Mechanisms Involved in Milk Endogenous Proteolysis Induced by a Lipopolysaccharide Experimental Mastitis

J. Dairy Sci. 85:2562–2570 © American Dairy Science Association, 2002.

Mechanisms Involved in Milk Endogenous Proteolysis Induced by a Lipopolysaccharide Experimental Mastitis F. Moussaoui,* I. Michelutti,† Y. Le Roux,* and F. Laurent* *Laboratoire de Sciences Animales, Unite´ sous contrat avec l’Institut National de la Recherche Agronomique (INRA), Ecole Nationale Supe´rieure d’Agronomie et des Industries Alimentaires (ENSAIA), 54 505 Vandoeuvre-le`s-Nancy, France †FNCL: Fe´de´ration Nationale des Coope´ratives Laitie`res 75 314 Paris Cedex 09

ABSTRACT

INTRODUCTION

Experimental mastitis has been induced by the lipopolysaccharide (LPS) of Escherichia coli on six dairy cows in order to study the mechanisms involved in milk endogenous proteolysis during the inflammatory process. Variations in somatic cell count (SCC), plasmin activity, and total casein (CN) content were measured, and proteose-peptone content and the percentage of pH 4.6 insoluble peptides including γ-CN have been considered as indicators of endogenous proteolysis. Furthermore, polymorphonuclear neutrophils (PMN) maturity has been evaluated by optical microscopy, and proteolysis by PMN proteinases has been studied at neutral and acidic pH in order to establish a link between caseinolysis, proteinase class, and PMN maturation. Two peaks of proteose-peptones content have been noticed for the six cows. First peak could be explained by both plasmin activity and SCC, while second peak was concomitant with a low plasmin activity but a SCC remaining high. The second peak of proteose-peptones content confirmed the role of cellular proteases in milk caseinolysis. Casein breakdown by cellular proteases was confirmed by SDS-PAGE electrophoresis, and a link between neutral proteinases activity and immature PMN recruitment was shown. Acidic proteinases activity was effective with mature PMN and during the recovery phase. (Key words: endogenous proteolysis, lipopolysaccharide, plasmin, somatic cells)

Mastitis is a complex inflammatory condition of the mammary gland that results in decreased milk synthesis (Kaartinen et al., 1988; Michelutti et al., 1999), increased permeability of mammary epithelial cell tight junctions, influx of serum constituents and somatic cells into milk, and altered milk composition (Guidry et al., 1983; Michelutti et al., 1999). Increased proteolytic activity in mastitic milk results in hydrolysis of CN and reduced yield and quality of dairy products (Andrews and Alichanidis, 1983; De Rham and Andrews, 1982; Le Roux et al., 1995a; Michelutti et al., 1999). Milk endogenous proteolysis has two main origins: plasmin activity and milk somatic cell proteases. Proteolysis of caseins by plasmin (EC 3.4.21.7) is well known and especially the cleavage points of β-CN leading to the formation of γ-CN and some components of the proteose-peptones (PP) fraction (Eigel, 1977b; Le Bars and Gripon, 1993; Auldist et al., 1996). Lysosomes in somatic cells contain a variety of proteolytic enzymes (Grieve and Kitchen, 1985; Jain, 1993). Cathepsin D (EC 3.4.23.5) is an intracellular aspartic proteinase identified in bovine milk as the major lysosomal proteinase, which displays optimum proteolytic activity at pH between 2.8 and 4.0. Cathepsin D is able to degrade purified αs1-, αs2-, β-CN, even κ-CN releasing the glycomacropeptide (f106-109), and α-LA (Larsen et al., 1996). Another principal enzyme originated from polymorphonuclear neutrophil cells (PMN) is the serine proteinase, elastase (EC 3.4.21.37) (Jain, 1993), which can hydrolyze αs1-CN (Considine et al., 2000), β-CN (Considine et al., 1999), and, less efficiently, whey proteins such as α-LA or β-LG (Jakobsson et al., 1983). The PP content in milk with high SCC is correlated with the intensity of proteolytic activity, so PP fraction is widely used as an indicator of caseinolysis (Le Roux et al., 1995a; Auldist et al., 1996; Michelutti et al., 1999). This fraction is a heat-stable, acid-soluble protein fraction of milk, which contains many peptides and proteins. The first group of PP is a complex mixture of

Abbreviation key: FPLC = fast-pressure liquid chromatography, LPS = lipopolysaccharide, PMN = polymorphonuclear neutrophils, PI = postinfusion, PP = proteose-peptones.

