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The Journal of Immunology, 2002, 168: 816-824.
Copyright © 2002 by The American Association of Immunologists

The Bacterial Peptide N-Formyl-Met-Leu-Phe Inhibits Killing of Staphylococcus epidermidis by Human Neutrophils in Fibrin Gels1

Yongmei Li*, John D. Loike*, Julia A. Ember{dagger}, P. Patrick Cleary{ddagger}, Emily Lu*, Sadna Budhu*, Long Cao§ and Samuel C. Silverstein*

* Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY 10032; {dagger} BD PharMingen, San Diego, CA 92121; {ddagger} Department of Microbiology, University of Minnesota Medical School, Minneapolis, MN 55455; and § New York Blood Center, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To study human neutrophil (polymorphonuclear leukocyte (PMN)) migration and killing of bacteria in an environment similar to that found in inflamed tissues in vivo, we have used fibrin gels. Fibrin gels (1500 µm thick) containing Staphylococcus epidermidis were formed in Boyden-type chemotaxis chambers. PMN migrated <300 µm into these gels in 6 h and did not kill S. epidermidis when the gels contained heat-inactivated serum, C5-deficient serum, a streptococcal peptidase specific for a fragment of cleaved C5 (C5a), or anti-C5aR IgG. In contrast, in gels containing normal human serum, PMN migrated ~1000 µm into the gels in 4 h and into the full thickness of the gels in 6 h, and killed 90% of S. epidermidis in 6 h. fMLP reduced PMN migration into fibrin gels and allowed S. epidermidis to increase by ~300% in 4 h, whereas leukotriene B4 stimulated PMN to migrate the full thickness of the gels and to kill 80% of S. epidermidis in 4 h. We conclude that both complement opsonization and C5a-stimulated chemotaxis are required for PMN bacterial killing in fibrin gels, and that fMLP inhibits PMN bactericidal activity in fibrin gels. The latter finding is surprising and suggests that in the presence of fibrin fMLP promotes bacterial virulence.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cellular mechanisms used by neutrophils (polymorphonuclear leukocytes (PMN)3) to locate and kill bacteria in tissues have, until recently, been largely unexplored. Abundant evidence indicates that chemoattractants or chemokines are required to promote PMN emigration from the vasculature, but few investigators have reported the role(s) of these substances in enabling PMN to locate and kill bacteria that gain access to tissue spaces (1, 2).

Under physiological conditions, tissue spaces contain three-dimensional arrays of matrix proteins (e.g., collagens). Following wounding or invasion of tissues by bacteria, fibrinogen-rich exudates lead to the formation of fibrin gels to which bacteria bind or in which they become embedded (3, 4). Fibrin also is deposited on prosthetic devices such as venous catheters, making them attractive substrates for adhesins expressed by Staphylococci and other bacteria (5). Staphylococcus aureus express a coagulase that promotes fibrin formation, while Streptococci express streptokinase, an enzyme that promotes fibrinolysis. Thus fibrin is involved in many aspects of bacterial infection.

We reported previously that PMN chemotaxis is under the dual regulation of chemoattractant and extracellular matrix protein components (6, 7). For example, PMN stimulated with fMLP or TNF-{alpha} form zones of close apposition on fibrin and do not migration into these gels. In contrast, PMN stimulated with leukotriene B4 (LTB4) or IL-8 form zones of loose apposition on fibrin and migrate effectively into these gels. In addition, fMLP or TNF-{alpha} inhibited PMN migration through fibrin gels in response to LTB4 or IL-8. These findings suggested that fMLP might exert an inhibitory effect on PMN killing of bacteria at sites of fibrin deposition by blocking PMN migration into and in these sites.

