Immunology 1977 33 231
Human polymorphonuclear leucocyte receptors for staphylococcal opsonins
J. VERHOEF *, P. K. PETERSON & P. G. QUIE Departments of Medicine and Pediatrics, University of Minnesota Medical School, Minneapolis, Minnesota, U.S.A.
Received 25 November 1976; accepted for publication 20 December 1976
ingestion as well as the attachment phase of phagocytosis.
Summary. The presence of receptors on the plasma membrane of human polymorphonuclear (PMN) leucocytes for factors related to complement and for the Fc region of immunoglobulin has not been clearly defined for opsonized bacteria. To separate the activity of these two receptors, the uptake of [3H]thymidine labelled staphylococci opsonized with normal serum or heat-inactivated serum was measured. Phagocytosis was depressed when bacteria opsonized with normal serum were incubated with trypsin-treated leucocytes, suggesting that complement receptors of human PMN leucocytes are trypsin-sensitive. Phagocytosis of bacteria opsonized with heat-inactivated serum was not depressed by trypsin, but was blocked by incubating PMN leucocytes with heat-aggregated IgG and by incubating opsonized bacteria with protein A. In experiments performed to quantify the number of bacteria attached to but not ingested by PMN leucocytes, it was shown that both complement and Fc receptors participate in the ingestion phase of phagocytosis. Cell membranes of human PMN leucocytes possess two receptors for opsonized staphylococci; a complement receptor which is utilized when bacteria are opsonized in normal serum and an Fc receptor when bacteria are opsonized in heatinactivated serum. Both receptors participate in the
INTRODUCTION The process of phagocytosis has been separated into two distinct phases: attachment and ingestion (Rabinovitch, 1967; Stossel, 1975). The attachment of particles to phagocytic cell membranes is generally believed to be mediated through receptors with certain immunological specificities. In the case of macrophages and mononuclear phagocytes, receptors have been described for particles coated with IgM, IgG and a modified component of complement (C3b) (Lay & Nussenzweig, 1968; Holland, Holland & Cohn, 1972; Mantovani, Rabinovitch & Nussenzweig, 1972; Speigelberg, 1974; Reynolds, Atkinson, Newball & Frank, 1974; Griffin, Bianco & Silverstein, 1975; Bianco, Griffin & Silverstein, 1975; Griffin, Griffin, Leider & Silverstein, 1975). Macrophage receptors for IgG-coated particles (Fc receptors) have been shown to mediate both attachment and ingestion, however, receptors specific for complement-coated particles are involved only in the attachment phase and do not independently participate in particle ingestion unless the macrophages are activated. Although there is evidence to suggest that the receptors on polymorphonuclear (PMN) leucocytes and macrophages have similar functions (Quie, Messner & Williams, 1968; Messner & Jellinek, 1970; Henson, Johnson & Spiegelberg, 1972;
* Present address: Laboratory for Microbiology, Catharijnesingel 59, Utrecht, The Netherlands. Correspondence: Dr Jan Verhoef, Laboratory for Microbiology, Catharijnesingel 59, Utrecht, The Netherlands.
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Mantovani, 1975; Scribner & Fahrney, 1976), this has not been established with certainty. Most studies of phagocyte receptors have been performed using either opsonized sheep erythrocytes or immune complexes. The functional existence and relative importance of Fc and complement receptors for the phagocytosis of bacteria is unclear. In this study the characteristics of PMN leucocyte receptors for staphylococci were investigated. Evidence is presented which suggests that bacteria opsonized in the presence of complement and immunglobulin are primarily phagocytized via the complement receptor, and that this receptor is involved both in the attachment and in the ingestion phases of phagocytosis by PMN leucocytes.
MATERIALS AND METHODS
Bacterial strains Two Staphylococcus aureus strains, Cowan I and Wood 46, and three Staphylococcus epidermidis strains, Her and Bra (isolates from patients with S. epidermidis endocarditis) and 407 (a commensal strain) were used. The S. epidermidis strains belonged to biotype 1 (Baird-Parker, 1974). Radioactive labelling The method used to measure bacterial attachment, ingestion and killing by PMN leukocytes has been reported in detail elsewhere (Verhoef, Peterson & Quie, 1977) and was modified slightly for the purposes of this study. One-tenth millilitre of an overnight culture of bacteria was inoculated into 10 ml Mueller-Hinton broth (Difco, Detroit, Michigan) containing 002 mCi thymidine methyl 3H-labelled (sp. act. 6-7 Cifmmol, New England Nuclear, Boston, Massachusetts). After 18 h growth at 370, the bacteria were washed three times in phosphate-buffered saline, pH 7-4 (PBS). A final bacterial concentration of 5 x 108 cfu/ml PBS was obtained using a spectrophotometric method confirmed by pour plate colony counts. Leucocytes Forty millilitres of blood were drawn from healthy donors in a syringe containing 200 u heparin. The erythrocytes were sedimented for 1 h in 6%. dextran '70' (Cutter Labs, Berkeley, California) in normal saline (10 ml blood: 3 ml saline). The leucocyte-rich plasma was withdrawn and centrifuged at 160 g for 5 min. The resulting pellet was washed twice in
heparinized saline (10 u heparin/10 ml saline). Using a standard method, total and differential leucocyte counts were performed, and the final leucocyte pellet was resuspended to a concentration of 107 PMN leucocytes/ml Hanks's balanced salt solution with 1 % gelatin (HBSS).
Treatment of PMN leucocytes with heat-aggregated IgG PMN leucocytes were incubated for 30 min at 370 in HBSS containing 5 mgy heat-aggregated IgG in HBSS. The leucocytes were then washed twice in HBSS. Control (untreated) leucocytes were likewise incubated and washed in HBSS. Trypsin treatment of PMN leucocytes Using a method similar to that described for macrophages (Bianco et al., 1975) PMN leucocytes were incubated with 1 mg/ml of trypsin (Difco) in HBSS for 30 min at 37°. The leukocytes were then washed twice in HBSS containing 1 mg/ml soya bean trypsin inhibitor (Worthington Biochemical Corporation, Freehold, New Jersey) to terminate trypsin digestion. Control (untreated) leucocytes were likewise incubated and washed in HBSS.
Opsonins Serum from five normal donors was pooled and kept frozen in 1-ml aliquots at -70°. C2-deficient serum was obtained from a patient with inherited complete C2 deficiency (Kim, Friend, Dresner, Yunis & Michael, 1977). Serum was obtained from an asymptomatic child with C3 deficiency (23 mg/100 ml C3) in which levels of all other complement components were normal (kindlyprovided by Dr Y. Kim, University of Minnesota). IgG deficient serum was obtained from a child with congenital rubella syndrome in which IgG, IgA and IgE were nondetectable by Mancini radial immunodiffusion and by Ouchterlony methods. This serum contained 39 mg% IgM. These sera were stored in 0-2-ml aliquots at -70°. Shortly before use the aliquots were thawed and diluted to a final concentration of 10% in HBSS. Heat-inactivated normal serum was prepared by heating aliquots at 560 for 1 h and then diluting to a final concentration of 10%.
Opsonization of bacteria 015 ml of a bacterial concentration of 5 x 108 cfu/ml was added to a plastic tube (12 x 75 mm, Falcon, Oxnard, California) containing 0 8 ml of opsonin. After 60 min incubation at 370, the mix-
Neutrophil receptors for staphylococci tures were centrifuged at 1600 g for 15 min. The supernatants were discarded and the bacterial pellets resuspended in 0 75 ml HBSS.
Treatment of opsonized bacteria with protein A In one series of experiments bacteria which had been opsonized with normal serum and with heatinactivated serum were incubated for 30 min at 370 with purified protein A (kindly provided by Dr A. Forsgren, University of Malmo, Sweden) in HBSS (150 jug/ml). After centrifuging at 1600 g for 15 min, the bacterial pellets were resuspended in HBSS. After opsonization, control bacteria were incubated in HBSS, centrifuged and resuspended in HBSS.