Received November 16, 2001. Accepted March 20, 2002. Corresponding author: F. Moussaoui; e-mail: fatima.moussaoui @ensaia.inpl-nancy.fr.

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protein hydrolysates, mainly issued from CN such as β-CN-5P (f1-105 and 1-107; noted f1-105/7), β-CN-4P (f1-28), and β-CN-1P (f29-105/7) resulting from proteolysis of the β-CN by endogenous plasmin (Andrews, 1983b; Le Roux et al., 1995a). Second group includes component PP3, a hydrophobic glycoprotein and a fragment of component PP3 (f54-135), resulting from hydrolysis of PP3 by plasmin (Girardet and Linden, 1996). Increase of PP content in milk is mainly explained by increase of β-CN proteolysis, but can also be explained by proteolysis of other CN (Le Bars and Gripon, 1993; Auldist et al., 1996). The proportion of γ-CN issued from β-CN proteolysis by plasmin is also considered as an indicator of proteolysis (Eigel, 1977b). The fraction of pH 4.6 insoluble peptides, precipitating with CN and including γ-CN, can also include peptides issued from other CN (Le Bars and Gripon, 1993). It has been reported by some authors that plasmin is mainly responsible for caseinolysis: Results from these studies were obtained from milks with high SCC until 2 × 106 cells/ml (Andrews and Alichanidis, 1983; Kaarrtinen et al., 1988; Le Roux et al., 1995a). Some others showed that the role of somatic cells in caseinolysis increases for SCC > 2 to 3 × 106 cells/ml (Andrews, 1983b; Michelutti et al., 1999; Saeman et al., 1988). The link between SCC and caseinolysis as far as the change in the nature of PMN recruited has not yet been studied. Neutral proteases could show significant activity because they are at their optimal pH when released in milk after degranulation of PMN. But what about acidic proteases activity when cathepsin D, for example, has previously been shown to have a significant role in proteolysis (Larsen and Benfeldt, 1996)? This work was an update of milk composition change during a clinical mastitis. It evaluated the respective roles of plasmin and PMN in native proteolysis during a kinetic after lipopolysaccharide (LPS) intramammary infusion. Several parameters have been studied, such as SCC, plasmin activity, PP content, the percentage of pH 4.6 insoluble peptides, including γ-CN and the other CN fractions of milk from inflamed quarters. In addition, another aim of this study was to link PMN maturity and proteolytic activity in both neutral and acidic conditions in order to clarify the mechanisms involved in cellular proteolysis. MATERIALS AND METHODS Treatment and Sampling Six Holstein cows without any major mammary pathogens and SCC <100,000 cells/ml (quarter milk) were used. Cows were between second and fourth lactation (they were challenged between 66 and 103 d) and