To explore mechanisms by which PMN locate and kill bacteria in tissues, and to examine the roles of various chemoattractants/chemokines/cytokines in this process, we adapted a fibrin-gel system used by Hurst et al. (8) and by Rotstein et al. (9) to study phagocytosis and killing, respectively, of bacteria in a tissue matrix-like environment in vitro. In this report, we describe a method for quantitative assessment of PMN chemotaxis and killing of Staphylococcus epidermidis in fibrin gels, and of the effects of complement and of the chemoattractants fMLP, LTB4, and a fragment of cleaved C5 (C5a) on these processes. We report that C5a, generated by complement activation, is essential for PMN migration and killing of these bacteria in fibrin gels. LTB4 enhanced, and fMLP inhibited, PMN migration and bacterial killing. The latter finding demonstrates that in the presence of fibrin, fMLP can protect bacteria from attack by PMN.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Thrombin, fMLP, carboxypeptidase Y, cytochalasin D, and Histopaque 1077 and 1119 were from Sigma-Aldrich (St. Louis, MO). D-phenylanalyl-L-propyl-L-arginine chloromethyl ketone and LTB4 were from Calbiochem-Novabiochem (San Diego, CA). Human fibrinogen was from American Diagnostica (Greenwich, CT). Cell culture inserts (0.4-µm pore size, 24-well plate format), tissue culture plates (24-well and 48-well format), agar, and trypticase soy broth (TSB) were from BD Biosciences (Franklin Lakes, NJ). Heparin was from Elkins-Sinn (Cherry Hill, NJ). Rabbit monoclonal anti-human C5aR (C85-4124) and an isotype control anti-keyhole limpet hemocyanin rabbit mAb were a generous gift from Dr. J. A. Ember. Purified streptococcal C5a peptidase (SCPA) was from Dr. P. P. Cleary.

Staphylococcus epidermidis

S. epidermidis H753, a clinical isolate from the cerebrospinal fluid (CSF) of a patient with an infected CSF shunt, was provided by the Diagnostic Microbiology Laboratory at Columbia-Presbyterian Hospital (New York, NY). For experiments, 3% TSB was inoculated with S. epidermidis from a single colony and incubated with shaking overnight at 37°C. The overnight culture was subcultured into fresh TSB, grown to late log phase, pelleted, washed three times in PBS, and resuspended in PBS. The OD of this suspension at 600 nm was monitored, and CFU of S. epidermidis were determined by reference to a standard curve relating OD at 600 nm to the CFU of S. epidermidis.

Human sera

C5-deficient human serum was from Sigma-Aldrich. Normal human serum (NHS) was prepared by incubating human AB plasma (New York Blood Center, New York, NY) with 1 U/ml thrombin at room temperature for 15 min and centrifuging the mixture at 8000 x g to remove fibrin. NHS was then filter sterilized using 0.22-µm filters (Pall Gelman Laboratory, Ann Arbor, MI). Heat-inactivated human serum (HIS) was prepared by heating NHS at 56°C for 30 min. Zymosan-activated serum (ZAS) was prepared as described (10). All sera were stored at -80°C until use.

Human PMN

PMN were prepared as described (6). Briefly, fresh heparinized blood was obtained from healthy adult volunteers after informed consent. PMN were isolated by centrifugation on Histopaque 1077 and 1119 gradients. Contaminating RBCs were removed by hypotonic lysis. The purity of PMN isolated by this method was >95%, as determined by Wright-Giemsa staining. Purified PMN were resuspended in PBSG-HSA (PBS containing 0.5 mM Mg2+, 1 mM Ca2+, 5 mM glucose and 0.1% human serum albumin).

Formation of fibrin gels containing S. epidermidis

Tissue culture inserts were filled sequentially with 5 µl of PBSG-HSA containing 0.1 U of thrombin and 100 µl of PBSG-HSA containing 1 mg/ml purified human fibrinogen, 1 x 104 CFU of S. epidermidis, the indicated percentage (v/v) of serum (e.g., 1–40% NHS, 10 or 40% HIS, or 40% C5-deficient serum), with or without SCPA (0.01–10 µg/ml). The inserts were incubated for 5 min at room temperature to allow the fibrin to gel. These gels were ~1500 µm in thickness. Once the fibrin gel formed, D-phenylanalyl-L-propyl-L-arginine chloromethyl ketone (10-7 M, 10 µl) was added to the top of the gels to inhibit thrombin.

PMN chemotaxis

Inserts containing fibrin gels formed as described above were placed in 24-well tissue culture plates to form modified Boyden-type chemotaxis chambers. A total of 5 x 105 PMN in 100 µl of PBSG-HSA were placed on top of each gel. Alternatively, before addition to the migration chamber, PMN (5 x 106/ml) were pretreated at 4°C for 40 min in PBSG-HSA containing 2 µg/ml mAb against human C5aR, or 2 µg/ml control Ab. A total of 500 µl of PBSG-HSA alone or PBSG-HSA containing fMLP (10-6 or 10-8 M) or LTB4 (10-7 M) was added to the bottom compartment. The chambers were incubated at 37°C in a humidified incubator containing 5% CO2/95% air for 4–6 h, at which time the distance PMN penetrated into the fibrin gel was measured visually by focusing first on the surface of the gel (as marked by a drop of RBCs added on top of the gel at the end of the incubation) and then on cells at the leading front, and measuring the distance between the gel surface and the leading front with a micrometer mounted on the focusing knob of a Nikon phase-contrast microscope (Nikon, Melville, NY).

