Immunology 1976 31 159

The effect of cytophilic IgG2 on phagocytosis by ovine polymorphonuclear leucocytes D. L. WATSON Veterinary Research Station, Glenfield, New South Wales, Australia

Received 24 September 1975; accepted for publication 2 February 1976

Summary. Non-lactating, multiparous ewes were immunized either by subcutaneous infection with live Staphylococcus aureus (seventeen ewes) or by intramuscular injections of a killed S. aureusoil adjuvant vaccine (seventeen ewes). Polymorphs which were subsequently collected from the mammary glands of the animals were used in in vitro phagocytosis assays against Pseudomonas sp. or S. aureus. There was no difference between polymorphs from the two groups of ewes in their ability to phagocytose Pseudomonas organisms. Polymorphs from the infected ewes showed significant phagocytic superiority over cells from ewes given the killed vaccine when S. aureus was the target organism. This phagocytic superiority could be abrogated by removal of cytophilic immunoglobulin from polymorphs and restored by replacement of cytophilic immunoglobulin. It was shown by staining polymorphs with FITC-conjugated anti-immunoglobulin sera that cytophilic immunoglobulin on the surface of polymorphs belonged to the IgG2 class of immunoglobulins. When 'neutral' polymorphs (from non-immunized ewes) were coated with IgG2 purified from the sera of infected ewes, they exhibited enhanced phagocytosis of staphylococci compared with 'neutral' polymorphs carrying IgG2 from the sera of ewes given the killed vaccine.

which suggested that polymorphonuclear leucocytes collected from ewes which had been infected with Staphylococcus aureus as well as receiving a killed S. aureus vaccine, exhibited enhanced phagocytosis of staphylococci in vitro. Phagocytosis by these cells was enhanced in comparison with polymorphs collected from ewes which had received only the killed S. aureus vaccine (Watson, 1975). These results raised the question of a mechanism for the observed enhancement of phagocytosis. One possibility was that polymorphs may be 'activated' by specifically committed lymphocytes in a manner analogous to the activation of macrophages during infection with intracellular bacteria (Mackaness, 1971). Alternatively, it was considered that cytophilic antibody on the surface of polymorphs may have a role in enhancement of phagocytosis (Fidalgo and Najjar, 1967). The present studies were designed to examine the hypothesis that enhanced phagocytosis by polymorphs from infected ewes was mediated by cytophilic antibody on the surface of these cells. To simplify interpretation of the results, ewes in these experiments were either infected with staphylococci or given the killed vaccine.

INTRODUCTION previous publication evidence was presented

Experimental animals A total of thirty-four non-lactating, non-pregnant, multiparous ewes were randomly allocated to two treatment groups. Only ewes which had ceased lactating at least 4 months previously were used in




Present address and correspondence: Department of Tropical Veterinary Science, James Cook University of North Queensland, Queensland 4811, Australia.



D. L. Watson

the experiments. They were kept in pens and fed a mixture of lucerne chaff, sorghum and oats. Bacteria and vaccine A coagulase positive strain of Staphylococcus aureus isolated from a case of acute clinical mastitis in a dairy cow was used in the experiments. The organisms were maintained on nutrient agar slopes and grown for 24 h in nutrient broth when required. A freeze-dried culture of Pseudomonas sp. was grown in nutrient broth for 24 h prior to dilution for use in the in vitro assay. The number of bacteria in broth cultures was determined spectrophotometrically. The oil adjuvant-S. aureus vaccine was prepared as described by McDowell and Watson (1974). An overnight broth culture of the bacteria was boiled in a water bath for 2 h. The killed culture contained 1 5 x 109 organisms per millilitre. Each dose of vaccine contained 2-85 ml of killed culture, 0 15 ml Tween 80, 6 3 ml Marcol 52 (Esso Petroleum Company) and 0 7 ml Mannide monooleate

employed. Briefly, the polymorphs were washed (x2) in Tris-Hanks's balanced salt solution containing 01 per cent gelatine (HBSS) and after counting the cells in a haemocytometer a suspension of 20 x 106 polymorphs per millilitre was prepared. A suspension of live S. aureus containing 107 organisms per millilitre was made by diluting a thrice washed broth culture in 0-01 per cent bovine albumin water. Mixtures of the staphylococci and polymorphs were made up in sterile, siliconed bijou bottles and sealed. Incubation mixtures were made up as follows:

Bacterial suspension Polymorph suspension HBSS

Test mixtures

Bacterial control



01 0-5 04

01 09

(Sigma). Immunization procedures The seventeen ewes in group 1 were injected subcutaneously in each hind leg with 5 x 108 live, washed ( x 3) staphylococci in 1 ml of sterile, isotonic saline. On the same day ewes in group 2 were immunized intramuscularly in each hind leg with 10 ml of the oil adjuvant vaccine. Two weeks later the ewes in group 2 were again immunized, intramuscularly in the forelegs, with the oil adjuvant vaccine.