Phagocytosis mixtures 0-75 ml of the leucocyte suspension was added to the plastic tubes containing 0 75 ml of opsonized bacteria. The final bacteria: PMN leucocyte ratio was approximately 10 : 1 in all experiments. The phagocytosis mixtures were tumbled at 10 r.p.m. at 370 in a rotating rack (Fisher Roto-Rack, Fisher Scientific Co., Chicago, Illinois).
Determination ofPMN leucocyte bacterial uptake and killing Immediately after the phagocytosis mixtures were constituted, duplicate 5-,ul samples were taken for plating to determine the total cfu added to the mixtures at time '0'. To determine the leucocyteassociated bacterial population, duplicate 100 pl samples were taken from the phagocytosis mixtures with an Eppendorf pipette at 3-, 10- and 20-min intervals and placed in 3 ml cold PBS in polypropylene vials (Bio-Vials, Beckman, Chicago, Illinois). The vials were centrifuged for 5 min at 160 g at 40 and the leucocyte pellets washed twice with ice cold PBS. The final leucocyte pellets were disrupted by vigorous mixing in 2 5 ml sterile distilled water. From these suspensions duplicate 5 ul samples were taken with an Eppendorf pipette to determine the viable leucocyte-associated bacterial population by plating in nutrient agar. Cfu were counted after 18 h incubation at 37°. The final 2-5-ml suspensions were centrifuged at 1600 g for 15 min, and the total leucocyte-associated population of bacteria (alive and dead) was then determined by solubilizing the pellets in 2*5 ml scintillation liquid (toluene containing fluoralloy (TLA, Beckman) and 20%Y Biosolve-3 (Beckman)) and counting in a liquid scintillation counter (Beckman LS-250). To determine the total F
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bacteria-associated c.p.m. (representing leucocyteassociated bacteria plus extracellular bacteria) duplicate 100-pl samples were taken at the end of the assay period, placed in 2-5 ml water and centrifuged at 1600 g for 15 min. The pellets were resuspended in scintillation liquid and counted. An average of the duplicate values was used for all calculations. The per cent of the total bacterial population that was leucocyte-associated at a given sampling time (per cent uptake) was calculated using the following formula: per cent uptake = c.p.m. in leucocyte pellet x I0. c.p.m. in total bacterial pellet (Formula 1) The leucocyte-associated bacterial population that was viable at a given sampling time was calculated according to this formula: per cent viable leucocyte-associated bacteria = per cent cfu in leucocyte pellet x 100. per cent uptake (Formula 2) where the denominator was obtained from Formula 1 and the numerator was derived using Formula 3: Cfu in leucocyte pellet at sampling time 100 cfu at time '0' (Formula 3) The killed population of bacteria was determined according to Formula 4: per cent killed leucocyte associated bacteria = per cent uptake (Formula 1)per cent viable leucocyte-associated bacteria (Formula 2).
Determination of the attached bacterial population To lyse the extracellular population of bacteria, including those bacteria that were attached to but not ingested by the leucocytes, duplicate 100 ,ul samples were placed in PBS containing 1 ,ug/ml lysostaphin (Schwarz-Mann, Orangeburg, New York). These samples were then incubated at 370 for 30 min followed by washing the leucocytes twice in PBS. Simultaneous samples were processed in the standard manner to determine the total leucocyteassociated bacterial population (attached plus ingested bacteria). Bacterial uptake was calculated as outlined above. The difference between the per
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cent uptake calculated from the samples placed in PBS and the samples placed in PBS containing lysostaphin was considered to represent the attached bacterial population. As less than 10% of the total c.p.m. were leucocyte-associated at 20 min when bacteria without opsonin were added to the phagocytosis mixtures, it appeared that washing of the leucocytes eliminated most of the extracellular bacteria and that lysostaphin lowered the leucocyteassociated cpm primarily by lysing the attached bacterial population. To confirm lysostaphin activity, control mixtures containing the same concentration of bacteria and serum but no leucocytes were also sampled into PBS and PBS containing 1 ug/ml lysostaphin followed by incubation at 370 for 30 min. After centrifuging at 1600 g, the bacterial pellets were suspended in scintillation liquid and counted.
RESULTS PMN leucocyte receptors for staphylococcal phagocytosis To study whether two distinct receptors for staphylococcal phagocytosis could be demonstrated, PMN leucocytes were (1) treated with trypsin to inactivate potential complement receptors and (2) incubated with heat-aggregated IgG to block potential Fc receptors. Staphylococci which had been opsonized in normal serum and in heat-inactivated serum were then added to these leucocytes as well as to untreated control cells. Fig. 1 shows the results of this experiment using S. aureus Wood 46. As can be seen in Fig. la, trypsinization of the leucocytes markedly reduced their ability to take up bacteria which had been opsonized in normal serum but did not significantly change uptake of bacteria opsonized with heat-inactivated serum. Whereas 85 %o of bacteria opsonized with normal serum were leucocyte-associated after 20 min incubation with untreated leucocytes, only 21 %o were taken up by trypsinized cells. When the bacteria were opsonized with heat-inactivated serum, 49%4 were leucocyteassociated when control cells were used and 44%/o when trypsinized leucocytes were studied. Therefore, the receptors for bacteria opsonized in the presence of complement are trypsin sensitive, and the receptors for bacteria opsonized without complement are not affected by trypsin. Although trypsinization of PMN leucocytes did not significantly affect uptake of bacteria opsonized
10°r
0X a
(~~~bac .0 + PMN normol)
-------
100 _ (b)
N + (bac ( PMN*normaI) bac.N+ \
80 -
PM Agg IqG) PMN
60 -
( bac -H + 4 PMN * norma I) (bac.H + k PMN- AggIgG)
20
0
3
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20 Time (min)
Figure 1. Uptake of S. aureus Wood 46 by trypsinized (a) and heat-aggregated IgG treated (b) polymorphonuclear (PMN) leucocytes. Bacteria were opsonized with normal serum (bac. N), heat-inactivated serum (bac. H) and Hanks's balanced salt solution (HBSS) (bac. 0) before being added to leucocytes which had been incubated in HBSS (PMN. normal), HBSS containing trypsin (PMN.tryp) and HBSS containing heataggregated IgG (PMN. Agg IgG).
with heat-inactivated serum, pre-incubation of cells with heat-aggregated IgG did reduce their ability to take up these bacteria (Fig. Ib). Whereas 49%4 of bacteria opsonized with heat-inactivated serum were leucocyte-associated after 20 min incubation with untreated leucocytes, only 26% were taken up by cells treated with heat-aggregated IgG. Treatment of leucocytes with heat-aggregated IgG did not, however, significantly affect their ability to take up bacteria opsonized with normal serum (Fig. lb). Approximately 80%Y of bacteria opsonized with normal serum were taken up after 20 min incubation with both control and heat-aggregated IgG treated leucocytes. Thus, PMN leucocyte receptors for bacteria opsonized in the absence of complement were
Neutrophil receptors for staphylococci blocked by incubation with heat-aggre.gated IgG, but the receptors for bacteria opsonized in the presence of complement remained intact. Results similar to the findings with S. aureus Wood 46 were obtained with S. aureus Cowvan I and with S. epidermidis Her and 407 (data Xnot presented). When S. epidermidis Bra was stucdied, however, trypsinization of the leucocytes had le.ss effect on the uptake of bacteria opsonized with normal serum. After 20 min incubation, 89%4 of S. epidermidis Bra were taken up by untreated lebucocytes compared with 75%. by trypsinized leucocytes. As was observed with the other four strain,s, treatment of leucocytes with heat-aggregated IgG did not significantly reduce their ability to take up S. epidermidis Bra opsonized with normal serum (dEata not shown). The effect of protein A on the function of PMN leucocyte receptors Results from the above experiments suzgested that although a distinct Fc receptor anid complement receptor for staphylococcal phagoc)itosis could be identified, when staphylococci were opsonized in normal serum, i.e. in the presence of immunoglobulin and complement, they were tak en up by PMN leucocytes primarily via the comple ment receptor To further study the nature of tihese receptors, opsonized bacteria were incubated in HBSS con100* 80 .