Enumeration of S. epidermidis in fibrin gels

Fibrin gels (100 µl) containing 40% NHS, 1 x 104 CFU of S. epidermidis, with or without 4 x 105–4 x 106 PMN were formed as described above. A total of 200 µl of PBS (no Ca2+ and Mg2+) containing 5 mg/ml trypsin, with or without 20 mM EDTA and 20 µM cytochalasin D (pH 10.4, 4°C), was added to each gel for 10 min to allow diffusion of phagocytosis inhibitors into the gel. The gels were then incubated at 37°C for 18 min. The liquefied gels were diluted with sterile distilled water and incubated for another 5 min at 37°C, as described (11), to completely lyse the PMN. Serially diluted samples were plated on TSB agar plates and incubated overnight at 37°C, and colonies were counted manually.

PMN killing of S. epidermidis embedded in fibrin gels

Two killing assays were used. In the first, fibrin gels (100 µl in volume) containing 1–2 x 104 CFU of S. epidermidis and 40% NHS were formed in culture inserts as described above. The inserts were then placed in a 24-well plate to form Boyden-type chemotaxis chambers. A total of 5 x 105 PMN in 100 µl of PBSG-HSA were overlaid on top of the gels, and the indicated chemoattractants in PBSG-HSA were placed in the bottom compartment. The chambers were incubated at 37°C for 4–6 h in a humidified incubator containing 5% CO2/95% air. The gels then were lysed and their content of viable S. epidermidis was assayed as described above. Thus, this assay measures the rate of PMN migration into the fibrin gels and killing of the embedded bacteria.

In the second assay, fibrin gels (100 µl in volume) containing 1 x 104 CFU of S. epidermidis, 5 x 105 PMN, 10% NHS, fMLP (0, 0.01 nM to 1 µM), and, where indicated, 1 U/ml carboxypeptidase Y were formed in 48-well tissue culture plates, incubated at 37°C for 90 min, and lysed, and the number of viable bacteria remaining was assayed, all as described above. Thus, the second assay measures PMN killing of bacteria within the fibrin gel.

PMN killing of S. epidermidis in suspension

Killing of S. epidermidis in suspension was assayed as described (12). Briefly, 500 µl of PBSG-HSA containing 10% NHS or 10% C5-deficient serum, 2.5 x 106 PMN, and 0.5 x 105 CFU of S. epidermidis with or without fMLP (10-6 M) or LTB4 (10-7 M) was placed in a sterile 1.5-ml Eppendorf tube, and the tube was incubated at 37°C on an Orbit Environ-shaker (Lab-Line Instruments, Melrose Park, IL) rotating at 200 rpm. After 90 min, a 100-µl sample was diluted in sterile distilled water to lyse PMN. Serial dilutions of the sample were plated on TSB agar plates and incubated at 37°C overnight, and the CFU of S. epidermidis was counted manually as described above.

Statistics

Experiments were performed at least three times in duplicate and are reported as the means ± SEM for the number of experiments indicated. Significance was obtained using a two-sample paired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Opsonization with IgG and the third component of complement are required for PMN to kill S. epidermidis H753

Preliminary experiments showed that >90% of S. epidermidis H753 was killed when incubated in suspension or in fibrin gels with 4 x 106/ml PMN in the presence of NHS, but not in the presence of NHS that had been preadsorbed with protein A to remove IgG class Abs (data not shown), or heated to inactivate complement (data not shown). Immunofluorescence studies using anti-human IgG and anti-human C3 confirmed that S. epidermidis incubated in NHS or in C5-deficient serum was coated with both IgG and C3 (data not shown) and that IgG directed against S. epidermidis found in NHS was needed to promote C3 fixation on these bacteria (data not shown). Thus, opsonization of S. epidermidis H753 with both IgG and complement was required for PMN to kill these bacteria under the conditions of these experiments.

