Phagocytic Activity of Milk Leukocytes During Chronic Staphylococcal Mastitis

Phagocytic Activity of Milk Leukocytes During Chronic Staphylococcal MastitisABSTRACT
Receptors for lgG on milk leukocytes were detected by rosette formation using sensitized erythrocytes. The percentage (41) of milk leukocytes from uninfected glands forming sensitized erythrocyte rosettes was significantly greater than the percentage (13) of leukocytes from glands with chronic staphylococcal mas- titis. A greater percentage of polymor- phonuclear leukocytes than macrophages formed sensitized erythrocyte rosettes, regardless of the infection status of the gland from which they were obtained. Both IgG-receptor and nonimmunologic r e c e p t o r - m e d i a t e d phagocytosis were greater for milk leukocytes from un- infected glands than for milk leukocytes from chronically infected glands. Pre- incubation of normal milk leukocytes in whey prepared from mastitic milk re- sulted in a decrease in their capacity to form sensitized erythrocyte rosettes as well as a reduction in their phagocytic capacity. Immune complexes prepared in vitro also reduced the phagocytic capa- city of normal milk leukocytes and inhibited their capacity to form sensitized erythrocyte rosettes. These data indicate a factor, possibly consisting of immune complexes, was present in secretions from glands chronically infected with staph- ylococci. This factor reduced the phago- cytic capacity of milk leukocytes. •
Received April 2, 1987. Accepted September 30, 1987.
Present address: Faculty of Veterinary Medicine, Department of Microbiology, Warsaw Agricultural University, Warsaw, Poland.
2 Reprint requests. 3 Deceased.
M. N I E M I A L T O W S K I , 1 B. J. NONNECKE, 2 and S. P. TARGOWSKI 3 National Animal Disease Center
Agricultural Research Service US Department of Agriculture
Ames, IA 50010
I N T R O D U C T I O N
Staphylococcus aureus is a frequent cause of chronic mastitis in dairy cows (13). After a short, acute phase, the infection usually be- comes chronic. The cow produces a small amount of antibody against bacterial cell-welt components (peptidoglycan, teichoic acid) and a relatively large amount of antibody against bacterial toxins and metabolites during the chronic stage preventing the development of gangrenous mastitis (1).
Macrophages and polymorphonuclear leu- kocytes (PMNL) constitute the first line of defense against intramammary infection by staphylococci. These leukocytes phagocytize bacteria via 1) receptors for the Fc portion of IgG (FcR), 2) nonimmunological receptors, and 3) receptors for complement (C3) (11, 12). Antibodies against cellular components may opsonize staphylococci, enhancing FcR-medi- ated phagocytosis. In the absence of specific antibody, bacteria can be phagocytized by n o n i m m u n o l o g i c a l r e c e p t o r s . Unopsonized S. aureus, strain 305, can be phagocytized via these receptors. However, some strains of S. aureus (i.e., encapsulated strain Smith) can be phagocytized only after opsonization (7). The role of complement in phagocytic processes by milk leukocytes is probably insignificant because bovine milk contains a negligible amount of complement (5).
Staphylococci often persist, despite the presence of antibody and phagocytes, in secretions from infected glands. Therefore, a factor in mastitic milk similar to that previously detected in secretion from cavian mammary glands inflamed by the infusion of lipopolysac- charide (7) may inhibit phagocytosis of staph- ylococci by blocking the FcR on the surface of the PMNL or macrophage. This factor may consist of antigen-antibody complexes referred to as immune complexes (IC). Endogenous as well as exogenous antigens can trigger IC formation. The IC, through the Fc portion Ig
1988 J Dairy Sci 71:780-787 780
PHAGOCYTIC FUNCTION 781
in the complex, can bind to the FcR of the phagocyte potentially- masking the recognition system of the phagocyte (4, 9). This study, investigated phagocytosis mediated by' FcR
and nonimmunological receptors on milk leukocytes from mammary glands chronically infected with S. a~treus.
M A T E R I A L S A N D METHODS
Milk Samples
Ten Holstein-Friesian cows in midlactation were used. Five cows were experimental- ly infected in a single gland with S. aureus
(strain 305, ATCC 27940), resulting in the establishment of a chronic infection. Five uninfected cows served as controls. Milk samples were collected aseptically" on 3 con- secutive d from both mastitic and control cows. Milk was diluted 1:3 with sterile phosphate- buffered saline (PBS) and centrifuged (250 y. g for 10 min) to separate milk. Leukocytes were washed three times in PBS and subsequently used in various assays. Approximately" 80% of the leukocytes were viable as determined by trypan-blue dye exclusion. Milk leukocyte smears were stained with Star-stain (Volu-Sol. Medical Industries, Inc., Las Vegas, NV) and examined microscopically" (200 cells/smear) for differential leukocyte counts by" the same observer throughout the study'. One hundred microliters of milk were inoculated onto blood agar plates which were subsequently" incubated at 37°C for 24 h. Colony-forming units of bacteria//al of milk were determined. Whey was prepared from milk by" precipitating casein with acid, as previously described (7).
