J. Dairy Sci. 2007. 90:2229-2240. doi:10.3168/jds.2006-653
© 2007 American Dairy Science Association ®
Effect of a Biological Response Modifier on Expression of Growth Factors and Cellular Proliferation at Drying Off
B. E. Dallard*,
V. Ruffino*,
S. Heffel* and
L. F. Calvinho*,
,1
* Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, Rvdo. Padre Kreder 2805, (3080) Esperanza, Santa Fe, Argentina
Estación Experimental Agropecuaria Rafaela, Instituto Nacional de Tecnología Agropecuaria (INTA), CC 22 (2300) Rafaela, Santa Fe, Argentina
1 Corresponding author: lcalvinho{at}rafaela.inta.gov.ar
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ABSTRACT
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Agents that increase natural protective mechanisms have been proposed for the prevention and treatment of intramammary infections. Staphylococcus aureus is a major pathogen causing primarily subclinical chronic mastitis that responds poorly to antibiotic therapy. The objectives of this study were to describe the effects of a single intramammary infusion of a lipopolysaccharide-based biological response modifier (BRM) on mammary epithelial cellular proliferation and expression of insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in uninfected and Staph. aureus-infected bovine mammary glands during involution. Three groups of 12 cows, 6 Staph. aureus-infected and 6 uninfected, were infused with BRM or placebo in 2 mammary quarters and killed at 7, 14, and 21 d of involution. The proportion of infected quarters, mammary cell proliferation, and IGF-I and VEGF expression were evaluated. Biological response modifier treatment decreased the proportion of Staph. aureus-infected mammary quarters at 7 d of involution, but a similar number of isolations were observed at 14 and 21 d of involution in either treated or control quarters. The percentage of proliferating mammary epithelial cells was higher in infected than uninfected quarters at every observation period, irrespective of the treatment administered, whereas uninfected BRM-treated quarters showed increased cell proliferation at 7 d of involution. Insulin-like growth factor-I expression in uninfected quarters was not affected by treatment and showed a decrease at 21 d of involution. Expression of IGF-I was greater in infected than uninfected quarters at every observation period, irrespective of the treatment received. Expression of VEGF was greater in BRM-treated uninfected quarters at 7 d of involution compared with controls. In infected quarters, VEGF expression was lowest in BRM-treated quarters at 7 d of involution and increased throughout the observation period. Conversely, untreated infected quarters showed the highest VEGF expression at 7 d and decreased at 21 d of involution. Mammary cell proliferation and expression of IGF-I and VEGF were increased in Staph. aureus-infected quarters. Increased mammary cell proliferation and VEGF expression were observed in BRM-treated quarters during the first week of involution.
Key Words: biological response modifier • mammary gland involution • Staphylococcus aureus • cellular proliferation
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INTRODUCTION
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Susceptibility of the bovine mammary gland to new IMI is markedly increased during early involution and the periparturient period. Intramammary infections are often associated with clinical mastitis during early lactation and can have a marked detrimental effect on subsequent milk yield and quality (Oliver and Sordillo, 1988; Nickerson, 1989). Although not fully understood, increased susceptibility to IMI has been related to changes during the involution process that may facilitate bacterial penetration of the streak canal, interfere with natural defense mechanisms, and enhance bacterial growth (Oliver and Sordillo, 1989). Conversely, fully involuted bovine mammary glands are markedly resistant to IMI (Nickerson, 1989). These observations led to the hypothesis that hastening mammary gland involution while increasing natural protective mechanisms during the early nonlactating period could favor prevention of IMI during the involution process (Oliver and Smith, 1982a).
A greater understanding of components implicated in the immunological response of the mammary gland has led researchers to address alternative approaches to classic mastitis control measures based on hygiene and antibiotic therapy, such as manipulation of local immune responses to mastitis pathogens. Biological response modifiers (BRM) or immunomodulators are compounds capable of interacting with the immune system to regulate specific aspects of the host response. The type of activity of these compounds depends on their mechanism of action, site of action, dose, and route and timing of administration (Tzianabos, 2000). Biological response modifiers have been used in an attempt to enhance nonspecific immune mechanisms against bovine mastitis pathogens (Campos et al., 1993; Zecconi, 2000; Takahashi et al., 2004). However, the exact mechanism of action and effect of compounds used on the bovine mammary tissue are not fully understood.