Quantitative recovery of S. epidermidis from fibrin gels

Rotstein et al. (9) reported that PMN killed 90% of Escherichia coli embedded in gels formed with 1 mg/ml fibrinogen. In their study, bacteria were recovered from fibrin gels after trypsin digestion. However, they did not report the efficiency of recovery of bacteria from these gels, and they did not report the effects of digestion of the gels on recovery of viable bacteria. Therefore, we examined the recovery of bacteria in our system of fibrin gels that contained S. epidermidis, NHS, and the indicated number of PMN (Table IGo). The gels were digested as described in Materials and Methods, and we compared recovery of S. epidermidis in the presence or absence of cytochalasin D and EDTA.


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  		Table I. Effect of EDTA and cytochalasin D on recovery of S. epidermidis from fibrin gels1

 
More than 99% of S. epidermidis was recovered from gels containing S. epidermidis alone or S. epidermidis and 4 x 105 PMN, whether or not EDTA, an inhibitor of integrins (13), and cytochalasin D, an inhibitor of chemotaxis and phagocytosis (14), were included in the lysis buffer (Table IGo). However, when using a 10-fold greater number of PMN (4 x 106), only 75% of S. epidermidis was recovered from gels lysed in the absence of EDTA and cytochalasin D, a significant decrease in recovery (p < 0.01) as compared with >99% recovery from gels lysed in the presence of these inhibitors (Table IGo). Further studies showed >99% recovery of S. epidermidis when cytochalasin D was the sole inhibitor in the lysis buffer (data not shown). Thus, inclusion of cytochalasin D in the lysis buffer ensures full recovery of S. epidermidis from fibrin gels, even when the gels contain PMN at a concentration 8-fold in excess of that used in subsequent experiments.

PMN migrate into fibrin gels in a complement-dependent manner

To examine the factors that influence PMN chemotaxis into fibrin gels, gels containing NHS or HIS without or with S. epidermidis were formed in Boyden-type migration chambers as described in Materials and Methods. PMN were placed into the upper compartment of these chambers, and migration was measured by the distance PMN penetrated into the gels after a 6-h incubation at 37°C. PMN migration into these gels depended on the presence and concentration of NHS in the gels. As shown in Fig. 1Go, PMN migrated through the full thickness of the gels (1500 µm) in 6 h when the gels contained 40% NHS and S. epidermidis, but only ~80% of the thickness of the gels (~1200 µm) when S. epidermidis was not added to the gels. In contrast, PMN penetrated only 20% of the thickness of the gels (~300 µm) into fibrin gels containing 40% HIS or low concentrations (1–5%) of NHS, regardless of whether they contained S. epidermidis.



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  		FIGURE 1. Effect of serum concentration on PMN chemotaxis into fibrin gels. Fibrin gels (1500 µm thick, 100 µl in volume) containing NHS (1, 5, 10, or 40%) or HIS (10 or 40%) with or without 1 x 104 CFU of S. epidermidis were formed in Boyden-type migration chambers. PMN (5 x 105) were placed on top of each gel. The bottom compartments were filled with PBSG-HSA buffer, and the migration chambers were incubated at 37°C for 6 h. PMN migration into fibrin gels was determined visually by focusing first on the surface of the gel and then on cells at the leading front, and measuring the distance between the gel surface and the leading front using a micrometer on the microscope as described in Materials and Methods. Data represent the means ± SEM from four experiments, each performed in duplicate.

 
These results indicate that components released following complement activation are required to stimulate PMN migration into fibrin gels under these conditions. These results are consistent with Hurst and Wilton’s observation (8) that heating plasma clots that contained Capnocytophaga ocharacea abolished PMN migration into the clots. These experiments indicate that if S. epidermidis produce chemoattractant substances (15, 16), they do not release them in quantities sufficient to promote PMN migration under these conditions.

To determine whether activated complement components stimulate PMN migration into fibrin gels, we placed 10% ZAS (a source of C3a and C5a) in the bottom compartment of chemotaxis chambers and examined PMN migration into gels containing HIS alone or HIS and S. epidermidis. PMN migrated 90% of the full thickness of these gels in the presence of ZAS regardless of the presence or absence of bacteria (data not shown).