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produced more than 12 L per milking. A single rear quarter was infused with 10 μg of Eschericia coli LPS (LPS 026:B6, Sigma Chemical Co., St Louis, MO) dissolved in 10 ml of pyrogen-free physiological (0.9%, vol/ vol) sodium chloride buffer. Rectal temperatures were recorded at least every 2 h during the first 12 h postinfusion (PI). Milks from infused quarters were collected after a complete milking, gently stirred and subsampled immediately after milking. Milk samples were collected at 0 h just before infusion, and at 4, 8, 12, 16, 25, 36, 52, 64, and 76 h PI; some samples were missed due to very low volumes of milk. Milk samples collected at 0 h were a control for conditions before infusion. After each 35ml milking, stored at 4°C, samples were collected for SCC determination. Fresh milk (4 ml) was immediately stored at −75°C to measure plasmin activity, 120 ml was dedicated to PMN extraction performed within the hour of collection, cells were used to count PMN by microscopy and for CN hydrolysis in vitro. Skim milk (6000 × g for 45 min at 4°C) was frozen at −20°C for CN and PP content determination. For frozen samples, the measurements were led within the week following the experiment to prevent proteolysis due to storage. Analyses Infectious status of quarters. The infectious status of quarters was evaluated according to the procedure of Harmon et al., 1990. Quarter foremilk samples were collected aseptically for diagnosis of IMI at d -5, -1, 2, and 10. A 25-μl sample of each fresh milk sample was plated on sheep blood agar (BioMerieux, Marcy l’Etoile, France) and incubated at least for 48 h at 37°C in order to detect major pathogens. Both major and minor pathogens were identified by the Laboratoire Interprofessionnel de Pixe´re´court, France. Somatic cells. Somatic cells were quantified by electronically counting nuclei after coloration (Fossomatic 5000, Foss Electric, Hillerød, Denmark) with, however, dilutions of milks expected to have a SCC >106 cells/ml (including times 4 to 36 h PI). The method for isolation of quarter milk cells was adapted from Dulin et al., (1982). Fresh milk (120 ml) was diluted 1:2 with PBS pH 7.2 and centrifuged at 350 × g for 10 min at 20°C. The supernatant was centrifuged again under the same conditions. The two pellets were pooled, washed in buffer, and centrifuged at 250 × g for 10 min at 20°C. The washed cell pellet was resuspended in Dulbecco’s modified Eagle medium (Sigma) supplemented with Lglutamine, 10% (wt/vol) fetal calf serum, insulin, hydrocortisone, penicillin, and streptomycin. Viability (%) and counts of cell suspensions were both determined using trypan blue staining (0.5%) in a Malassez chamJournal of Dairy Science Vol. 85, No. 10, 2002

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ber (Bessis, 1977). Cell suspensions adjusted to 500,000 cells/ml with supplemented Dulbecco’s modified Eagle medium were spun in a cytospin chamber for 10 min at 270 × g (Shandon-Southern Cytospin 3, Pittsburgh, PA). Cytospin smears were stained with May-Gru¨nwald-Giemsa (Sigma; St. Louis, MO; Bessis, 1977). On each slide from milk between 200 and 500 cells were counted and differentiated into PMN and mononuclear cells. The range 200 to 500 is due to an approximate dilution of cell pellets, as the precise SCC wasn’t known at this stage of the experiment. PMN nucleus was characterized by counting lobuli in order to evaluate cell maturity through the inflammation kinetic. Plasmin activity. Plasmin activity was measured by a method based on the release of a yellow compound (measured at 405 nm) when a synthetic substrate (DVal-Leu-Lys p-nitroanilide dihydrochloride; Sigma) that is sensitive to plasmin was hydrolyzed, after the addition of dissolving reagent (Linden et al., 1987). Total N, NPN, soluble N, total CN, and PP contents. Total N, NPN, soluble N, total CN, and PP contents were determined as described previously (Le Roux et al., 1995a). Protein and CN contents were calculated according to the method of Ribadeau-Dumas and Grappin (1989). CN fractions proportions. The proportions of each CN fraction in the total CN content was determined by fast-pressure liquid chromatography (FPLC) according to the method of Collin et al. (1991). Percentages of αs-, β-, κ-CN, and pH 4.6 insoluble peptides including γ-CN were determined on five cows. Electrophoresis. Somatic cell pellets, freshly extracted from milk, for each cow and for each point of the kinetic, were brought to a concentration of 107 cells/ ml with a dilution in sodium acetate buffer 0.2 M pH 7.2 or 5.6. Cell membranes were disrupted by seven cycles of freezing-thawing and added to total CN (Sigma): 10 mg/ml in 0.2 M sodium acetate pH 7.2 or 5.6, NaN3 0.04% (wt/vol). Each sample contained 5 times more CN than cell. The mixture of CN + cells at each point of the milking kinetic of each cow (two pH were tested: 7.2 and 5.6) were incubated at 37°C for 12 h. Samples of the ten points (0 to 76 h PI) of the kinetic were characterized by SDS-PAGE by to the method of Laemmli and Favre (1973). For each sample, 20 μg of casein per well were used. In the presence of 0.1% (wt/ vol) SDS and 5% (vol/vol) 2-mercaptoethanol, SDSPAGE was performed with 5% polyacrylamide for the stacking gel in 0.125 M Tris (pH 6.8) and with 15% polyacrylamide for the separating gel in 0.38 M Tris (pH 8.8). Proteins were stained by Coomassie blue R 250 0.1% (wt/vol) in ethanol 50% (vol/vol), acetic acid Journal of Dairy Science Vol. 85, No. 10, 2002