Studies performed in cows showed that intramammary inoculation of Escherichia coli LPS at drying off resulted in an increase in phagocytic cell number, lactoferrin, serum albumin, Ig concentration, and pH and a decrease in secretion volume, indicating acceleration of mammary gland involution (Oliver and Smith, 1982a). In addition, intramammary LPS infusion at drying off was associated with a 50% reduction of isolation of mastitis pathogens during the first 4 wk of the nonlactating period (Oliver and Smith, 1982b). However, little information is available describing changes occurring in the nonlactating bovine mammary gland as a result of intramammary injection of LPS (Nickerson et al., 1992). Furthermore, little is known about variations in the expression of growth factors (GF) in involuting bovine mammary glands.
The GF network in the bovine mammary gland has not yet been fully explored, and delineation of the network at different stages of involution is essential. Based on studies performed with infected lungs and liver from the human and rat, Sheffield (1997) concluded that dramatic changes in a variety of genes, including GF and stress-induced genes, are likely to occur during IMI and may play a role in the progression of infection and recovery. Milk IGF-I concentrations have been shown to increase during experimentally induced E. coli mastitis (Shuster et al., 1995). Mammary tissue is known to respond to a variety of peptide GF, including IGF and epidermal GF (EGF; Peri et al., 1992; Grosvenor et al., 1993). Expression of GF may be related to tissue repair or may serve to protect cells against adverse conditions (Schultz et al., 1991; Cioffi et al., 1994). Thus, although the exact role of GF during mammary gland infection is not entirely clear, analogy with other systems suggests a possible role in tissue protection (Sheffield, 1997).
Vascular endothelial GF (VEGF) is an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties (Pepper et al., 2000). Recently, other functions of VEGF have been discovered, such as antiapoptosis activity, lymphangiogenesis (Baldwin et al., 2002; Nagy et al., 2002), immunosuppression (Ohm et al., 2003), stimulation and recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells in angiogenesis (Lyden et al., 2001), and regulation of hematopoietic stem cell survival (Mann and Plant, 2002). Several laboratories have elucidated the pivotal role of VEGF in the regulation of normal and abnormal angiogenesis (Ferrara, 1993). Little has been reported concerning the regulation of VEGF development during normal mammary involution (Pepper et al., 2000; Hovey et al., 2001), and roles have not been ascribed to this GF during the mammary infectious process.
The objectives of this study were to describe the effects of a single intramammary infusion of a LPS-based BRM on mammary epithelial cellular proliferation and expression of IGF-I and VEGF in uninfected and Staph. aureus-infected bovine mammary glands during involution.
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MATERIALS AND METHODS
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BRM
The BRM contained LPS of an E. coli strain (LN02) at a 0.35 µmol concentration and 4.5 mg of membranous and ribosomal fractions of the same strain, incorporated into liposomes contained in 10 mL of aqueous-based vehicle (Laboratorio Neomar, Buenos Aires, Argentina). Liposome (The Liposystem Complex, I.R.A., Milan, Italy) composition was 40% phospholipids and 60% hydrophilic medium and active principle. Concentration of LPS was determined by quantification of 2-keto-3-deoxyoctonate by a colorimetric method (Osborn, 1963).
Animals
Holstein nonpregnant cows in late lactation from the Rafaela Experiment Station of the INTA herd producing approximately 8 kg of milk/d before experimentation were used. Based on previous bacteriological studies, animals were either uninfected or infected with Staph. aureus. Infections were naturally acquired either in the dry period or in the first 2 mo of the lactation preceding initiation of the study. Mammary quarters from either infected or uninfected animals were included in the experiment.