To identify the specific complement components involved, we performed two complementary experiments. First, we examined PMN migration into fibrin gels containing 40% C5-deficient serum and S. epidermidis. Under these conditions, PMN migrated only ~50 µm into these gels (Fig. 2Go). Second, we tested the effect of inhibiting C5aR function with a blocking mAb. PMN treated with anti-C5aR Ab appeared round and penetrated only ~100 µm after 6 h into gels containing 40% NHS and S. epidermidis (Fig. 2Go). PMN not treated with Ab or treated with an isotype control Ab appeared polarized and penetrated the full thickness (1500 µm) of the gels after 6 h. These experiments indicate that C5a is the primary chemoattractant mediating PMN migration into these gels.



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  		FIGURE 2. PMN migrate into fibrin gels in response to C5a. Fibrin gels (1500 µm thick, 100 µl in volume) containing 40% NHS or 40% C5-deficient serum and 1 x 104 CFU of S. epidermidis were formed in Boyden-type migration chambers. A total of 5 x 105 PMN in PBSG-HSA alone, or in PBSG-HSA with 2 µg/ml of anti-human C5aR Ab or 2 µg/ml isotype control Ab, were placed on top of the gels. The bottom compartments were filled with PBSG-HSA, and the migration chambers were incubated at 37°C. Shown is PMN migration into fibrin gels after 6 h, measured as described in Fig. 1Go. Data represent the means ± SEM of three experiments, each done in duplicate. **, p < 0.01 compared with control where PMN migrated into NHS-containing fibrin gels in the absence of Ab.

 
SCPA inhibits PMN migration and clearance of S. epidermidis in fibrin gels

SCPA is a serine protease that specifically cleaves and inactivates C5a (17). To determine whether degradation of C5a affects PMN bacterial killing in fibrin gels, we measured the effect of SCPA on PMN migration and bacterial killing in fibrin gels containing 40% NHS and S. epidermidis. In the absence of SCPA, PMN penetrated the full thickness of these gels (1500 µm) after 6 h (Fig. 1Go). SCPA reduced PMN migration in a dose-dependent manner (Fig. 3Go) with an IC50 of ~0.7 µg/ml. At 1 µg/ml SCPA maximally reduced PMN migration to ~400 µm (p < 0.01), a distance similar to that migrated by PMN into fibrin gels containing HIS (Fig. 1Go). Inhibition of PMN migration was associated with bacteria growth of 3-fold increase over the initial inoculum (Table IIGo). In contrast, at lower SCPA concentrations (0.01 and 0.1 µg/ml), PMN penetrated the full thickness of the gels (Fig. 3Go) and killed ~90% of the inoculum after 6 h (Table IIGo). These results suggest that SCPA inactivates C5a generated in these fibrin gels and, by doing so, reduces PMN migration into the gels and subsequent killing of bacteria. Thus, S. epidermidis are protected from PMN and are able to grow.



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  		FIGURE 3. SCPA reduces PMN chemotaxis into fibrin gels. Fibrin gels (1500 µm thick, 100 µl in volume) containing 40% NHS, 1 x 104 CFU of S. epidermidis, and SCPA at the indicated concentrations were formed in migration chambers. PMN (5 x 105) were placed on top of each gel. PBSG-HSA with the same concentration of SCPA as in the gels was placed in the bottom compartments, and the migration chambers were incubated at 37°C. Shown is the distance PMN migrated into fibrin gels after a 6-h incubation, measured as described in Fig. 1Go. Data represent the means ± SEM of three experiments, each done in duplicate.

 

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  		Table II. SCPA inhibits PMN bacterial killing in fibrin gels1

 
To test whether C5a per se is required for PMN to kill S. epidermidis, we mixed PMN and S. epidermidis in suspension in C5-deficient serum for 90 min at 37°C, as described in Materials and Methods. When PMN and bacteria were mixed together mechanically, PMN bactericidal activity occurred independent of chemotaxis because inhibitors of chemotaxis such as pertussis toxin did not affect bactericidal activity in suspension (data not shown). Under these conditions, PMN killed S. epidermidis in C5-deficient serum as effectively as in NHS (Table IIIGo). This indicates that C5a-mediated PMN activation is not needed for these cells to kill S. epidermidis. It strongly suggests that C5a serves an important role as a chemoattractant in PMN killing of S. epidermidis in fibrin gels.