-2
i(bacN +HBSS) (bac- N +PA)
60 .
c
(bac H+HBSS)
I(boc H +PA) -
Time (min)
Figure 2. Effect of protein A on uptake of S. aureus Wood 46 by polymorphonuclear leucocytes. Bacteria were opsonized with normal serum (bac.N) and heat-inactivated serum (bac.H) before being incubated in Hanks's Balanced Salt Solution (HBSS) and in HBSS containing protein A (PA). After centrifuging and resuspending the bacterial pellets in HBSS, normal leucocytes were added to the bacteria.
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taining staphylococcal protein A (a protein known to react with the Fc fragment of IgG (Forsgren & Sjdquist, 1966)) before adding the bacteria to PMN leucocytes. Fig. 2 demonstrates the results of this experiment using S. aureus Wood 46, a strain deficient in cell wall protein A. Whereas protein A treatment of bacteria opsonized with heat-inactivated serum significantly reduced bacterial uptake (I0%Y uptake after 20 min incubation compared with 40%4 uptake of untreated bacteria), the effect of protein A on bacteria opsonized with normal serum was minimal (55 % uptake compared with 67%4 uptake of untreated bacteria). Therefore, the receptor for bacteria opsonized with heat-inactivated serum is specific for the Fc fragment of immunoglobulin and does not appear toplayan important rolein the phagocytosis of bacteria opsonized with normal serum. The effect of opsonization with C2 apd C3 deficient sera on the function of PMN leucocyte receptors Two strains of staphylococci were selected to study the importance of complement factors on phagocytosis: S. aureus Cowan I, known to depend on the presence of the classical complement pathway for effective opsonization and S. epidermidis 407, known to be effectively opsonized by the alternative complement pathway (Verhoef, Petersen, Kim, Sabath & Quie, 1977). PMN leucocyte uptake of S. aureus Cowan I was decreased when bacteria were opsonized with both C2 deficient and C3 deficient sera (Fig. 3a). When opsonized with normal serum, 89%Y of the bacteria were leucocyte-associated after 20 min incubation compared with 51 Y. of bacteria opsonized with C3 deficient serum. The uptake of S. epidermidis 407 was also reduced when bacteria were opsonized with C3 deficient serum but was not significantly affected by opsonization with serum deficient in C2
(Fig. 3b). Approximately 90%. of bacteria were leucocyte-associated when opsonized with normal serum and C2 deficient serum compared with 52%0 uptake of bacteria opsonized with C3 deficient serum. Efficient attachment therefore required activated components of C3 achieved via the classical pathway (S. aureus Cowan I) or the alternative pathway (S. epidermidis 407). The role of PMN leucocyte Fc and complement receptors in the attachment and ingestion phases of
phagocytosis To study the role of the Fc and complement receptors in the ingestion as well as the attachment phases of
J. Verhoef, P. K. Peterson & P. G. Quie
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The size of the attached population of leucocyteassociated bacteria was also assessed by incubating samples from the phagocytosis mixtures in lysostaphin in order to lyse the extracellular (including attached) population of bacteria. The number of leucocyte-associated bacteria in these samples was then compared with the number of leucocyteassociated bacteria in samples processed without lysostaphin treatment. The validity of this method was confirmed when it was established that samples from control mixtures containing the same concentration of bacteria used in the phagocytosis mixtures but without leucocytes lost over 90%Y of the radio-
0a
@
100 _ (
100 _ (a)
(norma serum)
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*(bocN + k
c
80-o
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80 60-
60-
(03
deficient)
+ -*(bac-H PMN. normal)
serum
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40
( heat- inactivated serum
20
20
0
3
20
10
'
0 100 _
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Figure 3. Effect of opsonization of S. aureus Cowan I (a) and S. epidermidis 407 (b) with normal serum, C2-deficient serum, C3-deficient serum and heat-inactivated serum on uptake by normal polymorphonuclear leucocytes. Bacteria were incubated for 60 min in each opsonic source before being added to the leucocytes.
phagocytosis, experiments were performed to determine the per cent of leucocyte-associated bacteria that were killed at each sampling time. If attachment occurred without ingestion presumably a relatively small number of leucocyte-associated bacteria would be killed. Bacterial killing was determined after phagocytosis via the complement receptor by using leucocytes treated with heat-aggregated IgG in order to block Fc receptors and by using bacteria opsonized with IgG deficient serum. After 20 min incubation, approximately 95% of S. aureus Cowan I which had been opsonized with normal serum and with IgG deficient serum were killed when taken up by normal leucocytes; 84% were killed when taken up by leucocytes pre-treated with heat-aggregated IgG (data not given).
80
-
+ b(ac.IGdef \ ~~~~~PMN-normal
/
610
40
20
0
3
20
10 Time (min)
Figure 4. Effect of lysostaphin on measured PMN leucocyte uptake of S. aureus Cowan I opsonized with normal serum and heat-inactivated serum (a) and IgG-deficient serum (b). Bacteria were opsonized with normal serum (bac.N), heatinactivated serum (bac.H) and IgG-deficient serum (bac.IgG def) before being added to normal leucocytes (PMN.normal) and heat-aggregated IgG treated leucocytes (PMN.Agg IgG). Samples were placed in phosphate-buffered saline (closed symbols) and in phosphate-buffered saline containing lysostaphin (open symbols). Following incubation, the leucocytes were washed, and bacterial uptake was measured.
Neutrophil receptors for staphylococci activity from the bacteria after 30 min incubation in lysostaphin, and bacteria incubated without lysostaphin lost none. When S. aureus Cowan I which had been opsonized with normal serum was added to PMN leucocytes pre-treated with heat-aggregated IgG, 91% of the bacteria were leucocyte-associated after 20 min (Fig. 4a). Incubation of simultaneous samples in lysostaphin reduced the measured leucocyte-associated bacterial population by only 7%. When bacteria which had been opsonized in heatinactivated serum were added to untreated leucocytes there was uptake of 40% of bacteria after incubation of samples in lysostaphin resulting in only a slight decrease in the measured leucocyte-associated bacterial population (Fig. 4a). Fig. 4b shows the effect of lysostaphin treatment of samples taken from a phagocytosis mixture containing bacteria opsonized with IgG-deficient serum. After 20 min incubation 91% of the bacteria were determined to be leucocyteassociated when samples were not treated with lysostaphin compared with 71% following lysostaphin treatment. Again, lysostaphin did not have a marked effect on the measured leucocyte-associated bacterial population. Although 20% of bacteria opsonized with complement appeared to be attached and not ingested, these experiments suggest that ingestion as well as attachment is associated with both the complement receptor and the Fc receptor of human PMN leucocytes.