Experimental Design
Infection of mammary quarters with Staph. aureus was considered as a covariable. Infectious status of mammary quarters was determined within 6 mo before initiation of the experiment and confirmed 20 and 3 d before inoculation. Infected quarters were randomly selected from cows showing at least 2 quarters infected with Staph. aureus. From these cows, only 2 infected quarters were infused with either BRM or placebo (vehicle alone). Uninfected quarters were selected from cows free of infection at the time of sampling. Only 2 quarters from each cow were infused with either BRM or placebo. Cows whose mammary quarters became infected during the experimental period were excluded from the study. Cows were killed at 7, 14, and 21 d after infusion. Uninfected (n = 6) and Staph. aureus-infected (n = 6) mammary quarters were included in each group (7, 14, and 21 d). In all cases, milking was interrupted after intramammary infusion, and cows were fed only alfalfa hay and had free access to water for the remainder of the experiment. Mammary secretion samples were aseptically collected for bacteriological analysis using standard procedures (Hogan et al., 1999) 3 d before BRM administration, immediately before inoculation, and every 48 h after infusion. Teats were dipped in a 0.5% iodophor solution after samples were taken. Animals included in the 3 groups were slaughtered at 7, 14, and 21 d after inoculation at a local abattoir and samples for histological analysis were taken. According to the eligibility criterion used, 36 out of 44 cows were included in the study.
Bacteriological Examination
Ten microliters of mammary secretion samples were streaked onto blood agar plates supplemented with 5% bovine blood and incubated aerobically for 48 h. Plates were examined for bacterial growth at 24 and 48 h. Isolated colonies were identified according to standard procedures (Hogan et al., 1999). The presence of one colony of Staph. aureus on blood agar was considered as a positive identification. Intramammary infection was defined as isolation of the same organism from 2 consecutive samples. Absence of IMI in each group was defined as 2 negative bacteriological cultures before slaughter.
Tissue Sample Preparation
Immediately after cows were killed, samples of mammary parenchyma from each mammary quarter were taken and processed for light microscopy. Tissue samples were taken from the dorso-lateral portion of the gland (deep parenchyma) at a depth of 4 cm following previous descriptions (Nickerson et al., 1992). Tissue samples of approximately 1 cm3 were fixed in 10% neutral buffered formalin, washed in PBS, and processed for inclusion in paraffin (Woods and Ellis, 1994). Serial 5-µm sections were mounted on glass previously treated with 3-aminopropyltriethoxysilane (Sigma-Ald-rich, St. Louis, MO) and stained with hematoxylin-eosin for a preliminary observation.
Image Analysis
Image analysis was performed using an Image Pro-Plus 3.0.1 system (Media Cybernetics, Silver Spring, MA). Images were digitized by a charge-coupled device color video camera (Sony, Montvale, NJ) mounted on top of a conventional light microscope (Olympus BH-2, Olympus Co., Japan) using an objective magnification of 40x. Microscopic fields covering the entire area were digitized and stored in a 24-bit true color TIFF format. The resolution of the images was set to 640 x 480 pixels. Each pixel of the image corresponded to 0.26 µm, and each field represented a tissue area of 0.02 mm2. Details of image analysis as a valid method for quantifying expression levels and methodological details have been described previously (Dallard et al., 2005; Ellis et al., 2005). Briefly, the immunohistochemical stained area (IHCSA) for antibody reaction was calculated as a percentage of the total area evaluated through the color segmentation analysis, which extracts objects by locating all objects of the specific color (brown stain). The brown stain was selected with a sensitivity of 4 (maximum 5) and a mask was next applied to make the separation of colors permanent. The images were then transformed to a bilevel scale TIFF format. The IHCSA (percentage of black area) was calculated from at least 50 images in alveoli, ducts, and interstitial tissue.