Figure 1. Kinetics of SCC in inflamed quarter milk after an intramammary infusion of 10 μg of endotoxin of cows A (䊐), B (䊏), C (䊊), D (䊉), E (䉭), F (▲) at times 0, 4, 8,12,16, 25, 36, 52, 64, and 76 h. Time 0 h was considered as a standard before LPS infusion.

10% (vol/vol) and TCA 2% (wt/vol), and distained in ethanol 30% (vol/vol) and acetic acid 7.5% (vol/vol). RESULTS Clinical Response to Endotoxin All quarters were free of major pathogens and no quarter was infected during the entire experimental period. About 1 h PI, the gland started swelling markedly until a maximum towards 4 h PI. At this time, the udder was very firm and painful. From the fourth hour PI, milk changed from normal appearance to a serous fluid, and clots appeared in the samples until 25 h PI. As for fever, rectal temperature increased as early as 2 h and peaked significantly at 6 h, reaching a maximum of 40.6°C (± 0.89) with a range from 39 to 41.3°C. Return to initial values was observed at 12 h PI. Although there was a considerable variation among cows, the inflammatory responses to LPS infusion involved similar changes for all six cows. SCC and Identification in Milk Average SCC was 31,600 cells/ml at 0 h. SCC peaked a first time between 8 and 16 h PI, reaching a maximum of 27 × 106 cells/ml. A second increase of SCC was observed between 16 and 25 h PI for three cows, and a steady state for the others. Initial level wasn’t recovered yet at 76 h PI with an average SCC of 792 000 cells/ml (Figure 1). Before infusion, 12% (average value) of SCC from milk of infused quarters were PMN (Figure 2A). Percentage of PMN increased up to 4 h PI to reach maximum values between 8 and 25 h PI. A decrease was

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Figure 3. Plasmin activity change during a lipopolysaccharide (LPS) experimental mastitis, in quarter raw milk of cows A (䊐), B (䊏), C (䊊), D (䊉), E (䉭), and F (▲).

at 12 h PI. Young PMN percentage returned to initial level at 76 h PI. Plasmin Activity Before infusion, plasmin activity average level was 11.3 (±2.47) μmol p-nitroanilide/h per liter. It increased and peaked at 4 or 8 h PI (for six cows), a return to baseline (for five cows) was then noticed afterwards (Figure 3). Biochemical Composition Figure 2. Polymorphonuclear neutrophils (PMN) percentage of total somatic cells in milk (A) with cows A (䊐), B (䊏), C (䊊), D (䊉), E (䉭), and F (▲) and evolution of the stage of maturity of polymorphonuclear neutrophil (PMN; (2B) through time in inflamed quarter milk after intramammary infusion of 10 μg of endotoxin. Young PMN (䊐) and old PMN (䊏) have been considered as one to two and three to six lobuli cells, respectively, in optical microscopy, after a MayGru¨nwald and Giemsa staining of the nucleus. Where not visible, error bars are contained within the symbols.

Total CN content. Casein content just before infusion averaged 25.1 g/L (±2.0). The average maximum variation of CN content was −18.8% (±7.2). According to individual curves, tCN was reduced, reaching a minimum at 8 h PI for three cows, and at 12 h PI for the three others. Return to baseline was noticed at 25 or 36 h PI (five cows), except for one cow, where no return to baseline was observed after 52 h PI (Figure 4).