Sampling Eight weeks after the initial immunization, both mammary glands of each ewe were infused with 10 ml sterile, isotonic saline containing 5 pg bacterial endotoxin. Polymorphonuclear leucocytes were collected by flushing out the mammary glands with cold saline 12 h later (Watson, 1975). Blood samples were collected from the animals at this time and serum prepared. For sampling, each ewe in group 1 was randomly paired with a ewe in group 2. All subsequent laboratory assays were carried out on the paired samples. Assay of phagocytosis The standard assay for in vitro phagocytosis of staphylococci using normal, washed polymorphs (schedule A), as described by Watson (1975) was

The incubation mixtures were gently agitated in a shaking water bath at 370 for 1 h, after which pour plates were prepared and the number of bacterial colonies counted 24 h later (McDowell and Lascelles, 1971). As was shown previously, this assay provides a direct measure of intracellular killing (Watson, 1975). The assay for specificity of phagocytosis was similarly performed, using Pseudomonas sp. (106/ml) in place of S. aureus.

Removal of cytophilic immunoglobulin A suspension of polymorphs (20 x 106/ml) in HBSS was incubated for I h at 370 (Hsu and Mayo, 1973). The cells were then washed twice in cold HBSS

(schedule B). Alternatively, removal of cytophilic immunoglobulin was achieved by stripping with 0-25 per cent pronase (Koch-Light Laboratories Ltd) in HBSS according to the method of Jones, Cebra and Herzenberg (1973). The calls were then washed twice in cold HBSS (schedule E).

Replacement of cytophilic immunoglobulin Polymorphs from which cytophilic immunoglobulin had been removed by incubation at 370 were incubated at similar concentration in 5 ml autologous serum (schedule C) or pure ovine IgG2 (5 mg/ml)

Cytophilic IgG2 and phagocytosis by polymorphs for 20 min at 40 (Hsu and Mayo, 1973). These cells were then washed twice in cold HBSS.

Digestion of cytophilic immunoglobulin The Fab' fragments were removed from cytophilic immunoglobulin on the surface of polymorphs by incubation in HBSS containing 0 25 per cent trypsin (BDH Chemicals Ltd) according to the method of Hsu and Mayo (1973). The cells were then washed twice in cold HBSS (schedule D). Immunofluorescence studies Rabbit anti-ovine serum (RAS) and monospecific rabbit anti-ovine yl (RAyl), anti-ovine y2 (RAy2), anti-ovine a (RAa) and anti-ovine p (RAu) were prepared (Watson, Brandon and Lascelles, 1972). Pure ovine light chains were prepared from IgGI by reduction and alkylation according to the method of Fleischman, Pain and Porter (1962). A preparation of Fab' fragments was prepared by trypsin digestion (Schrohenloher, 1963). Antisera to the light chain (RALC) and Fab' (RAFab) preparations were raised in rabbits (Watson et al., 1972). The IgG in normal rabbit serum and in each antiserum was conjugated with fluorescein isothiocyanate (FITC) as described by Lee and Lascelles (1970). Preparations of polymorphs were stained with FITC-conjugated antisera (Hsu and Mayo, 1973) and the number of fluorescing cells per microscopic field (magnification x 400) was counted and expressed as a percentage of the total number of polymorphs per field. To test the specificity of fluorescence, the following procedure was adopted for each series of stainings. Preparations were treated with: (a) conjugated normal rabbit serum; (b) first treated with unconjugated specific antiserum then with specific conjugated antiserum; (c) first treated with unconjugated normal rabbit serum then with specific conjugated antiserum; and (d) specific conjugated antiserum. Only those preparations which showed fluorescence with (c) and (d), and which failed to fluoresce with (a) and (b) were regarded as showing specific fluorescence (Lee and Lascelles, 1970). Preparation of IgG2 Pure IgG2 was prepared from the sera of twelve ewes (six from each group) by fractionation on DEAE-Sephadex (Pharmacia) as described by


Mackenzie (1968). Purity of the preparations was tested by immunoelectrophoresis and double diffusion in agar using monospecific antisera (Watson et al., 1972).