Immunohistochemistry
Details and concentration of antibodies are summarized in Table 1
. Each antibody was assayed in at least 5 sections of each tissue sample. A streptavidin-biotin immunoperoxidase method was performed as described previously (Dallard et al., 2005). Briefly, sections were deparaffinized, hydrated, and microwave pretreated (antigen retrieval). The endogen peroxidase activity was inhibited with 1% H2O2 and nonspecific binding was blocked with 10% normal goat serum. All sections were incubated with the primary antibodies for 18 h at 4°C and then for 30 min at room temperature with rat-preabsorbed biotinylated secondary antibodies selected specifically against one of each of the 2 types of primary antibodies (monoclonal or polyclonal). The visualization of antigens was achieved by the streptavidin-peroxidase method (BioGenex, San Ramon, CA), and 3.3-di-aminobenzidine (Liquid DAB-Plus substrate kit, Zymed, San Francisco, CA) was used as the chromogen. Finally, the slides were washed in distilled water and counterstained with Mayer’s hematoxylin, dehydrated, and mounted. To verify specificity, adjacent control sections were subjected to the same immunohistochemical method, replacing primary antibodies by rabbit and mouse nonimmune serum. Each primary antibody was probed with an absorption test involving the respective antigen (15 µg/mL; Sigma-Aldrich). The specific effect of the secondary antibodies was tested by incubation with primary antibodies of proven negative reaction with rat antigen: anti-CD45 (clone: PD7/26 & 2B11; Dako, Carpinteria, CA) and anti-Ki-67 (polyclonal, rabbit antihuman Ki-67; Dako). To exclude the possibility of nonsuppressed endogenous peroxidase activity, some sections were incubated with DAB reagent alone.
Cellular Proliferation
Proliferation was evaluated by measuring expression of proliferation cell nuclear antigen (PCNA). Expression of PCNA was detected by immumohistochemistry using a specific monoclonal antibody (Table 1
). All immunostained mammary epithelial cells showing a characteristic nuclear staining pattern were considered positive. Cells were classified into 3 categories according to staining intensity: light (1+), moderate (2+), and intense (3+). A minimum of 1,000 cells in at least 20 microscopic fields were counted in each specimen. Cells showing an intense nuclear staining (PCNA 3+) were quantified and results were expressed as a percentage (Muñoz de Toro et al., 1998).
Statistical Analysis
Results were expressed as the mean ± standard error of the mean. Data were analyzed by one-way ANOVA using Duncan’s multiple range test. Statistical analysis was performed with SPSS software (version 11.0 for Windows, SPSS Inc., Chicago, IL). Statistical significance was fixed as P < 0.05.
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RESULTS
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Infection Status
In animals killed at 7 d of involution, Staph. aureus IMI was not detected in 4 BRM- and 1 placebo-treated quarter during the observation period, respectively. In animals killed at 14 d, Staph. aureus was not detected in 1 BRM-treated and 1 placebo-treated quarter at the end of the observation period, whereas in animals killed at 21 d, Staph. aureus was detected throughout the observation period in both BRM-treated and placebo-treated quarters.
Cellular Proliferation
The proliferation of mammary epithelial cells was evaluated with PCNA and is summarized in Table 2. The cellular proliferation pattern in uninfected quarters treated with BRM was similar across sampling days. The percentage of proliferation was lower, however, at 7 d in placebo-treated quarters (P < 0.05) and increased through the observation period to reach values similar to BRM-treated quarters (14 and 21 d). The proliferation percentage in infected quarters was greater at 7 d into the dry period in either BRM- or placebo-treated quarters (P < 0.05) and decreased throughout the observation period. Cellular proliferation percentages were similar for control and BRM-treated infected quarters at every observation period. Proliferation percentages in infected quarters were greater than in uninfected quarters at every observation period, irrespective of treatment (P < 0.05).
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Table 2. Effect of treatment with a biological response modifier (BRM) on expression of proliferation cell nuclear antigen (PCNA) in uninfected and Staphylococcus aureus-infected quarters during involution
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Pattern of GF Localization in Mammary Gland Structures
The positive immunohistochemical reaction with each protein (IGF-I and VEGF) was detected by brown cytoplasmic staining and evaluated by the IHCSA. Regional differences in the distribution and IHCSA for each GF were observed within the mammary gland. The effects of treatment on IHCSA for IGF-I and VEGF in uninfected and Staph. aureus-infected quarters during involution are summarized in Tables 3 and 4.