then observed at 12 h PI for three cows, followed by an increase, which reached at least 93% of somatic cells at 16 and 25 h PI. At 76 h PI, percentage of PMN was not different from initial level for all cows. PMN with three to six lobuli were considered as old cells, whereas one to two lobuli PMN were considered as young cells (Figure 2B). Thus, at initial level, old PMN percentage was significantly higher than that of young PMN, with 64 (±2.7) and 36% (±2.7), respectively (average value for six cows). Meanwhile, young PMN percentage increased sharply as soon as 4 h PI. It reached a maximum of 52% (±2.7) at 12 h PI (1.5 times the initial value), simultaneously with the significant drop of the percentage of PMN in somatic cells observed

Figure 4. Total casein content change after intramammary infusion of 10 μg of endotoxin in quarter skimmed milk of cows A (䊐), B (䊏), C (䊊), D (䊉), E (䉭), and F (▲). Journal of Dairy Science Vol. 85, No. 10, 2002

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Parts of αs-, β-, and κ-CN in total CN. Average percentage of αs1- and αs2-CN in total CN before infusion was 43.4% (±3.4). Average maximal decrease of the percentage of αs-CN was −21.8% (±21.9), with maximal individual variations between -8.5 and 60.8%. Individual curves showed a minimum reached at 4 (three cows) or 8 h PI (2 cows). Return to baseline was observed at 64 or 76 h PI (4 cows) except for one cow, where it was noticed at 12 h PI (Figure 5A). The percentage of β-CN averaged 39.5% (±2.7) just before infusion. It decreased with an average maximal variation of β-CN independently from time of −25.8% (±12.7). Individual curves showed a minimum reached at 4 (one cow), 8 (three cows) and 12 h (one cow). No return to baseline was observed after 76 h for all cows (Figure 5B). Before infusion, the percentage of κ-CN (Figure 5C) in total CN averaged 13.3% (±3.1). The percentage of κ-CN after infusion was not different from initial level except at 4 h PI (for four cows), where it increased with an average maximal variation of +53% (±41). Percentage of pH 4.6 insoluble peptides, including γ-CN. Just before infusion, percentage of pH 4.6 insoluble peptides averaged 3.82% (±0.92). Independently from time, the average maximal variation of percentage of pH 4.6 insoluble peptides was +380% (±333). Individual curves showed a maximum increase reached at 8 (four cows) or 12 h (one cow). Return to baseline was observed at 76 h PI for all cows (Figure 5D). Proteose-peptones content. Before infusion, PP content in milk averaged 1.02 g/L (±0.09). The PP content increased with an averaged maximum variation of +144% (±53). According to individual curves, first peak was noticed at 8 h PI (for the six cows) and second peak at 25 (four cows) or 36 h (two cows). The PP content returned to initial levels at 76 h PI for all cows (Figure 6). Electrophoresis SDS-PAGE electrophoregrams showed different patterns in function of the pH tested. In vitro proteolysis of CN with extract of somatic cells can occur at both pH 7.2 and 5.6. At pH 7.2, total CN fraction was strongly hydrolyzed after 12 h of incubation with milk somatic cells, but only from points 4 to 36 h. A maximum was reached at 12 h PI (Figure 7a), whereas proteolysis was very pronounced at pH 5.6 from 4 to 16 h PI (band intensity was lower than that of the reference at 0 h). The fraction then became different at points 25 and 36 h PI by apparition of several bands (Figure 7b). At 52 h the proteolysis is maximum with bands αs- and β-CN barely visible Journal of Dairy Science Vol. 85, No. 10, 2002

Figure 5. αs (A), β- (B), κ- (C), and pH 4.6 insoluble peptides including γ-caseins (D) percentage of total casein after intramammary infusion of 10 μg of endotoxin in quarter skimmed milk of cows A (䊐), B (䊏), C (䊊), D (䊉), and F (▲).

while other bands appeared beneath. The same conclusions were valuable for the six cows. DISCUSSION The response profiles for rectal temperature were in accordance with previous reports (Shuster et al., 1991).