Antibody assays Anti-staphylococcal direct agglutination assays were carried out on serial 2-fold dilutions of pure IgG2 (5 mg/ml) using phosphate-buffered saline, pH 7-2, containing 1:2500 sodium azide (PBS) as diluent. Staphylococci for use in the direct agglutination assay were grown on mannitol-salt agar and under these conditions of culture protein A does not develop (Haukenes, 1967; Lind, 1968), thereby precluding non-specific attachment to the bacteria of the Fc region of IgG. The organisms were killed with 1 per cent formalin (24 h at 370) and washed six times in PBS. Finally, a suspension containing 0*5 per cent staphylococci (v/v) was prepared in PBS (McDowell and Lascelles, 1971).

Determination of cell viability Polymorph viability was measured, after treatment in each of the schedules, by the eosin Y exclusion assay (Hanks and Wallace, 1958). RESULTS

Clinical findings after immunization Sixteen of the ewes from group 1 were slightly lame for 4-5 days after infection and developed small, chronic, suppurating abscesses at the site of injection. The remaining ewe in group 1 developed neither abscesses at the injection sites nor lameness. Four of the ewes in group 2 showed severe lameness in the forelegs for 4-5 weeks after the second injection of adjuvant vaccine. There were no obvious local reactions at the injection sites in any of the animals in group 2. Assays for specificity of phagocytic enhancement Polymorphs from four pairs of ewes were used in the in vitro assays for phagocytosis with Pseudomonas sp. as the target organism. The number of bacteria surviving with polymorphs from group 1 ewes was 3201 ± 1573 (mean ± s.e.) and for group 2 ewes was 3570 ± 1743. There was no significant difference between polymorphs from the two groups of ewes, in their ability to phagocytose Pseudomonas sp.


D. L. Watson

In vitro assays with staphyloccoci Results for in vitro assays for phagocytosis of staphylococci, after polymorphs were subjected to different treatments are shown in Table 1. The numbers of bacteria surviving after incubation with washed, untreated polymorphs from group 1 ewes were significantly less than for comparable polymorphs from group 2 ewes (schedule A). When Table 1. Colony counts (mean+ s.e.) of S. aureus in pour plates prepared after phagocytosis assays using polymorphs which had been subiected to various treatments

Schedule A: washed, untreated polymorphs (6) Schedule B: cytophilic immunoglobulin removed by incubation (6) Schedule C: cytophilic immunoglobulin replaced (6) Schedule D: trypsin digestion (4) Schedule E: pronase stripping

Group 1 Infected

Group 2 Killed vaccine

Difference Paired t-test

215+ 147

787+ 298





677+ 404

1415+ 535


9635+ 1631

5152+ 1626


6080+ 1033 5940+ 1076


infected ewes, in reducing the number of surviving bacteria, was restored. When Fab' fragments of cytophilic immunoglobulin were removed by trypsin digestion (schedule D), no differences in phagocytic ability were observed between polymorphs from the two groups. Similarly, when pronase was used to strip immunoglobulin from the surface of cells (schedule E), there was no difference between the two groups in the mean number of bacteria surviving. The use of either enzyme resulted in greater absolute numbers of bacteria surviving than with other treatments. Polymorphs from the one ewe in group 1 which did not develop subcutaneous abscesses after infection, exhibited no phagocytic enhancement in the schedule A assay, compared with cells from the group 2 partner ewe. Indeed, cells from the group 2 ewe were slightly superior in the in vitro assay. This pair of ewes was therefore considered unrepresentative and the results were not included in any of the mean values. Cell viability

Polymorphs were tested for viability after treatment in each of the schedules and in every case no less than 95 per cent of cells excluded the dye. Cells treated with trypsin however, showed a considerable degree of clumping, a feature not observed with other treatments.