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  		Table III. Killing of S. epidermidis in suspension is unaffected by the absence of C5 or the presence of fMLP or LTB41

 
fMLP inhibits, and LTB4 facilitates, C5a-stimulated PMN migration into S. epidermidis-containing fibrin gels

We previously reported (6) that PMN migration through fibrin gels is differentially regulated by chemoattractants. LTB4 and IL-8 stimulate PMN chemotaxis through fibrin gels. In contrast, fMLP and TNF-{alpha} inhibit PMN chemotaxis by activating {beta}1 integrins and causing the cells to adhere tightly to the underlying fibrin matrix (7). Those experiments were performed with albumin as the only serum protein in the gels and without bacteria.

To determine whether fMLP exerts similar effects in the presence of serum and/or C5a, we formed fibrin gels containing S. epidermidis and NHS in Boyden-type chemotaxis chambers. With no additional chemoattractant added in the bottom compartment (control), PMN migrated 1035 ± 66 µm (n = 6) or two-thirds the thickness of these gels after 4 h, and the full thickness of the gels (1465 ± 63, n = 5) after 6 h (Fig. 4Go). Placement of 10-6 M fMLP in the bottom compartment significantly (p < 0.001) reduced the migration distance to 536 ± 37 µm at 4 h (n = 8) and 843 ± 55 µm at 6 h (n = 5). At a 100-fold lower concentration (10-8 M), fMLP had no significant inhibitory effect (data not shown). In contrast, placement of LTB4 (10-7 M) in the bottom compartment enhanced the rate of PMN migration (Fig. 4Go). At 4 h, PMN penetrated the full thickness of fibrin gels incubated with LTB4. Thus, fMLP inhibits and LTB4 promotes C5a-mediated chemotaxis into S. epidermidis-containing fibrin gels.



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  		FIGURE 4. Effects of LTB4 and fMLP on C5a-stimulated PMN migration into fibrin gels. Fibrin gels (1500 µm thick, 100 µl in volume) with 1.9 x 104 CFU of S. epidermidis and 40% NHS were formed in Boyden-type migration chambers. PMN (5 x 105) were placed on top of each gel. PBSG-HSA alone or PBSG-HSA containing LTB4 (10 -7 M) or fMLP (10-6 M) was placed in the bottom compartments, and the migration chambers were incubated at 37°C. Shown is the distance migrated by PMN into fibrin gels after 4 and 6 h of incubation, measured as described in Fig. 1Go. Data represent the means and SEM from five to nine experiments, each done in duplicate. **, p < 0.01; ***, p < 0.001 compared with control where the bottom compartments contained buffer alone.

 
Extent of PMN migration into S. epidermidis-containing fibrin gels is a critical determinant of the ability of these cells to control S. epidermidis growth in these gels

We anticipated that enhanced PMN migration into S. epidermidis-containing fibrin gels would facilitate contact between the bacteria and the PMN, thereby promoting phagocytosis and killing. Conversely, we anticipated that inhibition of PMN migration into these gels would reduce contact between the PMN and the bacteria and thereby inhibit killing. We used LTB4 and fMLP to test these ideas.

Fibrin gels (1500 µm thick and 100 µl in volume) containing 1.9 x 104 S. epidermidis and 40% NHS were formed in Boyden-type chemotaxis chambers. Buffer alone or buffer containing LTB4 (10-7 M) or fMLP (10-6 or 10-8 M) were placed in the bottom compartment and the chambers were incubated for 4–6 h at 37°C. In the absence of added PMN, S. epidermidis grew to ~10 times the initial inoculum after 4 h of incubation, and to ~100 times the initial inoculum after 6 h (Fig. 5Go). The presence of 5 x 105 PMN initially added to the top of the gels markedly suppressed bacterial growth (Fig. 5Go). Under these conditions, the number of S. epidermidis grew to only two times the initial inoculum (from 1.9 x 104 to 4 x 104 CFU) after 4 h of incubation, and by 6 h had decreased by a log to 2 x 103 CFU (Fig. 5Go). LTB4 added to the bottom chamber significantly enhanced the rate at which PMN killed S. epidermidis in fibrin gels (Fig. 5Go). By 4 h, the number of bacteria in gels incubated with LTB4 had decreased to 20% of the initial inoculum and was 10 times lower than the number of bacteria in gels not incubated with LTB4 (3.7 x 103 CFU with LTB4 vs 4 x 104 CFU without LTB4). However, by 6 h there was no significant difference in the number of bacteria in gels incubated with or without LTB4. In contrast, 10-6 M fMLP added to the bottom compartment consistently inhibited PMN killing of S. epidermidis. By 4 h, significantly more S. epidermidis were recovered from gels incubated with fMLP than from gels incubated without fMLP (p < 0.05). By 6 h, the number of S. epidermidis in gels incubated with fMLP was about twice the initial inoculum, whereas only 10% of the initial inoculum remained viable in gels incubated without fMLP (p < 0.01) (Fig. 5Go).



