The role of leukotriene B₄ in cow metritis

Metritis is a common postpartum reproductive disorder of dairy cows around the world, which leads to an increase in treatment cost, extension of the calving interval and decline in milk production. It seriously constrains the development of the dairy industry (9). Bacterial contamination of the postpartum uterus of dairy cows is inevitable (7). It is generally believed that postpartum metritis is caused by the entry into and proliferation in the uterus of conditional pathogens (4). The invasion of pathogens causes the body to actively mobilise immune factors to resist infection, for example by recruiting immune cells and cytokines to strengthen the inflammatory response.

Mast cells (MC) are activated and degranulated to release pre-storage medium histamine in cow metritis (24). A type I hypersensitivity reaction of mast cells occurs during metritis, and local inflammation can cause histamine excitation. The increase of histamine 2 receptor expression and the decrease of histamine 1 receptor expression inhibit the inflammatory response and prevent excessive damage to the uterine tissue. In addition to histamine, the pre-storage medium of mast cells also includes serotonin, tumour necrosis factor alpha (TNF)α and tryptase. Mast cells also synthesise and secrete leukotrienes, prostaglandins, platelet activating factors and other compounds (27). Leukotriene B₄ (LTB₄), as a powerful chemokine, can recruit immune cells such as neutrophils, macrophages and T cells to inflammatory sites and play an important role in a variety of acute and chronic inflammatory reactions (13, 19). However, its role in metritis is still unclear.

Mast cells are an important link between nerves and the immune system, which is closely related to the activity of neuropeptides. It was found that the neuropeptide substance P (SP) could promote the synthesis of LTB₄ by mast cells by activating extracellular signal-regulated protein kinases 1 and 2 (17). Thermal, chemical and mechanical effects, bacteria and their secretions and other factors can cause changes in the potential on nerve endings and promote the opening of ion channels; calcium influx combines with vesicles, and SP is released after vesicles fuse with the cell membrane (14). Tryptase, as a pre-storage medium of mast cells, can promote the release of SP through protease-activated receptor-2 (PAR2) (16). Therefore, there is a positive feedback amplification effect between neuropeptide substance P (SP) and mast cells. The mutually promoting relationship between MC and SP is inhibited by the vasoactive intestinal peptide (VIP). The VIP neuropeptide can reduce organ damage by inhibiting mast cell degranulation (23). It can also inhibit the production of superoxide anion by monocytes and neutrophils and weaken the bactericidal activity (10).

The concentrations of inflammatory factors such as TNF-α, interleukin (IL)-1β, IL-6 and IL-8 increased significantly in cow metritis (6). Some of these cytokines came from mast cell degranulation and some from immune cells recruited by LTB₄. The gene promoter position of matrix metalloproteinase (MMP)-9 exists in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein 1, specificity protein 1 and other nuclear factor binding sites (1). Inflammatory factors can increase the expression of MMP-9 through these sites. Matrix metalloproteinase 9 participates in the activation of TNF-α (26). It can cleave IL-8 and enhance its chemotaxis at the specific site of the amino terminal (8). At the same time, inflammatory factors can enhance the synthesis and release of MMP-9 (21). Intact extracellular matrix molecules have no chemotaxis. When MMP-2 and MMP-9 degrade collagen in the extracellular matrix and form the proline-glycine-proline sequence, it can activate the CXC chemokine receptor 1/2 and lead to neutrophil chemotaxis (15). However, in the action of MMP-2 and MMP-9, the extracellular matrix in the basement membrane is degraded, the integrity of muscle fibres is destroyed, and inflammatory cells are immersed in muscle tissue, which may lead to a series of pathological inflammatory changes such as muscle fibre necrosis (5). Therefore, on the one hand, a small amount of collagenase is conducive to chemotaxis and promotes the activity of inflammatory factors, but on the other hand, excessive collagenase will cause tissue damage.

The LTB₄ receptor (BLT) is divided into high-affinity receptor BLT1 and low-affinity receptor BLT2. The former is expressed on the upper surface of immune cells and can induce leukocyte proliferation and chemotaxis, and the latter is expressed in almost all tissues, inducing tissue cells to produce superoxide ions and strengthening the bactericidal effect. The low-affinity BLT2 is expressed on the vascular endothelium to produce vascular endothelial growth factor, and mast cells release IL-8 to induce vascular regeneration (11). Leukotriene B₄ has a chemotactic action on fibroblasts, induces the synthesis of extracellular matrix proteins (22), and then participates in tissue remodelling. Therefore, the LTB₄ receptors can enhance the infection fighting ability of uterine cells and participate in uterine remodelling, and may also cause tissue damage. However, it is unclear how LTB₄ may affect the tissue structure of the endometrium in metritis. The expression of MMP-9 has not been reported in metritic uterine tissue.