Detection of Fc Receptors on Milk Leukocytes
Sheep erythrocytes (E) were washed three times with PBS. Erythrocytes were mixed with a subagglutinating dose of rabbit antieryth- rocyte-IgG (Cordis Laboratories, Miami, FLy. These sensitized ervthrocytes (EA) (2% vol/vol) were incubated (37°C for 45 min), washed three times with PBS, and resuspended in PBS to a final concentration of .5% vol/vol (6).
Milk leukocytes were distributed into two sets (triplicate) of f lat-bottom wells of micro- ti ter plates (Becton & Dickinson Company, Oxnard, CA). Fif ty microliters of leukocytes (1 x 1061ml) were mixed with 50 bd of either EA
(set 1) or E (set 2) and centrifuged (500 x g for 5 rain). Pelleted ceils were incubated 1 h at 4°C and subsequently' resuspended. Rosette-forming cells (3 or more erythrocytes/cell) /100 cells were determined microscopically. The per- centage of PMNL, macrophages, or lympho- cvtes forming EA rosettes were determined from differential smears prepared from milk leukocyte suspensions incubated with EA or E.
Detection of Blocking Factor in Milk
Immune complexes in wheys prepared from normal and mastitic milk were detected by" a procedure of Targowski and Milgram (6). Briefly-, routine lymphocyt ic leukemia L 1210 cells (ATCC CCL 219, a cell line expressing FcR), at a concentration of 5 × 106/mI (20 #i), were mixed with whey or its dilutions. Mixtures were incubated (4°C for 1 h), washed in PBS, and 50 ~1 of EA were added. Preparations were mixed and centrifuged (500 x g for 5 rain). Incubation of L 1210 cells with either in- activated serum (56°C for 30 min) or 1C formed in vitro (ovalbumin and bovine an- tiovalbumin serum) served as negative and positive controls, respectively-. Cells were incubated at 4°C for 1 h, gently" resuspended, and rosette-forming cells (three or more EA/- cell)/100 cells determined microscopically-.
Blocking of Fc Receptors by Mastitic Milk
Normal and mastitic wheys, undiluted and diluted in PBS at 1:2. 1:4, 1:8, and 1:16 (150 ~l), were mixed with 50 /ll of suspended leukocvtes (5 × 106 cells/ml) from normal milk. Mixtures were then incubated at 4°C for 1 h, washed in PBS, and 50 #1 of EA were added. Preparations were mixed, centrifuged (500 × g for 5 rain), incubated (4°C for 1 h), and gently" suspended. Rosette-forming cells/100 milk cells were determined. Smears from each suspension were prepared, stained, and differential cell counts performed.
In Vitro Preparation of Immune Complexes
Whey (source of lacteal antigen) from normal bovine milk and sera (source of anti- body) from cows with mastitis were used to generate IC (3, 7). Heat-inactivated serum was mixed with an equal volume of heat-inactivated
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782 NIEMIALTOWSKI ET AL.
whey subsequently incubated in a water bath (37°C for 40 min) and held overnight at 4°C. These preparations were mixed thoroughly after overnight incubation and used to block FcR on milk leukocytes.
Inhibition of Phagocytosis by Mastitic Milk
Leukocytes (50 /~1 of 5 × 106/ml) from normal and mastitic milk were distributed into flat-bottom wells of a microculture plate. To demonstrate phagocytosis mediated by FcR (set 1), 50 /ll of heat-killed S. aureus
(Smith strain, ATCC 1309, 1 × 107 cfu/ml) preopsonized with heat-inactivated bovine antistaphylococcal serum at dilution of 1:10 were added to each well. To demonstrate phagocytosis mediated by nonimmunological receptors (set 2), 50 /11 of heat-killed, non- opsonized S. aureus (strain 305, ATCC 27940, 1 × 107 cfu/ml) were added. Cultures were prepared in triplicate for each determination.
Inhibition of phagocytosis by mastitic milk or in vitro prepared 1C was determined as follows. Leukocytes (50 /al of 5 × 10 6 ceils/ ml) from normal milk were distributed into fiat-bottom wells. One hundred fifty microliters of undiluted or diluted normal or mastitic whey, or in vitro prepared blocking factor, were added to each well, mixed, and incubated at 4°C for 1 h. Opsonized or unopsonized staph- ylococci were added.
