Acta path. microbiol. scand. Sect. C , 84: 119-123, 1976

INFLUENCE O F IgG, F(ab’), AND IgM ON T H E PHAGOCYTIC AND BACTERICIDAL ACTIVITIES O F HUMAN NEUTROPHIL GRANULOCYTES KARENELINACHRISTIE, CLAUS0. SOLBERG, BODILLARSEN, h GROVand OLAVT 0 N D E R The University of Bergen, School of Medicine, Medical Department B, Broegelmann Research Laboratory for Microbiology, and Department of Microbiology, The Gade Institute, Bergen, Norway

Christie, K. E., Solberg, C. O., Larsen, B., Grov, A. & Tender, 0. Influence of IgG, F(ab’), and IgM on the phagocytic and bactericidal activities of human neutrophil granulocytes. Acta path. microbiol. scand. Sect. C, 84: 119-123, 1976. The phagocytosis and intracellular killing of Staphylococcus aureus by human granulocytes in the presence of immunoglobulin preparations have been examined. Isolated IgG from pooled human serum induced phagocytosis and intracellular killing of bacteria. F (ab’) fragments had no significant effect, indicating that the Fc piece of the IgG molecule is of importance not only for the phagocytosis but also for the intracellular killing of bacteria. Isolated IgM stimulated the phagocytosis to a minor extent, with no enhancement of the bactericidal activity. Key words: Granulocytes ; phagocytosis ; intracellular killing ; influence of immunoglobulins. C. 0. Solherg, Medical Department B, 5016 Haukeland sykehus, Bergen, Norway.

Received 25.viii.75

Accepted 23.x.75

The influence of immunoglobulins and fragments of IgG on the phagocytic activity of human leucocytes has been the subject of several investigations ( 1 , 2, 10, 16). The immunoglobulins attach to the surface of bacteria and usually enhance the phagocytosis by human leucocytes ( 1 , 7 ) . This attachment is specifically mediated by the Fab piece in an antigen-antibody reaction (4, l l ) , and the complex attaches to receptors for the Fc piece on the cell membrane of human granulocytes (2, 3, 8, 10). Adsorption of uncomplexed immunoglobulins to the Fc receptors (cytophilic antibodies) is also of importance for the phagocytosis of bacteria. I t is also claimed that immunoglobulins which are non-

specifically attached to bacteria by the Fc piece can act as opsonins (16). We have recently shown that the most important serum factors in the phagocytosis and killing of S. aureus by human granulocytes include specific antibodies against the bacteria (14, 15). Complement or other heat labile serum factors are also important for ingestion and intracellular killing of bacteria (5,14). In the present study we report further results concerning the influence of isolated IgG, IgM and pepsin digested IgG on the phagocytosis and killing of bacteria by human polymorphonuclear leucocytes.

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MATERIALS AND METHODS Leucocytes Leucocytes were obtained from normal individuals by Isopaque/dextran sedimentation of heparinized venous blood as described previously ( 12). Immunoglobulins Human IgG (Cohn's fraction 11) was purchased as a 16.5 per cent solution from AB Kabi, Stockholm. The solution was dialysed against Hanks' balanced salt solution (HBSS) containing 0.1 per cent gelatin and filtered through a Millipore filter (SWINNEX-13 filterunit, SXHA 013 0 s 0.45 p). IgG was digested with pepsin (2 x crystallized, Sigma Chemical Company, St. Louis, Mo., USA) at an enzyme to protein ratio of 2 : l O O in 0.1 M acetate buffer, pH 4.1 at 37" C for 16 h (9). The digestion was stopped by dialysis against phosphate buffered saline pH 7.2 (0.015 M phosphate, 0.15 M NaCl) at 4" C. The digest was finally dialysed against HBSS, filtered through a Millipore filter and stored at -20" C until used. The antigenbinding activity of the preparation did not change during digestion as the product agglutinated rabbit red cells to a titre similar to that of undigested IgG. The preparation did not sensitize rabbit red cells to agglutination by rheumatoid factor indicating that the Fc portion was destroyed (F(ab'), fragments). Human IgM was isolated from pooled normal serum by dialysation against distilled water in the cold and fractionation of the precipitate on Sephadex G-200 (2.3 x 90 cm) with phosphate buffer pH 7.2, containing 1 M NaCl as eluant. The fractions containing IgM without traceable contamination of IgG were pooled. The fractions were selected on the basis of their optical density at 280 nm, their ability to agglutinate rabbit erythrocytes before, but not after, treatment with 0.2 M mercaptoethanol, and their reactions with antisera against human IgG and IgM in double diffusion. Membrane filtration (Sartorius membrane filter, SM 13200, Gottingen) in HBSS was used to concentrate the fractions. IgM in the final preparation was quantitated by single radial immunodiffusion on Tri-Partigen immunodiffusion plates (Behringwerke AG, Marburg-Lahn) . The contamination of IgG did not exceed 0.02 mg/ml.