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Figure 6. Proteose peptones fraction content in inflamed quarter skimmed milk after intramammary infusion of 10 μg of endotoxin of cows A (䊐), B (䊏), C (䊊), D (䊉), E (䉭), and F (▲),

Similar responses of SCC have been elicited previously by both subclinical and clinical mild inflammations (Guidry et al., 1983; Shuster et al., 1991). Decrease in total CN content was maximum 8 h after infusion, while it reaches a minimum at 5 h PI according to Kaartinen et al. (1988). At 8 h PI, decrease of total CN content averaged 4.3 g/L, while increase of PP content was only 1.47 g/L. Indeed, reduction in total CN content could be explained both by reduced CN synthesis and proteolysis of CN. In previous work using E. coli experimental mastitis, a sequential proteolysis of αs- and β-CN in milk from infected quarters was observed through time (Michelutti et al., 1999). The current study used a model that mimicked an E. coli mastitis using LPS endotoxin without the whole bacteria. This model permitted to focus on sequential changes in the sources of endogenous proteolysis. Thanks to the absence of any bacterial proteolysis, the respective roles of plasmin and somatic cell proteases can be elucidated. Maximum increases in SCC and plasmin activity did not occur at the same time. SCC peaked initially at 8 or 16 h and again, even if less pronounced, at 16 or 25 h. This pattern agreed with previous reports (Guidry et al., 1983; Verdi and Barbano, 1991a). Percentage of milk PMN was mainly responsible for SCC variations, ¨ stensson (1990). The in accordance with Saad and O SCC response profile can be explained by the initial margination of mature leukocytes and their passage from blood into milk with a subsequent release of mature and immature PMN from bone marrow reserves after stimulation (Paape et al., 1991; Jain, 1993). The significant increase of plasmin activity can be explained by an increased influx of plasminogen from blood to milk (Kaartinen et al., 1988) with its activation into plasmin possibly mediated by somatic cells (Verdi and Barbano, 1991a; Heegaard et al., 1994). Variations of

Figure 7. SDS-PAGE electrophoregrams of casein hydrolysates by somatic cells pellets (107 cells/ml) freshly extracted from the milking kinetics. The proteolysis of caseins is evaluated by electrophoresis with total casein (tCN) as a standard. Cells from points 0 to 76 h postinfusion (PI) have been incubated with a total casein fraction at 37°C during 12 h in sodium acetate of 0.2 M buffer pH 7.2 (Figure 7a) and in sodium acetate 0.2 M buffer pH 5.6 (Figure 7b).

plasmin activity and SCC were not directly related in our study. The contribution of leukocytes to plasminogen activation was not self-evident as far as the kinetic Journal of Dairy Science Vol. 85, No. 10, 2002

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approach showed that plasmin activity declined significantly after 4 to 8 h, while SCC continued to increase. Kaartinen et al. (1988) have explained that the reduction in plasmin activity over 6 h resulted from lower levels of plasminogen contents after 8 h PI. The decrease of plasmin activity can also be explained by varying levels of other components that act as inhibitors or activators of the plasminogen and plasmin system (Grufferty and Fox, 1988; Heegaard et al., 1994). Some studies have suggested that between 200,000 and 1 to-2 × 106 cells/ml, plasmin activity is mainly responsible for CN breakdown (Le Roux et al., 1995a; Saeman et al., 1988). Our study is in agreement with this observation at 4 h PI whereas plasmin activity is maximum and SCC less than 2 × 106 cells/ml: a great caseinolysis was shown through decreases of αs- and βCN percentages, and increases of percentage of pH 4.6 insoluble peptides and PP content. However for SCC greater than 2 × 106 cells/ml, somatic cell proteases could be directly responsible for caseinolysis as suggested by the second peak of proteose-peptones content observed for all cows between 25 and 36 h PI. This peak was not attributed to plasmin activity, which decreased regularly until 52 h PI, but to SCC, which was higher than 5 × 106 cells/ml for the cows between 16 and 36 h PI. Therefore, we hypothesize that in the case of LPS infusion, caseinolysis was initially due to plasmin activity mainly, then to both plasmin activity and somatic cell proteases. From 16 to 52 h PI, caseinolysis was mainly due to cellular proteases with maximum activity at 25 and 36 h PI. The long time required for the return of PP content to initial values could be explained by the persistent elevation of SCC after LPS infusion. Variation in the profiles of individual CN breakdown confirmed the hypothesis of different sources of caseinolytic activity (De Rham and Andrews, 1982), including proteases from somatic cells. There was, however, an exception at 4 h PI where the percentage of κ-CN in total CN content was stable through time while total CN content varied. This result confirmed the potential resistance of κ-CN to the endogenous proteolysis. Peak of κ-CN percentage, noted at 4 h PI was observed previously (Michelutti et al., 1999) and could be explained by the recovery in κ-CN fraction of peptides issued from the proteolysis by plasmin of β- and αs2-CN. These peptides could have been eluted together with κ-CN by anion-exchange chromatography. Indeed, in vitro studies have shown that rate of proteolysis by plasmin is in the order β-, αs2- > αs1- >> κ-CN, whereas it is αs1- > β>> κ-CN for somatic cell enzymes (Grieve and Kitchen, 1985). Somatic cell proteolysis is mainly attributed to PMN. However, actions of PMN proteases have received limited study (Verdi and Barbano, 1991a, 1991b) and the rate of proteolysis of αs2-CN by somatic cells Journal of Dairy Science Vol. 85, No. 10, 2002