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  		FIGURE 5. Effects of fMLP and LTB4 on PMN killing of S. epidermidis in fibrin gels. Fibrin gels containing 1.9 x 104 S. epidermidis and 40% NHS were formed in Boyden-type migration chambers as described in Materials and Methods. PBSG-HSA alone (-) or PBSG-HSA containing 5 x 105 PMN (+) was placed on top of the gels. PBSG-HSA alone or PBSG-HSA with fMLP (10-6 M) or LTB4 (10-7 M) was placed in the bottom compartments, and the chambers were incubated at 37°C. Shown is the number of viable S. epidermidis in fibrin gels at 0, 4, and 6 h. Data represent the means ± SEM from at least five experiments, each done in duplicate. *, p < 0.05; **, p < 0.01 compared with the appropriate control value ({blacksquare}) at the indicated time point, in which no chemoattractant was added.

 
To confirm that the inhibitory effect of fMLP reflected its capacity to inhibit PMN migration within these gels, we formed fibrin gels with PMN, S. epidermidis, and fMLP embedded in them, as described in Materials and Methods, and measured their content of viable S. epidermidis after a 90-min incubation at 37°C. fMLP dose-dependently inhibited PMN killing of S. epidermidis with IC50 ~10 nM. Maximal inhibition occurred at a concentration of 1 µM (Fig. 6Go). In the absence of fMLP, PMN embedded together with S. epidermidis efficiently killed these bacteria (Table IVGo). When carboxypeptidase Y was added to the gels to degrade fMLP (18), the inhibitory effect of fMLP on PMN killing of S. epidermidis was abolished (Table IVGo). Control experiments reveal that carboxypeptidase Y alone did not affect killing of S. epidermidis in fibrin gels (Table IVGo).



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  		FIGURE 6. fMLP inhibits PMN killing of S. epidermidis in fibrin gels. Fibrin gels (1500 µm thick, 100 µl in volume) containing 4 x 105 PMN, 1 x 104 CFU of S. epidermidis, 10% NHS, and the indicated concentrations of fMLP were incubated at 37°C for 90 min. The gels were then digested and the number of viable S. epidermidis was assayed. Shown is the percent of the bacterial inoculum (1 x 104 CFU) killed. Data represent means ± SEM of three experiments.

 

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  		Table IV. Carboxypeptidase Y blocks fMLP’s inhibitory effect on killing of S. epidermidis in fibrin gels1

 
To be certain that neither fMLP nor LTB4 affected PMN killing of S. epidermidis per se, we measured PMN killing of these bacteria in suspension in the presence of fMLP (10-6 M) or LTB4 (10-7 M), as described in Materials and Methods. Neither fMLP nor LTB4 had any effect on the efficiency with which PMN killed S. epidermidis under these conditions (Table IIIGo). Taken together, the findings reported in Figs. 4–6GoGoGo and Tables III-IV show that fMLP inhibits and LTB4 enhances PMN bacterial killing in fibrin gels, and that these effects were due to the inhibitory or stimulatory effects of fMLP and LTB4, respectively, on PMN chemotaxis through fibrin matrices.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since Wright and Douglas (19, 20) first demonstrated that Abs and complement cooperate in opsonizing Staphylococci for phagocytosis by leukocytes, in vitro systems that mimic in vivo environments have profoundly influenced our understanding of the interactions of phagocytic leukocytes with microbial pathogens. While many investigators (12, 21, 22, 23) have used a "tumble" system to study the effects of opsonins and their cognate receptors on uptake and killing of bacteria by PMN in suspension, comparatively little attention has been given to in vitro systems that mimic tissue environments. The fibrin gel system described in this work has many of the properties of inflamed or injured tissue in vivo. It appears to be useful for examining the roles of chemoattractants and of PMN membrane proteins in regulating PMN migration in three-dimensional matrices and in binding and phagocytosis of bacteria embedded in these matrices.