DISCUSSION By studying the effects of trypsinization and of heataggregated IgG on PMN leucocyte uptake of [3H]-thymidine labelled staphylococci opsonized with normal serum and with heat-inactivated serum, two distinct leucocyte receptors for bacterial attachment could be identified. An Fc receptor appeared to play a major role in the attachment of bacteria opsonized in the absence of complement (heatinactivated serum). When bacteria were opsonized with heat-inactivated serum, trypsinization of leucocytes did not interfere with staphylococcal attachment. When bacteria were opsonized in the presence of both immunoglobulin and complement (normal serum), however, a receptor with complement specificity appeared to function as the major receptor for bacterial attachment. Trypsinization markedly reduced the ability of PMN leucocytes to
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take up bacteria opsonized with normal serum. Treatment with heat-aggregated IgG did not significantly impair the ability of PMN leucocytes to take up bacteria opsonized with normal serum but greatly reduced uptake of bacteria opsonized with heat-inactivated serum. Additional evidence for a receptor with Fc specificity and for the primacy of the complement receptor for attachment of bacteria opsonized with normal serum came from experiments in which opsonized bacteria were treated with protein A. These experiments were performed in an attempt to block attachment from the bacterial side of the staphylococcal-PMN leucocyte interaction. Protein A is known to have the capacity to combine with the Fc fragment of IgG (Forsgren & Sjbquist, 1966). When bacteria opsonized with heat-inactivated serum were treated with protein A prior to being added to PMN leucocytes, subsequent uptake was significantly reduced. When bacteria opsonized with normal serum were studied, however, uptake was not significantly affected. To determine the relative importance of the third component of complement for attachment, the phagocytosis of two staphylococcal strains with different opsonic requirements was studied. Whereas phagocytosis of only the S. aureus strain was significantly reduced after opsonization with C2 deficient serum, phagocytosis of both the S. aureus and S. epidernidis strains was decreased when opsonized with C3 deficient serum. Thus, activation of C3 at the bacterial cell surface appears to play a central role in staphylococcal phagocytosis. To determine whether the Fc and complement receptors participated in both the attachment and ingestion phases of phagocytosis, experiments were performed using S. aureus Cowan I opsonized with normal serum, IgG deficient serum (which contained normal complement levels) and heat-inactivated serum. Samples from phagocytosis mixtures were incubated with lysostaphin to eliminate the extracellular, attached leucocyte-associated bacterial population. Lysostaphin is an enzyme which is known to lyse S. aureus cell wall and which does not enter leucocytes (Tan, Watanakunakorn & Phair, 1971). Incubation of samples in lysostaphin did not significantly reduce the measured leucocyte-associated bacterial population when bacteria were opsonized with normal serum, IgG-deficient serum or heat-inactivated serum. Therefore, it appeared that both the Fc receptor and the complement receptor
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were associated with the attachment as well as with the ingestion phases of phagocytosis. Further evidence to support the hypothesis that complement receptors on PMN leucocytes are involved in ingestion as well as in attachment came from the observation that the great majority of bacteria opsonized with IgG deficient serum were rapidly killed. Furthermore, there was also rapid killing when bacteria opsonized with normal serum were incubated with PMN leucocytes treated with heat-aggregated IgG. These findings are in agreement with the report of Messner & Jellinek (1970) who showed normal killing of S. aureus by PMN leucocytes after blocking Fc receptors with Fc fragments and of Williams & Quie (1971) who reported normal staphylococcal killing by PMN leucocytes in the presence of agammaglobulinaemic serum. Somewhat different functions of the Fc and complement receptors of mononuclear phagocytes appear to be operative in the phagocytosis of sheep erythrocytes (Griffin et al., 1975; Bianco et al., 1975). In this system sheep erythrocytes are opsonized with IgG or with IgG and complement. The plasma membranes of mononuclear phagocytes have been shown to contain receptors for the Fc portion of IgG and for the activated form of the third component of complement (C3b). Internalization (ingestion) of particles is triggered by IgG. The Fc receptor mediates both the binding of particles to the plasma membrane and their ingestion. However, the macrophage complement receptor appears to play a role only in the attachment phase of phagocytosis. In contrast, complement receptors on PMN leucocytes participate in both attachment and ingestion of
staphylococci.
ACKNOWLEDGMENTS This study was supported in part by grants Al 08821 and Al 06931 from the National Institutes of Allergy and Infectious Diseases. Dr Verhoef was supported by the Netherlands Organization for the Advancement of Pure Research (ZWO). Present address: Laboratory for Micro-
biology, Catharijnesingel 59, Utrecht, Netherlands. Dr Peterson was a recipient of a Bristol Research Fellowship in Infectious Diseases at the time of these studies. Dr Quie is an American Legion Memorial Heart Research Professor.
REFERENCES BAIRD-PARKER A.C. (1974) The basis for the present classification of staphylococci and micrococci. Ann. N. Y. Acad. Sci. 236, 7. BIANCO C., GRIFFIN F.M. & SILVERSTEIN S.C. (1975) Studies of the macrophage complement receptor: alteration of receptor function upon macrophage activation. J. exp. Med. 141, 1278. FORSGREN A. & SJOQUIST J. (1966) 'Protein A' from S. aureus I. Pseudoimmune reaction with human y-globulin. J. Immunol. 97, 822. GRIFFIN F.M., BIANCO C. & SILVERSTEIN S.C. (1975) Characterization of the macrophage receptor for complement and demonstration of its functional independence from the receptor for the Fc portion of immunoglobulin G. J. exp. Med. 141, 1269. GRIFFIN F.M., GRIFFIN J.A., LEIDER J.E. & SILVERSTEIN S.C. (1975) Studies on the mechanisms of phagocytosis. J. exp. Med. 142, 1263. HENSON P.M., JOHNSON H.B. & SPIEGELBERG H.L. (1972) The release of granule enzymes from human neutrophils stimulated by aggregated immunoglobulins of different classes and subclasses. J. Immunol. 109, 1182. HOLLAND P., HOLLAND N.H. & COHN Z.A. (1972) The selective inhibition of macrophage phagocytic receptors by antimembrane antibodies. J. exp. Med. 135, 458. KIM Y., FRIEND P.S., DRESNER I.G., YUNIs E.J. & MICHAEL A.F. (1977) Inherited deficiency of the second component of complement with membranoproliferative glomerulonephritis. Amer. J. Med. (In press.) LAY W.H. & NUSSENZWEIG V. (1968) Receptors for complement on leukocytes. J. exp. Med. 128, 991. MANTOVANI B. (1975) Different roles of IgG and complement receptors in phagocytosis by polymorphonuclear leukocytes. J. Immunol. 115, 15. MANTOVANI B., RABINOVITCH M. & NUSSENZWEIG V. (1972) Phagocytosis of immune complexes by macrophages. J. exp. Med. 135, 780. MESSNER R.P. & JELLINEK J. (1970) Receptors for human yG globulin on human neutrophils. J. Clin. Invest. 49, 2165. QUIE P.G., MESSNER R.P. & WILLIAMS R.C., JR (1968) Phagocytosis in subacute bacterial endocarditis: Localization of the primary opsonic site to Fc fragment. J. exp. Med. 128, 553. RABINOVITCH M. (1967) The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp. Cell Res. 46, 19. REYNOLDS H.Y., ATKINSON J.P., NEWBALL H.H. & FRANK M.M. (1974) Receptors for immunoglobulin and complement on human and rabbit alveolar macrophages. Clin. Res. 22, 427A. SCRIBNER D.J. & FAHRNEY D. (1976) Neutrophil receptors for IgG and complement: Their role in the attachment and ingestion phases of phagocytosis. J. Immunol. 116, 892. SPIEGELBERG H.L. (1974) Biological activities of immunoglobulins of different classes and subclasses. Advanc. Immunol. 19, 259. STOSSEL T.P. (1975) Phagocytosis: recognition and ingestion. Semin. Hematol. 12, 83.
Neutrophil receptors for staphylococci TAN J.S., WATANAKUNAKORN C. & PHAIR J.P. (1971) A modified assay of neutrophil function: use of lysostaphin to differentiate defective phagocytosis from impaired killing. J. Lab. clin. Med. 78, 316. VERHOEF J., PETERSON P.K. & QUIE P.G. (1977) Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: a quantitative assay using 3H-thymidine labeled bacteria. J. Immunol. Methods. 14, 303.
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VERHOEF J., PETERSON P.K., KIM Y., SABATH L.D. & QUIE P.G. (1977) Opsonic requirements for staphylococcal phagocytosis: heterogeheity among strains. Immunology, 33, 191. WILLIAMS R.C., JR & QUIE P.G. (1971) Opsonic activity of agammaglobulinemic human sera. J. Immunol. 106, 51.