Microculture plates were centrifuged (500 × g for 5 min) and incubated (37°C for 30 min). Lysostaphin (32 IU; Sigma Chemical Co., St. Louis, MO) was added to each well, and plates
were incubated an additional 30 min at 37°C to lyse extracellular staphylococci. Cultures were subsequently washed and smears prepared. The percentage of leukocytes phagocytizing bacteria was calculated from observations on 100 cells. Phagocytized bacteria/lOO cells were also determined.
Statistical Analysis
Results were expressed as the mean -+ standard deviation. Significance was evaluated by Student's t test.
RESULTS
Milk Samples
Twenty-four milk samples from normal mammary glands with 1.7 × l0 s milk cells/ml or less served as controls, and 15 samples of milk from infected glands with 3.3 × 106/ml or more milk cells represented samples of mastitic milk. The cellular composition of milk from normal and infected glands is shown in Table 1. Bacteria were not isolated from normal milk, whereas 216 -+ 254 cfu S. aureus were cultured/ .1 ml of mastitic milk.
Blockage of FeReceptors on Normal Milk Leukocytes by Mastitic Whey
Leukocytes from normal milk formed more EA rosettes (P<.01) than celts from mastitic milk (41% vs. 13%). Polymorphonuclear leukocytes were the principal cells in normal and mastitic milk forming rosettes (Table 2). Rosette formation by normal milk leukocytes was blocked (P<.01) by preincubating them in whey from mastitic milk (Figure 1) or IC
TABLE 1. Characteristic of leukocytes obtained from normal and mastitic milk.
Differential cell counts 1 No. of 96 of Viable Milk samples cells/mt (X l0 s ) cells % PMNL % Macrophages % SML
Normal (n = 24) Mastitic
SE X SE X SE R SE X SE 1.7 .4 a 86 4 50 8 a 37 12 a 13 8
3.3 .9 b 93 3 87 5 b 5 3 b 8 3
a'bvalues in columns with different superscripts differ (P<.01). 1Epithelial cells were less than 1%, PMNL = polymorphonuclear leukocytes, SML = small mononuclear
leukocytes.
PItAGOCYTIC FUNCTION
TABLE 2. Roset te formation by milk leukocytes f rom normal and infected glands, x
783
Milk % Roset te format ion oo EA Roset te formation
samples E EA PMNL Macrophages SML
X, SE X SE X SE X, SE X, SE
Normal 1 1 41 2 a 37 4 a 3 1 6 4 (n = 24)
Mastitis 0 13 6 b 8 1 b 1 1 2 1 (n = 15)
a ' bva lues in columns with different superscripts differ (P<.01).
Z Calculated from observation of 200 polymorphonuclear leukocytes (PML), 200 macrophages, or 200 small mononuclear leukocytes (SML). E = Sheep erythrocytes, EA = erythrocytes mixed with antierythrocyte- lgG.
prepared in vitro (Figure 2). Preincubation of leukocytes in whey from uninfected glands did not reduce rosette formation (Figure 2).
Presence of Immune Complexes in Mastitic Milk
Immune complexes in mastitic milk were detected by the inhibition of rosette formation by L 1210 cells (a cell line expressing FcR). Immune complexes were detected in all masti- tic milk wheys up to a dilution of 1:8. In c o n t r a s t , IC w e r e n o t d e t e c t e d in u n d i l u t e d ,
Do 6O w
0 ~ m NORMAL UNDILUTED 1:2 1:4 MILK MASTITIC MILK
Figure 1. Percentage (X ± SD) o f normal milk leukocytes forming sensitized erythrocyte rosettes after preincubation in whey f rom normal or mastit ic milk.
n o r m a l m i lk w h e y . I m m u n e c o m p l e x e s p re - p a r e d in v i t ro w e r e d e t e c t e d a t a d i l u t i o n o f 1 : 1 6 (5 s a m p l e s ) b u t n o t in u n d i l u t e d sera o r w h e y s u s e d to g e n e r a t e t h e IC.
FcR-Mediated Phagocytosis and Its Inhibition
Percentages of phagocytizing PMNL and macrophages and numbers of bacteria phago- cytized/PMNL and macroptaages (phagocytic activities) from normal milk were greater
' ° °31 .
PBS WHEY SERUM BLOCKING FACTOR
Figure 2. Percentage (X _+ SD) of normal milk leukocytes forming sensitized erythrocyte rosettes after preincubation in phosphate-buffered saline, whey derived from normal milk, sera from mastit ic cows, and immune complexes prepared in vitro. The term "blocking factor" refers to immune com- plexes prepared in vitro.
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784 NIEMIALTOWSKI ET AL.