(4) * P< 0 05; t P< 0 01. Figures in parentheses are the number of pairs of ewes in each schedule. Each incubation mixture contained 106 staphylococci. Bacteria: polymorph ratio = 1:10. Maximum standardization of techniques was maintained between groups 1 and 2, within each schedule-statistical comparisons of horizontal differences in the table can therefore be considered realistically. Preliminary experiments showed that it would be invalid to attach significance to differences less than 5-fold between schedules.

polymorphs from both groups of ewes were preincubated for 1 h at 370 (schedule B) there was no difference between the two groups in the number of bacteria surviving in the subsequent in vitro assay. After replacement of cytophilic immunoglobulin by incubation of polymorphs in autologous serum (schedule C) the superiority of polymorphs from the

Immunofluorescence studies Results for the percentages of polymorphs fluorescing with four of the FITC-conjugated antisera, after treatment in the various schedules, are shown in Fig. 1. No significant differences were observed between the two groups for the number of polymorphs fluorescing with each antiserum after treatment in each of the Schedules. Values presented are therefore the pooled means for eight ewes (four from each group). More than 25 per cent of polymorphs fluoresced with FITC-RAy2 in schedule A. This staining was abrogated after incubation of the cells in schedule B and restored after treatment with autologous serum in schedule C. Treatment with trypsin only slightly decreased the percentage of cells staining with FITC-RAy2 but stripping with pronase resulted in less than 1 per cent of fluorescent cells.

Cytophilic IgG2 and phagocytosis by polymorphs 50r (b)

50r (a)

T 251-


_c Un

E 0





(e) IL


Properties of IgG2 Pure IgG2 was obtained by fractionation of blood sera from six ewes in each group. The twelve IgG2 preparations were then used in a standard antistaphylococcal agglutination assay. IgG2 from group 1 animals had a titre of 170+±4-2 antibody u/mg IgG2 (mean±s.e.) whereas for group 2 the titre was 10-4 ±19 antibody u/mg IgG2. The difference between the two groups of ewes was not statistically significant. Polymorphs were collected from an untreated ewe ('neutral' polymorphs), incubated for 1 h at 370 (as for schedule B), then washed suspensions were incubated separately with each of the twelve preparations of IgG2 at 4°. These polymorphs were then used in the standard in vitro assay and the results are shown in Table 2. The number of bacteria Table 2. Colony counts (mean+ standard error) of S. aureus in pour plates prepared after phagocytosis assays using 'neutral' polymorphs which had been incubated with IgG2 from either group I or group 2 animals

Colony counts


Figure 1. The mean percentage of polymorphs which specifically stained with four FITC-conjugated anti-sera, after treatment in schedule A (washed, untreated polymorphs), schedule B (cytophilic immunoglobulin removed by incubation), schedule C (cytophilic immunoglobulin replaced), schedule D (trypsin digestion) and schedule E (pronase stripping). Bars represent standard errors. The numbers of cells staining with RAyl-FITC, RAa-FITC and RApFITC were trivial (a) RAy2-FITC; (b) RAS-FITC; (c) RALC-FITC; (d) RAFAB-FITC.

Similar results were observed for cells stained with FITC-RAS after the various treatments. Preparation in schedule A resulted in 16 per cent of cells staining with FITC-RALC and 24 per cent with FITC-RAFab. These figures were sharply reduced after treatment in schedule B and restored after schedule C preparation. When treated with trypsin, less than 1 per cent of cells fluoresced with FITC-RALC and less than 3 per cent with FITCRAFab. The numbers of polymorphs fluorescing with either of these antisera after pronase stripping were negligible. After treatment in each of the schedules the numbers of polymorphs staining with FITC-RAyl, FITC-RAx and FITC-RA/u were insignificant.

Neutral polymorphs/group 1 IgG2 Neutral polymorphs/group 2 IgG2 Neutral polymorphs Bacterial control

82+22 (a) 111 + 22 (b) 421 4500

Each incubation mixture contained 106 staphylococci; bacteria: polymorph ratio = 1:10; t-test: (a) vs (b), P< 0 05.

surviving with 'neutral' polymorphs coated with IgG2 from group 1 ewes was significantly less (P

The effect of cytophilic IgG2 on phagocytosis by ovine polymorphonuclear leucocytes.

Immunology 1976 31 159 The effect of cytophilic IgG2 on phagocytosis by ovine polymorphonuclear leucocytes D. L. WATSON Veterinary Research Station,...
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