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Table 3. Effect of treatment with a biological response modifier (BRM) on immunohistochemical stained area (IHCSA) for IGF-I in uninfected and Staphylococcus aureus-infected quarters during involution
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Table 4. Effect of treatment with a biological response modifier (BRM) on immunohistochemical stained area (IHCSA) for vascular endothelial growth factor (VEGF) in uninfected and Staphylococcus aureus-infected quarters during involution
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IGF-I.
Immunostaining for IGF-I was seen in every studied period in both infected and uninfected quarters, ranging from weak to intense, and was mainly associated with the structures of the mammary parenchyma. Uninfected and infected quarters in control and BRM-treated groups showed strong IGF-I staining in the epithelium of the alveoli and ducts (Figure 1). In quarters with IMI, arteries and veins showed a strong reaction in the endothelium, vascular smooth muscle cells, and content. In contrast, in uninfected quarters IGF-I expression ranged from weak to moderate in those structures. In the 3 involution stages and treatments studied, macrophages frequently occurred in the lumen of the alveoli and in the stroma, with strong IGF-I cytoplasmic staining. Both the intralobular connective tissue and interlobular stroma displayed negative IGF-I staining. Expression of IGF-I in uninfected quarters followed a similar pattern in both the BRM- and placebo-treated groups and showed a significant reduction at 21 d compared with 7 and 14 d (P < 0.05). Conversely, IGF-I expression in infected quarters increased from 7 to 14 d of involution in both BRM- (P < 0.05) and placebo-treated groups and decreased by 21 d. A decrease in expression from 14 to 21 d, however, was significant only for the placebo-treated group (P < 0.05). Expression of IGF-I was greater in infected than in uninfected quarters at every observation period (P < 0.05), irrespective of treatment.
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Figure 1. Immunohistochemical identification of IGF-I. A) Bovine mammary gland infected with Staphylococcus aureus, treated with biological response modifier at 14 d of involution. Strong IGF-I staining in the epithelium of the alveoli and ducts. B) The positive areas were identified by color segmentation analysis.
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VEGF.
The specific cellular localization of VEGF was similar in uninfected and infected quarters during involution. Expression of GF was primarily associated with the epithelium of the alveoli and ducts of the mammary parenchyma with strong VEGF staining (Figure 2). The blood vessels expressed VEGF in their endothelium and smooth muscle cells. Macrophages in the lumen of the alveoli and in the stroma showed strong immunostaining in their cytoplasm. Both the intralobular connective tissue and interlobular stroma displayed negative VEGF staining. The percentages of IHCSA in uninfected quarters treated with BRM were greatest at 7 d of involution (P < 0.05) and then decreased throughout the observation period. Meanwhile, expression of VEGF in placebo-treated uninfected quarters did not show differences. Differences between BRM- and placebo-treated groups were significant at 7 d of involution (P < 0.05).
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Figure 2. Immunohistochemical identification of vascular endothelial growth factor (VEGF). A) Bovine mammary gland infected with Staphylococcus aureus, treated with biological response modifier at 21 d of involution. Strong VEGF staining in the epithelium of the alveoli and ducts. Arrows indicate macrophages in the lumen of the alveoli and in the stroma with a strong immunostaining in their cytoplasm. B) The positive areas were identified by color segmentation analysis.
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In infected quarters, the percentage of IHCSA showed a different pattern in either BRM- or placebo-treated groups. Whereas in the placebo-treated group, the greatest expression values were observed at 7 d of involution, followed by a decrease (P < 0.05) at 14 and 21 d, in the BRM-treated group, lesser expression was observed at 7 d of involution, followed by an increase at 14 and 21 d (P < 0.05). The percentages of IHCSA were greater in infected quarters for both treatment groups at every sampling period (P < 0.05).