Human polymorphonuclear leucocyte receptors for staphylococcal opsonins
J. VERHOEF *, P. K. PETERSON & P. G. QUIE Departments of Medicine and Pediatrics, University of Minnesota Medical School, Minneapolis, Minnesota, U.S.A.
Received 25 November 1976; accepted for publication 20 December 1976
ingestion as well as the attachment phase of phagocytosis.
Summary. The presence of receptors on the plasma membrane of human polymorphonuclear (PMN) leucocytes for factors related to complement and for the Fc region of immunoglobulin has not been clearly defined for opsonized bacteria. To separate the activity of these two receptors, the uptake of [3H]thymidine labelled staphylococci opsonized with normal serum or heat-inactivated serum was measured. Phagocytosis was depressed when bacteria opsonized with normal serum were incubated with trypsin-treated leucocytes, suggesting that complement receptors of human PMN leucocytes are trypsin-sensitive. Phagocytosis of bacteria opsonized with heat-inactivated serum was not depressed by trypsin, but was blocked by incubating PMN leucocytes with heat-aggregated IgG and by incubating opsonized bacteria with protein A. In experiments performed to quantify the number of bacteria attached to but not ingested by PMN leucocytes, it was shown that both complement and Fc receptors participate in the ingestion phase of phagocytosis. Cell membranes of human PMN leucocytes possess two receptors for opsonized staphylococci; a complement receptor which is utilized when bacteria are opsonized in normal serum and an Fc receptor when bacteria are opsonized in heatinactivated serum. Both receptors participate in the
INTRODUCTION The process of phagocytosis has been separated into two distinct phases: attachment and ingestion (Rabinovitch, 1967; Stossel, 1975). The attachment of particles to phagocytic cell membranes is generally believed to be mediated through receptors with certain immunological specificities. In the case of macrophages and mononuclear phagocytes, receptors have been described for particles coated with IgM, IgG and a modified component of complement (C3b) (Lay & Nussenzweig, 1968; Holland, Holland & Cohn, 1972; Mantovani, Rabinovitch & Nussenzweig, 1972; Speigelberg, 1974; Reynolds, Atkinson, Newball & Frank, 1974; Griffin, Bianco & Silverstein, 1975; Bianco, Griffin & Silverstein, 1975; Griffin, Griffin, Leider & Silverstein, 1975). Macrophage receptors for IgG-coated particles (Fc receptors) have been shown to mediate both attachment and ingestion, however, receptors specific for complement-coated particles are involved only in the attachment phase and do not independently participate in particle ingestion unless the macrophages are activated. Although there is evidence to suggest that the receptors on polymorphonuclear (PMN) leucocytes and macrophages have similar functions (Quie, Messner & Williams, 1968; Messner & Jellinek, 1970; Henson, Johnson & Spiegelberg, 1972;
* Present address: Laboratory for Microbiology, Catharijnesingel 59, Utrecht, The Netherlands. Correspondence: Dr Jan Verhoef, Laboratory for Microbiology, Catharijnesingel 59, Utrecht, The Netherlands.
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Mantovani, 1975; Scribner & Fahrney, 1976), this has not been established with certainty. Most studies of phagocyte receptors have been performed using either opsonized sheep erythrocytes or immune complexes. The functional existence and relative importance of Fc and complement receptors for the phagocytosis of bacteria is unclear. In this study the characteristics of PMN leucocyte receptors for staphylococci were investigated. Evidence is presented which suggests that bacteria opsonized in the presence of complement and immunglobulin are primarily phagocytized via the complement receptor, and that this receptor is involved both in the attachment and in the ingestion phases of phagocytosis by PMN leucocytes.
MATERIALS AND METHODS
Bacterial strains Two Staphylococcus aureus strains, Cowan I and Wood 46, and three Staphylococcus epidermidis strains, Her and Bra (isolates from patients with S. epidermidis endocarditis) and 407 (a commensal strain) were used. The S. epidermidis strains belonged to biotype 1 (Baird-Parker, 1974). Radioactive labelling The method used to measure bacterial attachment, ingestion and killing by PMN leukocytes has been reported in detail elsewhere (Verhoef, Peterson & Quie, 1977) and was modified slightly for the purposes of this study. One-tenth millilitre of an overnight culture of bacteria was inoculated into 10 ml Mueller-Hinton broth (Difco, Detroit, Michigan) containing 002 mCi thymidine methyl 3H-labelled (sp. act. 6-7 Cifmmol, New England Nuclear, Boston, Massachusetts). After 18 h growth at 370, the bacteria were washed three times in phosphate-buffered saline, pH 7-4 (PBS). A final bacterial concentration of 5 x 108 cfu/ml PBS was obtained using a spectrophotometric method confirmed by pour plate colony counts. Leucocytes Forty millilitres of blood were drawn from healthy donors in a syringe containing 200 u heparin. The erythrocytes were sedimented for 1 h in 6%. dextran '70' (Cutter Labs, Berkeley, California) in normal saline (10 ml blood: 3 ml saline). The leucocyte-rich plasma was withdrawn and centrifuged at 160 g for 5 min. The resulting pellet was washed twice in
heparinized saline (10 u heparin/10 ml saline). Using a standard method, total and differential leucocyte counts were performed, and the final leucocyte pellet was resuspended to a concentration of 107 PMN leucocytes/ml Hanks's balanced salt solution with 1 % gelatin (HBSS).
Treatment of PMN leucocytes with heat-aggregated IgG PMN leucocytes were incubated for 30 min at 370 in HBSS containing 5 mgy heat-aggregated IgG in HBSS. The leucocytes were then washed twice in HBSS. Control (untreated) leucocytes were likewise incubated and washed in HBSS. Trypsin treatment of PMN leucocytes Using a method similar to that described for macrophages (Bianco et al., 1975) PMN leucocytes were incubated with 1 mg/ml of trypsin (Difco) in HBSS for 30 min at 37°. The leukocytes were then washed twice in HBSS containing 1 mg/ml soya bean trypsin inhibitor (Worthington Biochemical Corporation, Freehold, New Jersey) to terminate trypsin digestion. Control (untreated) leucocytes were likewise incubated and washed in HBSS.
Opsonins Serum from five normal donors was pooled and kept frozen in 1-ml aliquots at -70°. C2-deficient serum was obtained from a patient with inherited complete C2 deficiency (Kim, Friend, Dresner, Yunis & Michael, 1977). Serum was obtained from an asymptomatic child with C3 deficiency (23 mg/100 ml C3) in which levels of all other complement components were normal (kindlyprovided by Dr Y. Kim, University of Minnesota). IgG deficient serum was obtained from a child with congenital rubella syndrome in which IgG, IgA and IgE were nondetectable by Mancini radial immunodiffusion and by Ouchterlony methods. This serum contained 39 mg% IgM. These sera were stored in 0-2-ml aliquots at -70°. Shortly before use the aliquots were thawed and diluted to a final concentration of 10% in HBSS. Heat-inactivated normal serum was prepared by heating aliquots at 560 for 1 h and then diluting to a final concentration of 10%.
Opsonization of bacteria 015 ml of a bacterial concentration of 5 x 108 cfu/ml was added to a plastic tube (12 x 75 mm, Falcon, Oxnard, California) containing 0 8 ml of opsonin. After 60 min incubation at 370, the mix-
Neutrophil receptors for staphylococci tures were centrifuged at 1600 g for 15 min. The supernatants were discarded and the bacterial pellets resuspended in 0 75 ml HBSS.
Treatment of opsonized bacteria with protein A In one series of experiments bacteria which had been opsonized with normal serum and with heatinactivated serum were incubated for 30 min at 370 with purified protein A (kindly provided by Dr A. Forsgren, University of Malmo, Sweden) in HBSS (150 jug/ml). After centrifuging at 1600 g for 15 min, the bacterial pellets were resuspended in HBSS. After opsonization, control bacteria were incubated in HBSS, centrifuged and resuspended in HBSS.