immunoglobulins. The bacteria were then centrifuged at 10,000 g for 20 min, twice washed and resuspended in HBSS to a final concentration of 10-14 x 107 colony-forming units per ml. Test Procedure The test procedure has previously been described in detail (12, 13, 14). T o 0.5 ml leucocyte suspension, 0.1 ml untreated bacteria suspension was added and 0.4 ml immunoglobulin preparations diluted in HBSS, 0.1 ml bacteria coated with immunoglobulins and 0.4 ml HBSS. This provided 2-3 bacteria per neutrophil granulocyte. In the tests using untreated bacteria, the final concentrations of immunoglobulin will be indicated. The tubes were incubated at 37" C and samples were removed at prescribed intervals for determinations of the total number of viable bacteria and the number of viable intracellular bacteria. Controls consisted of bacteria in HBSS mixed with granulocytes and bacteria mixed with the serum preparations to be tested in order to detect any effect of the granulocytes or serum preparations on the bacteria. Statistical Method The Wilcoxon-test for two samples was used.

RESULTS

Influence of IgG Leucocyte-bacteria suspensions with 0.064.0 mg IgG/ml were incubated and the total number of viable bacteria and the number of viable intracellular bacteria were determined after various lengths of time. Maximum phagocytosis and killing of bacteria occurred after 2 h a t the final IgG concentration of 1 mg/ml (Fig. 1). The number of colony forming units was reduced by 80 per cent, but still, 20 per cent of the bacteria were not phagocytized. Raising the concentration of IgG did not increase the activity of the granulocytes. Whether this lack of response was due to exBacteria Staphylococcus aureus (Heatley strain) was cul- cess of TgG molecules unspecifically blocking tured and prepared as earlier (12). The suspen- the attachment of sensitized bacteria to the sions of bacteria used contained 10-14 x lo7 colo- leucocytes was further investigated. Bacteria ny-forming units per ml. were sensitized in solutions of 0.15-10.0 mg IgG/ml and then incubated with leucocytes. Sensitization of Bacteria Maximum phagocytosis and killing occurred S.aureus suspended in HBSS was incubated at at the highest concentration of IgG used 37" C for 30 min with various concentrations of 120

The number of colony forming units was reduced by 57 per cent but, since the control without granulocytes showed a reduction by 50 per cent, the enhancement of phagocytosis was almost negligible. There was no stimulation of intracellular killing of bacteria. Even at concentrations as low as 0.25 mg/ml, the number of viable intracellular bacteria was more than 3 times as high as that to be seen when intact IgG was used, i.e. similar to the numbers obtained in the absence of immunogIobulins. The results strongly suggest that the Fc piece of the IgG molecule is decisive not only for the phagocytosis but also for the intracellular killing of bacteria by IgG. Bacteria sensitized with F (ab’) fragments were tested in the same way as described

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Fig. 1 . Counts of viable bacteria during incubation of granulocyte-bacteria suspensions and IgG or Hanks’ balanced salt solution (mean of 5 experiments). (The difference between 1 and 4 mg IgG/ ml was not significant. The difference between the other groups at 120 min was significant ( ~ 2 0 . 0 2 5) )

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(4 mg/ml), and the number of colony forming units was reduced by 95 per cent (Fig. 2 ) . Very few bacteria escaped engulfment. However, the pronounced reduction of viable bacteria was not only due to the activity of the leucocytes since controls without granulocytes showed a reduction by 40 per cent. The results indicated that the relative reduction in phagocytic activity in the presence of high amounts of I g G occurred at the level of granulocytes.