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DISCUSSION
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Escherichia coli LPS has been used extensively to study events that take place during mammary gland inflammation, and less frequently to stimulate bovine mammary gland nonspecific defense mechanisms after cessation of milking (Oliver and Smith, 1982a,b; Nickerson et al., 1992). In the present study, the effect of a BRM-containing E. coli LPS and cellular fractions incorporated into liposomes administered at the end of lactation in bovine mammary glands free of infection and infected with Staph. aureus was determined. Incorporation of LPS into liposomes reduces part of its biological activity and therefore diminishes potential toxic effects (Erridge et al., 2002a). In addition, liposomes function mainly to deliver associated antigens into macrophages that are target cells for LPS (Alving, 1993; Erridge et al., 2002a). The mechanism by which incorporation of LPS into liposomes changes the interaction with the innate immune system is not fully understood. Membrane-bound receptors CD14 and TLR4 are involved in activation of mononuclear cells by LPS, and this activation can be enhanced by soluble LPS-binding protein (Erridge et al., 2002b; Akira and Hemmi, 2003). A recent study showed that binding of phospholipids to soluble LPS inhibited LPS-induced tumor necrosis factor-
production by mononuclear cells because of interference with the presentation of LPS to the LPS-binding protein (Mueller et al., 2005). Phospholipids included in liposomes can therefore reduce the potency of LPS to stimulate immune cells (Mueller et al., 2005). The doses of BRM used in the present study proved to be safe for cows in a previous study, causing only mild inflammation and no systemic adverse effects (Calvinho et al., 2004).
The intramammary infusion of BRM decreased the proportion of Staph. aureus-infected quarters at 7 d of involution. However, a similar proportion of infected quarters was observed in both treatment groups at 14 and 21 d of involution. Early studies showed a similar frequency of isolation of coagulase-positive staphylococci during the first week of involution in mammary quarters infused with colchicine, endotoxin, or a combination of both compounds, compared with uninfused controls (Oliver and Smith, 1982b). In addition, recent studies showed that intramammary infusion of recombinant bovine granulocyte-macrophage colony-stimulating factor and recombinant bovine IL-8 in midlactation cows both recently and chronically infected with Staph. aureus caused a decrease in the number of organisms isolated from mammary secretions within 2 d following administration. However, quarters remained infected and the number of Staph. aureus colony-forming units per milliliter in mammary secretions rose thereafter (Takahashi et al., 2004, 2005). Lipopolysaccharide-based BRM are recognized by monocytes and macrophages, which react and release a range of proinflammatory mediators that, in moderate levels, benefit the host by promoting inflammation and priming the immune system (Erridge et al., 2002a). Evaluation of the efficacy of BRM to eliminate IMI was beyond the scope of this study. The effect on bacterial isolation achieved in this experiment through administration of BRM appeared to be short term and insufficient to resolve chronic gram-positive infections. Further research will be needed to evaluate the effect of this and similar compounds on recently acquired infections.
Mammary involution in the dairy cow takes place with minor loss of epithelial cells, as determined by morphological studies (Holst et al., 1987; Sordillo and Nickerson, 1988). It has therefore been suggested that a nonlactating period is important to enhance replacement of senescent mammary epithelial cells before the next lactation. In addition, mammary epithelial cells showed greater incorporation of [3H]thymidine during the nonlactating period, indicating augmented cell division and replacement of mammary cells (Capuco et al., 1997). Because previous studies have shown that E. coli LPS and Staph. aureus -toxin inhibit proliferation of bovine mammary epithelial cells in vitro (Matthews et al., 1994; Calvinho et al., 2001), PCNA was used to determine whether mammary epithelial cell proliferation was affected by either treatment with BRM or infectious status of the mammary quarter. Lack of differences in expression of PCNA between BRM-treated and control in infected quarters indicated that the BRM did not interfere with cell proliferation.