This study took advantage of the postpartum metritis state in dairy cows to analyse the concentration changes of endometrial SP, VIP and LTB₄ neuropeptides and detect the granular state of mast cells and the expression characteristics of the LTB₄ receptor, collagen and collagenase. It discusses the SP-LTB₄-MMP mechanism in postpartum metritis of dairy cows.

Animals. A total of 20 Holstein cows aged 3 to 6 years and 6 to 10 days postpartum were selected, of which 10 were healthy as the control group and 10 were diagnosed with acute suppurative postpartum metritis according to clinical symptoms and rectal examination as the experimental group.

Sample collection. Bovine endometrial samples were collected using an endometrial biopsy sampler (2). Endometrial tissue blocks were stored in 2.5% glutaraldehyde (pH 7.4) for transmission electron microscope observation, at 4℃ for ELISA detection, and in liquid nitrogen for quantitative real-time fluorescence PCR.

Transmission electron microscope observation. The sample was first fixed at 4℃ with phosphate buffer containing 2.5% glutaraldehyde (pH 7.2) for 2 h, and then a 1 mm × 2 mm section was prepared from it. This was then washed with sucrose phosphate buffer three times for 10 min each time, fixed with 1% osmic acid at 4℃ for 1 h, successively dehydrated with gradient dilution in ethanol, washed with propylene oxide and embedded in EPON epoxy resin. Subsequently it was subjected to toluidine blue staining and microscopic observation.

ELISA. The concentrations of SP, VIP and LTB₄ were measured using the ELISA kits (Uscn Life Science, USA) according to the manufacturer’s instructions. Standard reagents were used to construct a calibration curve. The sample OD value and the calibration curve were used to calculate the product concentration.

Quantitative real-time PCR (qPCR). Primer premier 5.0 (Premier Biosoft, San Francisco, CA, USA) was used to design primers based on Bos taurus reference sequences. The primer sequences, expected product length and accession numbers in GenBank are shown in Table 1. Total RNA was extracted from endometrium samples with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 1 μg of total RNA was subjected to reverse transcription to form cDNA with transcriptase according to the manufacturer’s instructions (Invitrogen). The mRNA expression levels of BLT2, MMP-2, MMP-9 and β-actin were quantified by real-time PCR with a LightCycler (Roche Life Sciences, Indiana, IN, USA) using a commercial kit (TaKaRa, Kusatsu, Japan). The values were normalised using β-actin as an internal standard.

Immunohistochemical staining of types I and IV collagen in the endometrium. The material used was prepared through routine procedures. The tissues were trimed to the appropriate size and shape. Then they were dehydrated and subsequently embedded into paraffin blocks. The paraffin blocks were trimmed to an optimal cutting surface. Slices were cut to a thickness of 5 μm, then rehydrated and rinsed with distilled water.

All the sections from different groups were processed simultaneously for immunohistochemistry. A gentle 0.01M phosphate-buffered saline solution (PBS; pH 7.2–7.4) was used to rinse between each step and the next. The steps were: incubation for 5 min in 3% hydrogen peroxide; 60 min in a blocking solution of 3% normal goat serum in PBS; overnight in primary antisera of types I and IV collagen (Boster Biological Technology, Wuhan, China) in blocking solution at 4℃; 30 min in diluted biotinylated species-specific anti-IG sera (Boster); and 20 min in streptavidin-biotin complex (Boster). Negative sections were incubated without primary antibodies. The sections were then stained for 4 min in 0.05% 3,3-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide. The reaction was halted by rinsing in cold PBS. The sections were counterstained with haematoxylin, dehydrated with alcohol, transparent with xylene and then coverslipped. Five sections spaced 500 μm apart were prepared from each group’s tissue samples.

Statistical analysis. The results were analysed by one-way analysis of variance. Data are presented as mean ± SD unless otherwise stated. The SPSS 25.0 statistics software package (IBM, Armonk. NY, USA) was used to process the experimental data. P-values < 0.05 were considered significant and P < 0.01 markedly significant.