No. phagocytized Source of % Phagocytizing staphylococci/lO0 leukocytes leukocytes PMNL Macrophages PMNL Macrophages
Normal milk (n = 24)
Mastitic milk (n = 15)
SE X SE X SE X SE 77 5 a 27 4 a 554 40 a 158 36 a
24 8 b 2 2 b 77 40 b 3 3 b
a 'bvalues in columns with different superscripts differ (P<.01).
( P < . 0 1 ) t h a n for l eukocy te s o b t a i n e d f r o m mast i t ic milk (Table 3). Phagocy t ic act ivi t ies of PMNL and mac rophages f rom n o r m a l milk were inh ib i t ed by p r e incuba t i ng t h e m wi th w h e y f rom mast i t ic milk or IC prepared in vi t ro, b u t n o t wi th n o r m a l milk w h e y (Tables 4 and 5). I n h i b i t i o n of phagocy tos i s by d i lu t ions of mas t i t i c w h e y was no t evident .
Nonimmunologic Receptor-Mediated Phagocytosis by Normal and Mastitic Milk Leukocytes
Phagocytos i s of u n o p s o n i z e d s t aphy lococc i by Ieukocy tes f rom n o r m a l and mas t i t i c mi lk is compared in Table 6. Percentages of phago- cyt iz ing PMNL and m ac r ophages and t h e
n u m b e r of bac te r ia phagocy t i zed by PMNL and mac rophages f rom n o r m a l mi lk were grea ter ( P < . 0 1 ) t han for l eukocy tes f rom mas t i t i c milk. Phagocytos is was inh ib i t ed b y p re incuba- t ion of l eukocy tes f r o m n o r m a l mi lk wi th IC prepared in vi t ro (Table 7).
D ISCUSSION
In the p resen t s tudy , FcR or n o n i m m u n o l o g i c r ecep to r -med ia t ed phagocy tos i s by milk leuko- cytes f rom glands wi th ch ron ic S. aureus
mast i t i s was reduced f rom t h a t of milk leu- kocy te s f r o m n o n i n f e c t e d glands. I nh ib i t i on of F c R - m e d i a t e d phagocy tos i s by mas t i t i c mi lk l eukocy tes was associated wi th a r e d u c t i o n in the i r capac i ty to f o r m EA rose t tes in vi t ro. We
TABLE 4. Effect of milk whey from normal or infected glands on FcR-mediated phagocytosis by normal milk polymorphonuclear leukocytes (PMNL) and macrophages.
Preincubation of leukocytes with whey or its dilutions
% Phagocytizing No. phagocytized
staphylococci/100 leukocytes PMNL Macrophages PMNL Macrophages
SE X SE .X SE X SE Whey from normal milk 76 4 a 25 3 543 37 a 170 8 a (n = 24)
Whey from mastitic milk 54 5 b 17 2 288 15 b 68 18 b (n = 15)
Dilution 1:2 71 3 a 26 2 455 34 a 138 15 a Dilution 1:4 75 3 a 25 2 523 19 a 158 7 a
a 'bvalues in columns with different superscripts differ (P<.05).
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PHAGOCYTIC FUNCTION 785
TABLE 5. Effect of immune complexes prepared in vitro on FcR-mediated phagocytosis by normal milk poly- morphonuclear leukocytes (PMNL) and macrophages.
No. phagocytized Preincubation of % Phagocytizing staphylococci/100 leukocytes leukocytes with PMNL Macrophages PMNL Macrophages
X SE ,X SE X SE X, SE PBS ~ 62 10 a 24 4 a 393 31 a 68 10 a (n = 3)
Whey 54 3 a 21 3 a 342 20 a 51 7 a (n = 3)
Serum 50 7 a 17 3 a 305 37 a 48 5 a (n = 3)
Immune complexes 9 1 b 4 1 b 39 4 b 12 i b (n = 3)
a 'bvalues in columns with different superscripts differ (P<.01). 1 Phosphate-buffered saline.
have previously d e m o n s t r a t e d t h a t F c R on phagocy t i c cells are b locked by IC presen t in milk f rom glands m a d e mas t i t i c by i n t r amam- mary in fus ion of l ipopolysacchar ide (7). In a s imilar fashion, FcR on milk l eukocy tes f rom glands chron ica l ly in fec ted wi th S. aureus
may be b locked by IC in the whey f rac t ion of the milk. Such a b lockage would impai r the phagocy t i c f u n c t i o n of the milk leukocytes , p reven t ing the e l imina t ion o f the bac ter ia f rom the gland. A similar b lockage of FcR on phago- cyt ic cells f r om pa t i en t s wi th IC disease has been observed (9).
Phagocytos is of opson ized s taphy lococc i by no rma l milk l eukocy tes was reduced by pre- i ncuba t ing t h e m wi th w h e y f rom chronica l ly…