has not yet been described. Moreover, a concentration of αs1-CN that is three- to fivefold higher than that of αs2-CN in normal milk (Ribadeau-Dumas and Grappin, 1989) could explain, in part, the different impact of proteolysis on global αs-CN proportion that is dependent upon the source of proteolysis. While plasmin is partly linked to CN micelles (Grufferty and Fox, 1988), somatic cell proteases are located in granules of mature PMN and must be released from the cells. The fact that bacterial toxins can induce lysosomal rupture or generalized degranulation of PMN (Jain, 1993) might explain the caseinolysis that was noticed. Therefore, exposure of PMN to cytokines and chemoattractants results in rapid mobilization of azurophil granules (containing elastase and cathepsin G mainly) to the cell surface. As far as acidic proteases such as cathepsin D, it is possible that at sites of inflammation, the pericellular pH may be sufficiently low to allow cathepsin D to degrade CN (Owen and Campbell, 1999). In addition, it can be hypothesized that small CN fragments issued from previous degradation in milk by neutral proteases could be engulfed in PMN and submitted to another hydrolysis in the acidic milieu of the phagolysosomal compartment. Recently, Larsen et al. (1996) reported that bovine cathepsin D in milk is able to cleave all CN in a pattern different from that of plasmin. This indigenous enzyme is present in the primary granules of mature PMN (Jain, 1993). Differences in maturity of PMN could also have influenced the proteolytic capacities of the cells (Owen and Campbell, 1999; Bank and Ansorge, 2001). Thus, with incubating CN and somatic cells at two pH (7.2 and 5.6), two protease groups have been tested: neutral (serine proteases such as elastase and cathepsin G and metalloproteases such as collagenase) and acidic proteases (aspartic protease such as cathepsin D). All samples had the same PMN concentration (107 cells/ml) which supposed that differences regarding to proteolytic pattern on electrophoregrams could only be attributed to cell type, and explicitly to PMN stage of maturation. In this context, indentations of the nucleus (two and more lobuli) are features of mature neutrophils, and the more it is segmented and the more the cell stage of maturity is high. During an inflammatory reaction, there is an influx of juvenile cells as a result of the bone marrow stimulation (Jain, 1993). Because changes in neutrophil maturity commonly are associated with inflammation, an LPS infusion induces large numbers of circulating immature and juvenile PMN that are stimulated by activators (McDermott and Fenwick, 1992). These activated PMN have also an increased enzymatic activity not resulting in de novo synthesis of the enzymes but rather from a boost of enzymatic activities from both lysosomes and granules