An experimental challenge of lactating cows with E. coli has clearly documented the resulting stimulation of apoptosis and cell proliferation (Long et al., 2001). The authors found that the number of apoptotic epithelial cells, as determined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, increased from 1.8 ± 0.5 to 8.8 ± 2.8 cells. Interestingly, an increase in cell proliferation (epithelial, stromal, and lumenal) was also observed, and this might be a restorative mechanism to maintain alveolar integrity. Whether these cells represent scar tissue formation or go on to produce milk is unknown. In the present study, cellular proliferation was greater in Staph. aureus-infected than uninfected quarters throughout the observation period, which may serve as a mechanism to ameliorate tissue damage after infection or contribute to cellular repair mechanisms or both. Whereas bacterial toxins, or the proinflammatory mediators, may cause cell death by apoptosis, increased cell proliferation may compensate for cell loss during inflammation. Wesson et al. (2000) have described mechanisms of induction of apoptosis by Staph. aureus in mammary epithelial cells in vitro. However, the signaling events involved in mammary cell apoptosis and proliferation induced by Staph. aureus infection are still not fully understood.
Aspects of mammary growth during the dry period have been investigated (Capuco et al., 1997). The overall rate of [3H]thymidine incorporation by mammary tissue was 80% greater in dry cows than in lactating cows and tended to increase with advancing stages of the dry period. Autoradiographic localization of incorporated [3H]thymidine indicated that the replicating cells were primarily (>90%) epithelial. In cows, therefore, the dry period may be important for replacing senescent cells prior to the next lactation. Our observation that cellular proliferation increased from 7 to 14 d in uninfected control quarters suggests greater cell division for the purpose of cell renewal or turnover. In addition, treatment of the uninfected quarters with BRM produced a transient increase in PCNA expression at 7 d of mammary involution compared with placebo-treated quarters; thereafter, cellular proliferation was similar for both treatment groups. These results suggest that in uninfected quarters, BRM can contribute to increased mammary cell turnover during the first week of involution.
Studies involving induced infections have been used to determine some of the functional changes caused by mastitis. Such studies have shown that mastitis induces disruption of the normal secretory functions of the mammary gland (Sordillo et al., 1989). The changes in GF expression that are associated with mastitis are not well understood, and little information is available describing the variation in GF expression in involuting bovine mammary glands. Mammary tissues from different species, including the bovine, are known to respond to a variety of peptide GF, including IGF and EGF (Peri et al., 1992; Grosvenor et al., 1993). Various GF, such as EGF, IGF-I, acidic fibroblast GF, and basic fibroblast GF, have also been reported to increase in other tissues and cells during various stressing situations, including heat shock, wound repair, infection, and oxidative stress (Aloe et al., 1994; Vivekananda et al., 1994). Such expression may be related to tissue repair or may serve to protect cells (Schultz et al., 1991; Cioffi et al., 1994). Although the exact role of these factors during udder infection is not entirely clear, analogy with other systems suggests a possible role in tissue protection (Sheffield, 1997).
The IGF-I protein was primarily associated with the epithelium of alveoli and ducts, tissue elements of the parenchyma. The epithelial cells displayed strong staining in both uninfected and infected quarters during involution. This finding is consistent with previous studies that showed immunostaining for IGF-I in the cubic epithelium of small and large acini in bovine mammary glands during involution (Plath-Gabler et al., 2001). Mammary-specific expression of IGF-I in transgenic mice demonstrated that IGF-I promoted cell survival and delayed mammary gland involution (Hadsell et al., 1996). Based on expression of IGF-I in both uninfected and infected control quarters, our results suggest that IGF-I is probably an important local factor for remodeling of the bovine mammary gland during the first 2 wk of involution.
Cohick et al. (1995) demonstrated that incubation of inflammatory cytokines with the bovine mammary epithelial cell line MAC-T increases synthesis of IGF-binding proteins. This observation, as well as findings that synthesis of IGF-binding proteins are increased in the infected mammary gland, where concentrations of inflammatory cytokines are high (Shuster et al., 1995), suggest a causal association between local cytokine production and increased IGF-binding proteins in the milk of mastitic glands. Milk IGF-I concentrations have been shown to increase during E. coli-induced mastitis (Shuster et al., 1995). In the present investigation, the expression of IGF-I was greater in Staph. aureus-infected than in uninfected quarters. This result agrees with an earlier observation that IGF increases in the milk of endotoxin-infused glands (Schultz et al., 1991), which can be partially explained by permeability changes that would allow passive diffusion of IGF-I into milk from serum, because the concentration gradient is nearly 25-fold greater in serum. Furthermore, normal mammary epithelial cells do not synthesize IGF-I (Yee et al., 1989).