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Influence of F(ab’), Fragments Leucocyte-bacteria suspensions with 0.064.0 mg pepsin digested IgG/ml were tested in the same way as described above for I@. 9 Acta path. microbiol. scand. Sect. C, 84, 2

Fig. 2. Counts of viable bacteria during incubation of granulocyte suspension and bacteria sensitized with IgG (mean of 4 experiments). (The differences between the groups a t 120 min were significant ( p z 0 . 0 5 ) ).

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above for bacteria sensitized with intact IgG. There was no enhancement of either phagocytosis or intracellular killing of bacteria. Influence of IgM The influence on the phagocytic activity of the granulocytes of IgM at concentrations of 0.01-0.25 mg/ml was investigated. At a final IgM concentration of 0.25 mgjml, the reduction in the number of colony forming units was approximately 60 per cent. However, controls showed that IgM alone, in the absence of granulocytes, reduced the number of colony forming units by as much as 50 per cent. Furthermore, the number of viable intracellular bacteria was even higher than that in the controls where immunoglobulins were substituted by HBSS. Accordingly, IgM did not stimulate the bactericidal activity, but seemed to stimulate the phagocytic activity to a minor extent. The phagocytic and bactericidal activities did not change when the bacteria were sensitized with IgM before addition to the leucocytes. DISCUSSION

Two procedures were used to study the phagocytosis and intracellular killing of S. aureus by human granulocytes in the presence of immunoglobulin preparations. In one procedure, the immunoglobulins were added to the suspension of leucocytes and bacteria, in the other the bacteria were incubated with the immunoglobulins, washed, and then added to the leucocytes. By both procedures, isolated human IgG induced phagocytosis and intracellular killing of bacteria by human neutrophils. However, the phagocytosis was more pronounced if procedure 2 was used. As the number of colony forming units in the suspensions is a crucial parameter in the tests, agglutination of the bacteria by antibodies may result in a reduction in this number without killing the bacteria. However, the reduction in the number of colony forming units in a suspension of bacteria containing IgG was significantly higher in the pre-

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sence of leucocytes than in the absence of leucocytes. I n addition, the enhancing effect of IgG could easily be exhausted by absorption of the preparation with bacteria (14). This strongly suggests that the influence of IgG on the phagocytic and bactericidal activities of human neutrophils is due to absorption of the immunoglobulins on to the surface of the bacteria, followed by attachment of the sensitized bacteria to the cell membrane of the granulocytes (Fc receptors). The pronounced killing of bacteria to be seen if procedure 2 was used and the relatively decreased phagocytosis at concentrations greater than 1 mg IgG/ml if procedure 1 was used, indicate that non-antistaphylococcal IgG may occupy the Fc receptors on the surface of the granulocytes. The results obtained by F (ab') fragments which showed no difference in phagocytosis whether procedure 1 or 2 had been used are in keeping with this suggestion. F(ab'), fragments had very little, if any, enhancing effect on the phagocytosis, and the reduction in the number of colony forming units was apparently due to agglutination of the bacteria. The bactericidal activity was not enhanced at all. This indicates that the Fc piece of the IgG molecule is important, not only for the phagocytosis, but also for the intracellular killing of the bacteria. This is in agreement with the hypothesis that the attachment of sensitized bacteria to Fc receptors on the cell membrane of the granulocytes may also influence the bactericidal activity of the granulocytes, probably by acting as a stimulus by which the bactericidal enzymes may be released into the phagocytic vacuole (14, 15). Isolated human IgM had very little influence on the phagocytosis of bacteria by human neutrophils. The reduction in the number of colony forming units was mainly due to IgM alone. The bactericidal activity was not increased. The conclusion to be drawn on this basis is that the phagocytosis enhancing effect of IgM without complement is very low. This is in accordance with the observation that monocytes and macrophages have

very few surface receptor sites for IgM ( 6 ) . This may also explain the lack of bactericidal activity of the granulocytes in the presence of IgM and why complement is of such importance for phagocytosis and intracellular killing of bacteria (5, 14).