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(McDermott and Fenwick, 1992; Karimbakas et al., 1998). The cytokines secreted during an inflammation involved by LPS are responsible of both degranulation of PMN and lysosomal rupture in a lesser extent leading to a greater proteolytic activity (Jain, 1993; Owen and Campbell, 1999). At pH 7.2, significant proteolysis was noticed during the increase of percentage of young PMN (between 4 and 16 h PI). On the contrary, at pH 5.6, a steady proteolysis (in comparison with the reference at 0 h and with total CN sample) was noticed from 4 to 16 h PI and then became different at points 25 and 36 h PI because of an apparition of several bands. As far as PMN proteolytic activity at acidic pH, no reference in literature could have explained or supported the phenomenon observed at point 52 h. In vitro PMN proteolytic activity was representative of the caseinolysis in vitro as far as after membrane rupture; all proteases were released in the medium containing CN. The neutral pH in vitro that was close to mastitis milk pH recreated the situation of a degranulation. The acidic pH in vitro mimicked the lysosomal environment. The incubation time of 12 h recreated the lag between two following regular milkings when cellular proteases were in contact with CN. At neutral pH, no proteolytic activity was noticed with a basic percentage of old PMN (the most mature). However, proteolysis became effective during the increase of younger PMN number and also during the acute phase of the inflammatory reaction, which means that collagenase, elastase and cathepsin G might have been involved in extracellular proteolysis and during degranulation (Paape et al., 1991; Bank and Ansorge, 2001). As for acidic PMN proteases (e.g., cathepsin D) whose activity is effective after PMN stimulation but more pronounced with a basic percentage of old PMN, the main role is intracellular protein digestion, which occurs within the acidic environment of lysosomes (Owen and Campbell, 1999). Furthermore, events that occur during leukocyte chemotaxis could explain why somatic cells from blood and milk have different proteolytic capacities (Verdi and Barbano, 1991b; Fang et al., 1996). Casein breakdown takes place in the udder before milking, and it must be considered when interpreting CN proteolysis kinetics. During the inflammation of the udder, the increase of temperature (fever) couldn’t have had a significant effect on proteolysis as far as a negative effect was expected (decrease of proteolytic activity), while plasmin activity and PP content reached a maximum after the peak of body temperature. Furthermore, milk from unchallenged quarters didn’t show any significant variation during the kinetic for all parameters (data not shown). Location of milk storage in the udder might also be important. At 4-h milking inter-

vals, 90% of the recovered milk had been stored in the alveolar lumen (Knight et al., 1994). Thus, proteolysis from 4 h until 25 h PI could be underestimated since the reduced milking intervals would have decreased the length of milk storage in the udder and thereby decreased the length of contact between CN and proteases. Nevertheless, the irregular milking intervals didn’t have any impact on proteolysis in our findings according to previous study (Michelutti et al., 1999) on the one hand and to results for all parameters obtained from milk from the homolateral quarter in the other hand (data not shown). CONCLUSIONS Variation of protein composition of quarter milk after LPS intramammary infusion highlighted the sequential origins of endogenous proteolysis of CN, as well as the direct impact of somatic cell enzymes on caseinolysis. This study confirmed that proteolysis due to plasmin activity occurred mainly in the very early acute stage of inflammation, while a maximum plasmin activity is concomitant with SCC < 1 to 2 106 cells/ml. Then, caseinolysis is explained by both plasmin activity and somatic cell proteases, being mainly due to somatic cell proteases during recovery stage. Accurate assignment of caseinolysis products to plasmin activity or somatic cell enzymes is, however, not possible, as some products could be due to both sources of proteolysis. During the inflammatory process, a massive recruitment of immature PMN and cell stimulations can be involved in a mechanism of degranulation responsible of extracellular caseinolysis by neutral proteases such as elastase, collagenase, and cathepsin G. Beside this, mature PMN contained in their lysosomes acidic proteases like cathepsin D responsible of an intracellular caseinolysis. This CN breakdown was accompanied by a general reduction in CN content in milk from all quarters, which suggested reduced protein synthesis in addition of caseinolysis as it was previously observed in LPS and E. coli models. Further studies are required to determine the precise enzymatic roles of somatic cells in CN breakdown, using identification of some peptides in PP fraction resulting of cellular proteases during the acute phase of inflammation, and following specific enzymatic activities through the same kinetic. Also, blood proteolytic enzymes activities, which are different from that of plasmin, should be pursued. Finally, similar studies should be conducted in case of milk from quarters infected by various major pathogens in order to compare kinetics parameters regarding to their magnitude, mechanisms, and persistence. Journal of Dairy Science Vol. 85, No. 10, 2002

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