A previous study showed that mRNA for several GF, including acidic fibroblast GF, basic fibroblast GF, EGF, transforming GF-, IGF-I, and IGF-II, increased during Streptococcus agalactiae-induced mastitis (Sheffield, 1997). Those results indicated that mastitis induced changes in mRNA encoding a variety of GF and agreed with increased expression of GF that has been observed in recent studies of mammary infection (Chockalingam et al., 2005). Our results showed that treatment of infected quarters with BRM did not influence IGF-I expression during the first 2 wk of involution. Only at d 21 of involution were percentages of IHCSA in treated quarters greater than those of untreated controls. This response may be consistent with a possible effect of BRM for maintaining elevated IGF-I expression and extending the tissue remodeling in infected quarters. Conversely, treatment of uninfected quarters with BRM did not affect IGF-I expression compared with controls.
Northern blot analysis of RNA isolated at various stages of the mammary cycle in rodents demonstrated that VEGF is expressed predominantly by epithelial cells, and VEGF mRNA decreased progressively during involution (Pepper et al., 2000). In cleared mouse mammary gland devoid of epithelial components, VEGF was reduced by approximately 75%, demonstrating that although the epithelial component is the major source of VEGF, approximately 25% of VEGF is derived from stroma (Pepper et al., 2000). In contrast to these reports, analysis of VEGF expression in mouse mammary cleared fad pad across development stages by reverse-transcription PCR, combined with in situ hybridization and immunohistochemical analyses, indicated that the stroma is a primary source of VEGF within the developing mammary gland (Hovey et al., 2001). Our findings in the bovine mammary gland during involution demonstrated that VEGF expression was primarily associated with the epithelium of the alveoli and ducts of the mammary parenchyma. Expression of VEGF increased in BRM-treated infected quarters as mammary involution progressed, whereas control quarters showed greater VEGF expression at 7 d of involution and progressively decreased on 14 and 21 d. In addition, increased VEGF expression was observed at 7 d of involution in BRM-treated uninfected quarters that coincided with greater expression of intensely stained PCNA cells (data not shown), suggesting an intense cellular turnover both in endothelial cells and in epithelial cells from ducts and alveoli during this period. Conversely, uninfected control quarters did not show changes in VEGF expression during involution, suggesting a minor role during normal mammary gland involution. Nevertheless, the activity of VEGF during bovine mammary involution remains to be defined.
A recent immunohistochemical study showed prominent expression of VEGF in macrophages during mouse mammary gland development (Hovey et al., 2001). McLaren et al. (1996) demonstrated expression of VEGF and its receptors by macrophages. In the present study, strong immunostaining of VEGF in the cytoplasm of macrophages in both infected and uninfected quarters was recognized. Early studies have documented that the VEGF receptor fit-1 is expressed in human monocytes and that VEGF induces the activation and migration of human myocytes via this receptor (Barleon et al., 1996). Thus, monocyte-derived VEGF may induce chemotaxis of monocytes or macrophages in both the inflammatory and remodeling process and may act in an autocrine or paracrine fashion.
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CONCLUSIONS
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The presence of Staph. aureus IMI during mammary gland involution resulted in an increase in mammary epithelial cell proliferation and IGF-I and VEGF expression that was not affected by BRM treatment. In addition, in uninfected quarters, BRM treatment produced an increase in both cellular proliferation and VEGF expression during the first week of involution. The BRM treatment reduced the proportion of Staph. aureus-infected quarters at 7 d of involution, but a similar number of isolations was observed at 14 and 21 d of involution in either treated or control quarters.
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ACKNOWLEDGEMENTS
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The authors wish to express their appreciation to O. Warnke, M. Marín, V. Canavesio, and V. Neder for technical assistance and to R. Almeida for manuscript review. This work was supported by CAI+D (Universidad Nacional del Litoral) and Fundación ArgenINTA.
Received for publication October 9, 2006.
Accepted for publication December 29, 2006.
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