripheral blood, could be the cause of an increase in the concentration of inflammatory mediators in the blood serum of pyometra cows. Shortly after birth, the uterus is inhabited by many types of bacteria, which in the normal course of the post-partum period, are quickly eliminated by properly functioning local immunity mechanisms (Sheldon et al. 2006, 2009). In the period in which the study was conducted (70–90 days post-partum), the uterus should have diminished the load of pathogenic bacteria (Sheldon et al. 2009). When the number of pathogens is low or when they are absent, local immune mechanisms are not activated. The concentration of inflammatory mediators (cytokines, chemokines, APPs) does not increasing, which is observed in healthy cows. In contrast, cows with pyometra have persistent inflammation of the uterus and show an increase in these mediators. Research conducted by Kasimanickam et al. (2013) are consistent with our results and confirm the higher levels of proinflammatory cytokines (TNF-α, IL-1β and IL-6) in blood serum of cows with endometritis compared to healthy cows. However, the study by Kim et al. (2014) showed no difference in the concentrations of these cytokines in the serum of cows with endometritis and healthy animals. Ishikawa et al. (2004) also did not observe significant changes in the quantity of IL-6 in the serum of cows with endometritis. The cited studies were conducted on cows with endometritis, and not as our study on cows with pyometra. We refer to the literature concerning endometritis, because there is no data related to the level of cytokines and APPs in the course of pyometra in cows.
In our study, we have shown significantly higher levels of IL-6, IL-10 and Hp in uterine washings of cows with pyometra compared to cows without the disease. In addition, we have also demonstrated that cows with pyometra had a nearly 2-fold higher concentration of IL-10 in uterine washings than in blood serum. One of the reasons for the high concentration of IL-6 was the secretion of this protein by uterine endometrium cells, as a result of recognition of pathogen associated molecular patterns (PAMPs) by specific Toll-like receptors (TLR) (Beutler et al. 2003; Sheldon et al. 2009; Turner et al. 2012). This reaction is a key activation element of the local immune response of the uterus, leading to leucocyte chemotaxis and enhancement of the phagocytic activity of neutrophils and macrophages already present in the uterus. Leucocytes present in the uterus, and those flowing in with blood activated in this manner or directly stimulated by PAMPs of microorganisms (mainly phagocytes), also secrete cytokines, contributing to an increase in their concentration in the uterus (Singh et al. 2008). Our previous study (Brodzki et al. 2014c) confirmed the presence of pathogens and the consequently very high number of leucocytes (mainly neutrophils) in the uterus of cows with pyometra. This is reflected in our results in the form of elevated levels of proinflammatory cytokines TNF-α and IL-6 in uterine washings of cows with pyometra. Studies by other authors also showed a higher level of IL-6 mRNA in the uterus of cows with endometritis compared to healthy cows (Loyi et al. 2013; Sheldon et al. 2014). Likewise, the study by Kim et al. (2014) showed an increase of TNF-α and IL-6 in the uterine washings of cows with clinical endometritis. The reason for a high increase in the level of IL-10 in the uterus of cows with pyometra remains a matter of conjecture, because there is no data available in the literature concerning this issue. It is possible that the high concentration of IL-10 during the persistent inflammation of the uterus results from an increasing level of T CD8+ cells or other regulatory cells, which suppressor function may be associated with the production of IL-10 (Tangi et al. 2005; Seo et al. 2007; Askenasy et al. 2008; Hoek et al. 2009; Banos et al. 2013). Our previous studies confirmed the percentage increase of CD8+ cells in the uterine washings in cows with endometritis (Brodzki et al. 2014a,b). Perhaps similar correlations occur in the case of pyometra; however, further investigations are required to confirm them. High concentrations of IL-10 of anti-inflammatory activity suggest induction of a protective response of the tissues of the uterus against the overly aggressive activity of immunocompetent cells and inflammatory mediators secreted by them. Unfortunately, our studies also showed that such an interaction may be associated with the weakening of local anti-infectious immunity and lack of elimination of microorganisms from the uterus. Our previous studies have shown a significant weakening of the phagocytic function of phagocytic cells, both neutrophils and macrophages (Brodzki et al. 2014c). The effect of these changes can be a long-term persistence of pyometra.
Acute-phase proteins (APPs) play an important role in different stages of the inflammatory response and thus may serve as markers of various types of cattle diseases (Maden et al. 2012; Tothova et al. 2012). Of various APPs occurring in cows, haptoglobin (Hp) and serum amyloid A (SAA) are primary positive biomarkers (Tothova et al. 2014). SAA is an apolipoprotein occurring 24–48 h after infection, as a protein of the first line of response, and its secretion is dependent on IL-1 and/or TNF-α (Petersen et al. 2004; Tothova et al. 2014). Hp in turn is a protein of the second line of response, whose secretion is regulated by IL-6, and its high level is characteristic of long and less severe inflammatory processes (Petersen et al. 2004; Tothova et al. 2014). Our findings seem to confirm these relationships. The level of SAA was significantly higher in the serum of cows with pyometra compared to healthy animals. At the same time, high levels of TNF-α were detected in cows with pyometra. The level of haptoglobin (Hp) was significantly higher both in serum and uterine washings in cows with pyometra. Such changes in the concentration of APPs can suggest persistence of chronic inflammation of the uterus. Very interesting from a clinical point of view is the persistence of high concentrations of Hp in the serum and uterine washings of cows with pyometra. This protein can be considered a marker of inflammation of the uterus in this species. The increase of Hp in uterine washings is difficult to explain, because local production of this protein in the endometrium cells has not been confirmed. However, a significant increase of its level may suggest local production of Hp in the course of endometritis. Another possibility is the penetration of the protein into the lumen of the uterus from the blood, where significantly higher levels of this protein were recorded compared to uterine washings.
Conclusion
Our results indicate that based on the evaluation of the concentrations of cytokines and Hp in serum and uterine washings, it is possible to monitor the course of pyometra in cows. To evaluate the direction of inflammation development and closely related mechanisms of local immunity of the uterus, simultaneous measurements of several related cytokines with antagonistic interactions are required. This way, it is possible to accurately determine the current state of the local resistance of the uterus, which largely facilitates the determination of further prophylactic or therapeutic treatment. With an increase in the level of proinflammatory cytokines and anti-inflammatory cytokines (such as IL-10), incorporation of immunostimulatory remedies to the standard pyometra therapy may give a positive effect. However, when only the concentration of inflammatory cytokines is increased, such as IL-6 or TNF-α, there is no need to apply such remedies, because the local immunity mechanisms of the uterus are sufficiently activated.
Conflict of interest
None of the authors have any conflict of interest to declare.
Author contributions
PB obtained funding sources and supervised all stages of the study. Moreover, PB was responsible for the study design and participated in patient management, data collection interpretation and writing the manuscript. KK and JZ participated in data collection interpretation and critical revision of the manuscript. AB and JZ were responsible for statistical analysis, data interpretation, preparation and critical revision of the manuscript. All authors have read and approved the manuscript.