Phagocytosis mixtures 0-75 ml of the leucocyte suspension was added to the plastic tubes containing 0 75 ml of opsonized bacteria. The final bacteria: PMN leucocyte ratio was approximately 10 : 1 in all experiments. The phagocytosis mixtures were tumbled at 10 r.p.m. at 370 in a rotating rack (Fisher Roto-Rack, Fisher Scientific Co., Chicago, Illinois).
Determination ofPMN leucocyte bacterial uptake and killing Immediately after the phagocytosis mixtures were constituted, duplicate 5-,ul samples were taken for plating to determine the total cfu added to the mixtures at time '0'. To determine the leucocyteassociated bacterial population, duplicate 100 pl samples were taken from the phagocytosis mixtures with an Eppendorf pipette at 3-, 10- and 20-min intervals and placed in 3 ml cold PBS in polypropylene vials (Bio-Vials, Beckman, Chicago, Illinois). The vials were centrifuged for 5 min at 160 g at 40 and the leucocyte pellets washed twice with ice cold PBS. The final leucocyte pellets were disrupted by vigorous mixing in 2 5 ml sterile distilled water. From these suspensions duplicate 5 ul samples were taken with an Eppendorf pipette to determine the viable leucocyte-associated bacterial population by plating in nutrient agar. Cfu were counted after 18 h incubation at 37°. The final 2-5-ml suspensions were centrifuged at 1600 g for 15 min, and the total leucocyte-associated population of bacteria (alive and dead) was then determined by solubilizing the pellets in 2*5 ml scintillation liquid (toluene containing fluoralloy (TLA, Beckman) and 20%Y Biosolve-3 (Beckman)) and counting in a liquid scintillation counter (Beckman LS-250). To determine the total F
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bacteria-associated c.p.m. (representing leucocyteassociated bacteria plus extracellular bacteria) duplicate 100-pl samples were taken at the end of the assay period, placed in 2-5 ml water and centrifuged at 1600 g for 15 min. The pellets were resuspended in scintillation liquid and counted. An average of the duplicate values was used for all calculations. The per cent of the total bacterial population that was leucocyte-associated at a given sampling time (per cent uptake) was calculated using the following formula: per cent uptake = c.p.m. in leucocyte pellet x I0. c.p.m. in total bacterial pellet (Formula 1) The leucocyte-associated bacterial population that was viable at a given sampling time was calculated according to this formula: per cent viable leucocyte-associated bacteria = per cent cfu in leucocyte pellet x 100. per cent uptake (Formula 2) where the denominator was obtained from Formula 1 and the numerator was derived using Formula 3: Cfu in leucocyte pellet at sampling time 100 cfu at time '0' (Formula 3) The killed population of bacteria was determined according to Formula 4: per cent killed leucocyte associated bacteria = per cent uptake (Formula 1)per cent viable leucocyte-associated bacteria (Formula 2).
Determination of the attached bacterial population To lyse the extracellular population of bacteria, including those bacteria that were attached to but not ingested by the leucocytes, duplicate 100 ,ul samples were placed in PBS containing 1 ,ug/ml lysostaphin (Schwarz-Mann, Orangeburg, New York). These samples were then incubated at 370 for 30 min followed by washing the leucocytes twice in PBS. Simultaneous samples were processed in the standard manner to determine the total leucocyteassociated bacterial population (attached plus ingested bacteria). Bacterial uptake was calculated as outlined above. The difference between the per
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J. Verhoef, P. K. Peterson & P. G. Quie
cent uptake calculated from the samples placed in PBS and the samples placed in PBS containing lysostaphin was considered to represent the attached bacterial population. As less than 10% of the total c.p.m. were leucocyte-associated at 20 min when bacteria without opsonin were added to the phagocytosis mixtures, it appeared that washing of the leucocytes eliminated most of the extracellular bacteria and that lysostaphin lowered the leucocyteassociated cpm primarily by lysing the attached bacterial population. To confirm lysostaphin activity, control mixtures containing the same concentration of bacteria and serum but no leucocytes were also sampled into PBS and PBS containing 1 ug/ml lysostaphin followed by incubation at 370 for 30 min. After centrifuging at 1600 g, the bacterial pellets were suspended in scintillation liquid and counted.
RESULTS PMN leucocyte receptors for staphylococcal phagocytosis To study whether two distinct receptors for staphylococcal phagocytosis could be demonstrated, PMN leucocytes were (1) treated with trypsin to inactivate potential complement receptors and (2) incubated with heat-aggregated IgG to block potential Fc receptors. Staphylococci which had been opsonized in normal serum and in heat-inactivated serum were then added to these leucocytes as well as to untreated control cells. Fig. 1 shows the results of this experiment using S. aureus Wood 46. As can be seen in Fig. la, trypsinization of the leucocytes markedly reduced their ability to take up bacteria which had been opsonized in normal serum but did not significantly change uptake of bacteria opsonized with heat-inactivated serum. Whereas 85 %o of bacteria opsonized with normal serum were leucocyte-associated after 20 min incubation with untreated leucocytes, only 21 %o were taken up by trypsinized cells. When the bacteria were opsonized with heat-inactivated serum, 49%4 were leucocyteassociated when control cells were used and 44%/o when trypsinized leucocytes were studied. Therefore, the receptors for bacteria opsonized in the presence of complement are trypsin sensitive, and the receptors for bacteria opsonized without complement are not affected by trypsin. Although trypsinization of PMN leucocytes did not significantly affect uptake of bacteria opsonized
10°r
0X a
(~~~bac .0 + PMN normol)
-------
100 _ (b)
N + (bac ( PMN*normaI) bac.N+ \
80 -
PM Agg IqG) PMN
60 -
( bac -H + 4 PMN * norma I) (bac.H + k PMN- AggIgG)
20
0
3
10
20 Time (min)
Figure 1. Uptake of S. aureus Wood 46 by trypsinized (a) and heat-aggregated IgG treated (b) polymorphonuclear (PMN) leucocytes. Bacteria were opsonized with normal serum (bac. N), heat-inactivated serum (bac. H) and Hanks's balanced salt solution (HBSS) (bac. 0) before being added to leucocytes which had been incubated in HBSS (PMN. normal), HBSS containing trypsin (PMN.tryp) and HBSS containing heataggregated IgG (PMN. Agg IgG).
with heat-inactivated serum, pre-incubation of cells with heat-aggregated IgG did reduce their ability to take up these bacteria (Fig. Ib). Whereas 49%4 of bacteria opsonized with heat-inactivated serum were leucocyte-associated after 20 min incubation with untreated leucocytes, only 26% were taken up by cells treated with heat-aggregated IgG. Treatment of leucocytes with heat-aggregated IgG did not, however, significantly affect their ability to take up bacteria opsonized with normal serum (Fig. lb). Approximately 80%Y of bacteria opsonized with normal serum were taken up after 20 min incubation with both control and heat-aggregated IgG treated leucocytes. Thus, PMN leucocyte receptors for bacteria opsonized in the absence of complement were
Neutrophil receptors for staphylococci blocked by incubation with heat-aggre.gated IgG, but the receptors for bacteria opsonized in the presence of complement remained intact. Results similar to the findings with S. aureus Wood 46 were obtained with S. aureus Cowvan I and with S. epidermidis Her and 407 (data Xnot presented). When S. epidermidis Bra was stucdied, however, trypsinization of the leucocytes had le.ss effect on the uptake of bacteria opsonized with normal serum. After 20 min incubation, 89%4 of S. epidermidis Bra were taken up by untreated lebucocytes compared with 75%. by trypsinized leucocytes. As was observed with the other four strain,s, treatment of leucocytes with heat-aggregated IgG did not significantly reduce their ability to take up S. epidermidis Bra opsonized with normal serum (dEata not shown). The effect of protein A on the function of PMN leucocyte receptors Results from the above experiments suzgested that although a distinct Fc receptor anid complement receptor for staphylococcal phagoc)itosis could be identified, when staphylococci were opsonized in normal serum, i.e. in the presence of immunoglobulin and complement, they were tak en up by PMN leucocytes primarily via the comple ment receptor To further study the nature of tihese receptors, opsonized bacteria were incubated in HBSS con100* 80 .