8.

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REFERENCES 1. Dossett, J . H., Kronva!l, G., Williams, R . C . Jr. & Quie, P . G.: Antiphagocytic effect of staphylococcal protein A. J. Immunol. 103: 1405-1410, 1969. 2. Fidalgo, B. V . & Najjar, V . A.: The physiological role of the lymphoid system. 111. Leucophilic y-globulin and the phagocytic activity of the polymorphonuclear leucocyte. Proc. Nat. Acad. Sci. U.S.A. 57: 957-964, 1967. 3. Fidalgo, B. V . & Najjar, V . A.: The physiological role of the lymphoid system. VI. The stimulatory effect of leucophilic y-globulin (leucokinin) on the phagocytic activity of human polymorphonuclear leucocyte. Biochemistry 6: 3386-3392, 1967. 4. Lenhart, N . A., Mudd, S., Yoshida, A . & Li, I . W . : The common protein agglutinogen of Staphylococcus aureus. I. Distribution in international serotypes and corresponding antibody in human populations. J. Immunol. 92: 771776, 1963. 5. Li, I . W . & M udd, S.: The heat-labile serum factor associated with intracellular killing of Staphylococcus aureus. J. Immunol. 94: 852857, 1965. 6. LoBuglio, A . F., Cotran, R . S . & Jandl, J . H.: Red cells coated with immunoglobulin G : Binding and sphering by mononuclear cells in man. Science 158: 1582-1585, 1967. 7. Miescher, P . A., Spiegelberg, H . & Benacerraf, B.: Studies on the mechanism of immune phagocytosis of sensitized bacteria and red

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cells by the reticuloendothelial system in mice. In: Halpern, B. N. (Ed.) : R81e du Systhme RCticulo-EndothClial d a m 'ImmunitC Antibactkrienne et Antitumorale'. CNRS, Paris, 1963. Messner, R . P . & Jelinek, J . : Receptors for human y-G globulin on human neutrophils. J. Clin. Invest. 49: 2165-2171, 1970. Nisonoff, A., Wissler, F. C . & Lipman, L . N.: Properties of the major component of a peptic digest of rabbit antibody. Science 132: 17701771, 1960. Quie, P . G., Messner, R . P . & Williams, R . C., Jr.: Phagocytosis in subacute bacterial endocarditis. Localization of the primary opsonic site to Fc fragment. J. Exp. Med. 228: 553570, 1968. Shayegani, M . G., Hisatsune, K . & M u d d , S.: Cell wall component which affects the ability of serum to promote phagocytosis and killing of Staphylococcus aureus. Infect. Immun. 2 : 750-756, 1970. Solberg, C. 0.: Enhanced susceptibility to infection. A new method for the evaluation of neutrophil granulocyte functions. Acta path. microbiol. scand. Sect. B, 80: 10-18, 1972. Solberg, C. 0.: Protection of phagocytized bacteria against antibiotics. A new method for the evaluation of neutrophil granulocyte functions. Acta med. scand. 191: 383-387, 1972. Solberg, C. O., Christie, K . E., Larsen, B. & T m d e r , 0.: Influence of antibodies and thermolabile serum factors on the bactericidal activity of human neutrophil granulocytes. Acta path. microbiol. scand. Sect, C, 84: 112-118, 1976. Solberg, C . 0. & Hellurn, K . B.: Influence of serum on the bactericidal activity of neutrophi1 granulocytes. Acta path. microbiol. scand. Sect. B, 81: 621-626, 1973. van Oss, C. J,, Woeppel, M . S . & Marquart, S . E . : Immunoglobulins as aspecific opsonins. 111. The opsonizing power of fragments of polyclonal and monoclonal immunoglobulin G. J. Reticuloendothel. SOC. 13: 221-230, 1973.

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Influence of IgG, F(ab')2 and IgM on the phagocytic and bactericidal activities of human neutrophil granulocytes.

The phagocytosis and intracellular killing of Staphylococcus aureus by humans granulocytes in the presence of immunoglobulin preparations have been ex...
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