Mast cell ultrastructure. In the experimental group, MC granules were unevenly distributed, and the cavities were enlarged and appeared to be vacuolated (Fig. 1A). In the control group, MC granules were of different sizes and were homogeneously distributed (Fig. 1B). Compared with the control group, MC degranulation increased significantly in the experimental group.

Fig. 1

Substance P, vasointestinal peptide and leukotriene B₄ concentrations. The levels of endometrial SP, VIP and LTB₄ are shown in Table 2. The SP and LTB₄ concentrations were significantly higher in the experimental group than in the control group (P < 0.01 respectively), while the VIP concentrations were significantly lower (P < 0.01, respectively).

Levels of leukotriene B₄ receptor 2 and matrix metalloproteinases 2 and 9 mRNA expression. Expression of BLT2, MMP-2 and MMP-9 mRNA in the endometrium of the experimental group samples was significantly higher than that of the control group samples (P < 0.01) (Fig. 2).

Fig. 2

Immunohistochemical staining results of type I collagen and type IV collagen in the endometrium. The expression of collagens I and IV in the endometrium of the experimental group samples was significantly lower than that of the control group samples (P < 0.05) (Table 3).

It is inevitable for bacteria to enter a cow’s uterus and proliferate after parturition. Bacteria and their secretions stimulate the release of SP from uterine nerve axon terminals. Substance P can activate mast cells and cause Ca²⁺ mobilisation and cell degranulation (12). After activation, the tryptase released by mast cells can bind with PAR2 on the nerve surface containing neuropeptides to promote the release of SP and other neuropeptides. There are other ways to promote mast cell degranulation, such as the IgE-dependent histamine release process (28). However, only SP does not need extracellular Ca²⁺, and only SP has a mutual amplification effect on mast cell degranulation and plays a role in a limited time (3). This study found that the concentration of SP in uterine tissue increased significantly in metritis, and promoted the degranulation of mast cells through the receptor independent pathway, which is consistent with the phenomenon of mast cell activation and degranulation. Mast cells release pre-stored mediators such as histamine and tryptase after activation. This is consistent with the finding of our previous study that mast cells are activated and degranulated to release histamine in metritis (24). In addition to SP content, the content of leukotriene B₄ increased significantly in metritis, which is also the main new synthetic medium synthesised and released by mast cells after activation. Leukotriene B₄ recruits leukocytes to fight infection with its powerful chemotactic function. The effect of neuropeptide VIP on mast cells is opposite to that of neuropeptide SP, which can inhibit mast cell degranulation and change the content of intracellular particles (20). This is primarily achieved by promoting the generation and accumulation of cyclic adenosine monophosphate. This study revealed that the concentration of neuropeptide VIP in the endometrium decreased significantly in metritis, indicating that its inhibitory effect on SP decreased. The weaker inhibitory effect resulted in excessive activation of mast cells, which is also related to the significant increase of LTB₄ concentration. Although it is beneficial for the body to recruit immune cells to fight infection, it may also lead to excessive immunoactivity and histopathological damage.

In recent years, the importance of the regulatory effect of MMPs on inflammation has been recognised. Matrix metalloproteinases may not only affect the activity of some cytokines, but also continuously reshape the extracellular matrix, in order to facilitate the migration of immune cells to the disease site (25). Therefore, the roles of MMP-2 and MMP-9 in the inflammatory response must not be underestimated, and Opdenakker et al. (18) regarded MMP-9 as a regulator of the inflammatory response. The expression of MMP-2 and -9 mRNA increased significantly, which not only strengthened the auxiliary effect on cytokines, but also increased the remodelling of extracellular matrix, both of which are beneficial to the uterus in metritis in fighting infection. However, the expression of uterine tissue protein in the experimental group decreased significantly, indicating that the degradation of collagenase is extensive and may cause tissue damage.

In conclusion, bacteria and their secretions stimulate the release of neuropeptide SP from uterine nerve axon terminals. Significantly elevated SP promotes the activation of mast cells and the synthesis and release of LTB₄ to recruit immune cells such as granulocytes. The uterine tissue expresses large amounts of collagenase MMP-2 and MMP-9, which promotes the hydrolysis of the extracellular matrix to facilitate the migration of immune cells to inflammatory sites. The low expression of the VIP neuropeptide weakened its inhibitory effect on mast cell activation and LTB₄ synthesis. Thus, the overexpression of LTB₄ may be closely related to the tissue damage in metritis.