-2
i(bacN +HBSS) (bac- N +PA)
60 .
c
(bac H+HBSS)
I(boc H +PA) -
Time (min)
Figure 2. Effect of protein A on uptake of S. aureus Wood 46 by polymorphonuclear leucocytes. Bacteria were opsonized with normal serum (bac.N) and heat-inactivated serum (bac.H) before being incubated in Hanks's Balanced Salt Solution (HBSS) and in HBSS containing protein A (PA). After centrifuging and resuspending the bacterial pellets in HBSS, normal leucocytes were added to the bacteria.
235
taining staphylococcal protein A (a protein known to react with the Fc fragment of IgG (Forsgren & Sjdquist, 1966)) before adding the bacteria to PMN leucocytes. Fig. 2 demonstrates the results of this experiment using S. aureus Wood 46, a strain deficient in cell wall protein A. Whereas protein A treatment of bacteria opsonized with heat-inactivated serum significantly reduced bacterial uptake (I0%Y uptake after 20 min incubation compared with 40%4 uptake of untreated bacteria), the effect of protein A on bacteria opsonized with normal serum was minimal (55 % uptake compared with 67%4 uptake of untreated bacteria). Therefore, the receptor for bacteria opsonized with heat-inactivated serum is specific for the Fc fragment of immunoglobulin and does not appear toplayan important rolein the phagocytosis of bacteria opsonized with normal serum. The effect of opsonization with C2 apd C3 deficient sera on the function of PMN leucocyte receptors Two strains of staphylococci were selected to study the importance of complement factors on phagocytosis: S. aureus Cowan I, known to depend on the presence of the classical complement pathway for effective opsonization and S. epidermidis 407, known to be effectively opsonized by the alternative complement pathway (Verhoef, Petersen, Kim, Sabath & Quie, 1977). PMN leucocyte uptake of S. aureus Cowan I was decreased when bacteria were opsonized with both C2 deficient and C3 deficient sera (Fig. 3a). When opsonized with normal serum, 89%Y of the bacteria were leucocyte-associated after 20 min incubation compared with 51 Y. of bacteria opsonized with C3 deficient serum. The uptake of S. epidermidis 407 was also reduced when bacteria were opsonized with C3 deficient serum but was not significantly affected by opsonization with serum deficient in C2
(Fig. 3b). Approximately 90%. of bacteria were leucocyte-associated when opsonized with normal serum and C2 deficient serum compared with 52%0 uptake of bacteria opsonized with C3 deficient serum. Efficient attachment therefore required activated components of C3 achieved via the classical pathway (S. aureus Cowan I) or the alternative pathway (S. epidermidis 407). The role of PMN leucocyte Fc and complement receptors in the attachment and ingestion phases of
phagocytosis To study the role of the Fc and complement receptors in the ingestion as well as the attachment phases of
J. Verhoef, P. K. Peterson & P. G. Quie
236
The size of the attached population of leucocyteassociated bacteria was also assessed by incubating samples from the phagocytosis mixtures in lysostaphin in order to lyse the extracellular (including attached) population of bacteria. The number of leucocyte-associated bacteria in these samples was then compared with the number of leucocyteassociated bacteria in samples processed without lysostaphin treatment. The validity of this method was confirmed when it was established that samples from control mixtures containing the same concentration of bacteria used in the phagocytosis mixtures but without leucocytes lost over 90%Y of the radio-
0a
@
100 _ (
100 _ (a)
(norma serum)
a.
def ~~A ~~~~~~~~ (02 serum) ent)
*(bocN + k
c
80-o
\ PMN -AggIgGJ
80 60-
60-
(03
deficient)
+ -*(bac-H PMN. normal)
serum
40
40
( heat- inactivated serum
20
20
0
3
20
10
'
0 100 _
Time (min)
Figure 3. Effect of opsonization of S. aureus Cowan I (a) and S. epidermidis 407 (b) with normal serum, C2-deficient serum, C3-deficient serum and heat-inactivated serum on uptake by normal polymorphonuclear leucocytes. Bacteria were incubated for 60 min in each opsonic source before being added to the leucocytes.
phagocytosis, experiments were performed to determine the per cent of leucocyte-associated bacteria that were killed at each sampling time. If attachment occurred without ingestion presumably a relatively small number of leucocyte-associated bacteria would be killed. Bacterial killing was determined after phagocytosis via the complement receptor by using leucocytes treated with heat-aggregated IgG in order to block Fc receptors and by using bacteria opsonized with IgG deficient serum. After 20 min incubation, approximately 95% of S. aureus Cowan I which had been opsonized with normal serum and with IgG deficient serum were killed when taken up by normal leucocytes; 84% were killed when taken up by leucocytes pre-treated with heat-aggregated IgG (data not given).
80
-
+ b(ac.IGdef \ ~~~~~PMN-normal
/
610
40
20
0
3
20
10 Time (min)
Figure 4. Effect of lysostaphin on measured PMN leucocyte uptake of S. aureus Cowan I opsonized with normal serum and heat-inactivated serum (a) and IgG-deficient serum (b). Bacteria were opsonized with normal serum (bac.N), heatinactivated serum (bac.H) and IgG-deficient serum (bac.IgG def) before being added to normal leucocytes (PMN.normal) and heat-aggregated IgG treated leucocytes (PMN.Agg IgG). Samples were placed in phosphate-buffered saline (closed symbols) and in phosphate-buffered saline containing lysostaphin (open symbols). Following incubation, the leucocytes were washed, and bacterial uptake was measured.
Neutrophil receptors for staphylococci activity from the bacteria after 30 min incubation in lysostaphin, and bacteria incubated without lysostaphin lost none. When S. aureus Cowan I which had been opsonized with normal serum was added to PMN leucocytes pre-treated with heat-aggregated IgG, 91% of the bacteria were leucocyte-associated after 20 min (Fig. 4a). Incubation of simultaneous samples in lysostaphin reduced the measured leucocyte-associated bacterial population by only 7%. When bacteria which had been opsonized in heatinactivated serum were added to untreated leucocytes there was uptake of 40% of bacteria after incubation of samples in lysostaphin resulting in only a slight decrease in the measured leucocyte-associated bacterial population (Fig. 4a). Fig. 4b shows the effect of lysostaphin treatment of samples taken from a phagocytosis mixture containing bacteria opsonized with IgG-deficient serum. After 20 min incubation 91% of the bacteria were determined to be leucocyteassociated when samples were not treated with lysostaphin compared with 71% following lysostaphin treatment. Again, lysostaphin did not have a marked effect on the measured leucocyte-associated bacterial population. Although 20% of bacteria opsonized with complement appeared to be attached and not ingested, these experiments suggest that ingestion as well as attachment is associated with both the complement receptor and the Fc receptor of human PMN leucocytes.
DISCUSSION By studying the effects of trypsinization and of heataggregated IgG on PMN leucocyte uptake of [3H]-thymidine labelled staphylococci opsonized with normal serum and with heat-inactivated serum, two distinct leucocyte receptors for bacterial attachment could be identified. An Fc receptor appeared to play a major role in the attachment of bacteria opsonized in the absence of complement (heatinactivated serum). When bacteria were opsonized with heat-inactivated serum, trypsinization of leucocytes did not interfere with staphylococcal attachment. When bacteria were opsonized in the presence of both immunoglobulin and complement (normal serum), however, a receptor with complement specificity appeared to function as the major receptor for bacterial attachment. Trypsinization markedly reduced the ability of PMN leucocytes to
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take up bacteria opsonized with normal serum. Treatment with heat-aggregated IgG did not significantly impair the ability of PMN leucocytes to take up bacteria opsonized with normal serum but greatly reduced uptake of bacteria opsonized with heat-inactivated serum. Additional evidence for a receptor with Fc specificity and for the primacy of the complement receptor for attachment of bacteria opsonized with normal serum came from experiments in which opsonized bacteria were treated with protein A. These experiments were performed in an attempt to block attachment from the bacterial side of the staphylococcal-PMN leucocyte interaction. Protein A is known to have the capacity to combine with the Fc fragment of IgG (Forsgren & Sjbquist, 1966). When bacteria opsonized with heat-inactivated serum were treated with protein A prior to being added to PMN leucocytes, subsequent uptake was significantly reduced. When bacteria opsonized with normal serum were studied, however, uptake was not significantly affected. To determine the relative importance of the third component of complement for attachment, the phagocytosis of two staphylococcal strains with different opsonic requirements was studied. Whereas phagocytosis of only the S. aureus strain was significantly reduced after opsonization with C2 deficient serum, phagocytosis of both the S. aureus and S. epidernidis strains was decreased when opsonized with C3 deficient serum. Thus, activation of C3 at the bacterial cell surface appears to play a central role in staphylococcal phagocytosis. To determine whether the Fc and complement receptors participated in both the attachment and ingestion phases of phagocytosis, experiments were performed using S. aureus Cowan I opsonized with normal serum, IgG deficient serum (which contained normal complement levels) and heat-inactivated serum. Samples from phagocytosis mixtures were incubated with lysostaphin to eliminate the extracellular, attached leucocyte-associated bacterial population. Lysostaphin is an enzyme which is known to lyse S. aureus cell wall and which does not enter leucocytes (Tan, Watanakunakorn & Phair, 1971). Incubation of samples in lysostaphin did not significantly reduce the measured leucocyte-associated bacterial population when bacteria were opsonized with normal serum, IgG-deficient serum or heat-inactivated serum. Therefore, it appeared that both the Fc receptor and the complement receptor
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were associated with the attachment as well as with the ingestion phases of phagocytosis. Further evidence to support the hypothesis that complement receptors on PMN leucocytes are involved in ingestion as well as in attachment came from the observation that the great majority of bacteria opsonized with IgG deficient serum were rapidly killed. Furthermore, there was also rapid killing when bacteria opsonized with normal serum were incubated with PMN leucocytes treated with heat-aggregated IgG. These findings are in agreement with the report of Messner & Jellinek (1970) who showed normal killing of S. aureus by PMN leucocytes after blocking Fc receptors with Fc fragments and of Williams & Quie (1971) who reported normal staphylococcal killing by PMN leucocytes in the presence of agammaglobulinaemic serum. Somewhat different functions of the Fc and complement receptors of mononuclear phagocytes appear to be operative in the phagocytosis of sheep erythrocytes (Griffin et al., 1975; Bianco et al., 1975). In this system sheep erythrocytes are opsonized with IgG or with IgG and complement. The plasma membranes of mononuclear phagocytes have been shown to contain receptors for the Fc portion of IgG and for the activated form of the third component of complement (C3b). Internalization (ingestion) of particles is triggered by IgG. The Fc receptor mediates both the binding of particles to the plasma membrane and their ingestion. However, the macrophage complement receptor appears to play a role only in the attachment phase of phagocytosis. In contrast, complement receptors on PMN leucocytes participate in both attachment and ingestion of
staphylococci.
ACKNOWLEDGMENTS This study was supported in part by grants Al 08821 and Al 06931 from the National Institutes of Allergy and Infectious Diseases. Dr Verhoef was supported by the Netherlands Organization for the Advancement of Pure Research (ZWO). Present address: Laboratory for Micro-
biology, Catharijnesingel 59, Utrecht, Netherlands. Dr Peterson was a recipient of a Bristol Research Fellowship in Infectious Diseases at the time of these studies. Dr Quie is an American Legion Memorial Heart Research Professor.
REFERENCES BAIRD-PARKER A.C. (1974) The basis for the present classification of staphylococci and micrococci. Ann. N. Y. Acad. Sci. 236, 7. BIANCO C., GRIFFIN F.M. & SILVERSTEIN S.C. (1975) Studies of the macrophage complement receptor: alteration of receptor function upon macrophage activation. J. exp. Med. 141, 1278. FORSGREN A. & SJOQUIST J. (1966) 'Protein A' from S. aureus I. Pseudoimmune reaction with human y-globulin. J. Immunol. 97, 822. GRIFFIN F.M., BIANCO C. & SILVERSTEIN S.C. (1975) Characterization of the macrophage receptor for complement and demonstration of its functional independence from the receptor for the Fc portion of immunoglobulin G. J. exp. Med. 141, 1269. GRIFFIN F.M., GRIFFIN J.A., LEIDER J.E. & SILVERSTEIN S.C. (1975) Studies on the mechanisms of phagocytosis. J. exp. Med. 142, 1263. HENSON P.M., JOHNSON H.B. & SPIEGELBERG H.L. (1972) The release of granule enzymes from human neutrophils stimulated by aggregated immunoglobulins of different classes and subclasses. J. Immunol. 109, 1182. HOLLAND P., HOLLAND N.H. & COHN Z.A. (1972) The selective inhibition of macrophage phagocytic receptors by antimembrane antibodies. J. exp. Med. 135, 458. KIM Y., FRIEND P.S., DRESNER I.G., YUNIs E.J. & MICHAEL A.F. (1977) Inherited deficiency of the second component of complement with membranoproliferative glomerulonephritis. Amer. J. Med. (In press.) LAY W.H. & NUSSENZWEIG V. (1968) Receptors for complement on leukocytes. J. exp. Med. 128, 991. MANTOVANI B. (1975) Different roles of IgG and complement receptors in phagocytosis by polymorphonuclear leukocytes. J. Immunol. 115, 15. MANTOVANI B., RABINOVITCH M. & NUSSENZWEIG V. (1972) Phagocytosis of immune complexes by macrophages. J. exp. Med. 135, 780. MESSNER R.P. & JELLINEK J. (1970) Receptors for human yG globulin on human neutrophils. J. Clin. Invest. 49, 2165. QUIE P.G., MESSNER R.P. & WILLIAMS R.C., JR (1968) Phagocytosis in subacute bacterial endocarditis: Localization of the primary opsonic site to Fc fragment. J. exp. Med. 128, 553. RABINOVITCH M. (1967) The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp. Cell Res. 46, 19. REYNOLDS H.Y., ATKINSON J.P., NEWBALL H.H. & FRANK M.M. (1974) Receptors for immunoglobulin and complement on human and rabbit alveolar macrophages. Clin. Res. 22, 427A. SCRIBNER D.J. & FAHRNEY D. (1976) Neutrophil receptors for IgG and complement: Their role in the attachment and ingestion phases of phagocytosis. J. Immunol. 116, 892. SPIEGELBERG H.L. (1974) Biological activities of immunoglobulins of different classes and subclasses. Advanc. Immunol. 19, 259. STOSSEL T.P. (1975) Phagocytosis: recognition and ingestion. Semin. Hematol. 12, 83.
Neutrophil receptors for staphylococci TAN J.S., WATANAKUNAKORN C. & PHAIR J.P. (1971) A modified assay of neutrophil function: use of lysostaphin to differentiate defective phagocytosis from impaired killing. J. Lab. clin. Med. 78, 316. VERHOEF J., PETERSON P.K. & QUIE P.G. (1977) Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: a quantitative assay using 3H-thymidine labeled bacteria. J. Immunol. Methods. 14, 303.
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VERHOEF J., PETERSON P.K., KIM Y., SABATH L.D. & QUIE P.G. (1977) Opsonic requirements for staphylococcal phagocytosis: heterogeheity among strains. Immunology, 33, 191. WILLIAMS R.C., JR & QUIE P.G. (1971) Opsonic activity of agammaglobulinemic human sera. J. Immunol. 106, 51.