Three technical details about this fibrin gel system merit consideration here. The first is the importance of adding phagocytosis inhibitors during lysis of the fibrin gel. In the few studies in which PMN killing of bacteria in fibrin gels has been examined (8, 9), phagocytosis inhibitors were not used. As shown in Table IGo, in the absence of such inhibitors, recovery of viable bacteria from gels containing 4 x 107 PMN/ml was incomplete. In these studies, we used both cytochalasin D and EDTA as phagocytosis inhibitors. In subsequent studies (data not shown) we have found that cytochalasin D in the absence of EDTA blocks PMN bactericidal activity during gel lysis. Because EDTA has a deleterious effect on the viability of Gram-negative bacteria, this finding indicates that this system also can be used to analyze PMN killing of Gram-negative bacteria. Indeed, we have used it to study PMN killing of Pseudomonas aeruginosa (Y. Li and S. C. Silverstein, unpublished observations). The second is the use of chemotaxis chambers formed with filters that do not permit bacteria to diffuse into the lower compartment. By this means we achieved virtually complete recovery of bacteria (Table IGo). The third is the apparently spontaneous formation of C5a when NHS was placed in chambers containing fibrin gels. Formation of C5a is evidenced by the large number of PMN that migrated into fibrin gels containing NHS but lacking S. epidermidis (Fig. 1Go) and the dearth of PMN that migrated into fibrin gels containing S. epidermidis and C5-deficient serum (Fig. 2Go). We have not investigated the mechanism(s) responsible for generation of chemoattractants by NHS in fibrin gels lacking S. epidermidis. It seems likely that contact of serum with the plastic and/or filter that form the chemotaxis chambers, and/or the matrix proteins they contain, promotes complement activation. Clearly, future studies would be facilitated by use of chambers formed of materials and/or containing matrix proteins that do not cause such activation.

In vivo, PMN also encounter bacteria in tissues such as bone and cartilage, which are collagen-rich and are preferential sites of Staphylococcal infections (24, 25). In principle, it should be relatively easy to form three-dimensional matrices with proteins other than fibrin (e.g., collagens) and to examine the effects of these proteins on a variety of leukocyte functions (7, 26, 27, 28).

Roles of complement

Complement played at least two roles in PMN killing of S. epidermidis in fibrin gels. First, C3 opsonization of S. epidermidis was required for PMN to kill them. Immunofluorescence studies confirmed that S. epidermidis incubated in HIS were coated with IgG but not C3, while S. epidermidis incubated in NHS serum were coated with both IgG and C3 (data not shown). PMN were incapable of killing S. epidermidis in suspensions or gels containing HIS, but killed these bacteria efficiently when incubated with them in suspension or in fibrin gels containing NHS (Fig. 5Go). C3b and C3bi bound to the surfaces of Staphylococci have been identified as key ligands for PMN phagocytosis of these bacteria (29, 30).

Second, C5 was required to promote PMN migration into fibrin gels. Addition of SCPA, a C5a-specific peptidase, or of anti-C5aR IgG, blocked PMN migration into fibrin gels containing S. epidermidis and NHS (Figs. 2Go and 3Go). These findings strongly suggest that C5 was required to generate C5a, and that C5a was the principal chemoattractant that stimulated PMN to migrate into fibrin gels. These results also indicate that substances such as fibrin peptides generated during clotting (31, 32), C3a generated during C3b fixation (33), or products released from S. epidermidis (15, 16) play an insignificant role, compared with C5a, in stimulating PMN migration under the condition of these experiments. They lend support to the concept that SCPA is a virulence factor (34) and suggest that Streptococci expressing SCPA may facilitate infections mediated by bacteria that do not express this C5a-specific peptidase by degrading C5a.

C5a could enhance PMN phagocytosis by activating complement and FcR (13, 35). S. epidermidis incubated in C5-deficient serum were coated with C3 (data not shown). However, the finding that PMN kill S. epidermidis in suspension in the absence of C5, fMLP, or LTB4 as efficiently as in their presence (Table IIIGo) indicates that these chemoattractants are not required to activate PMN for phagocytosis or bacterial killing under the conditions of these experiments. We conclude that in contrast to bacterial killing in suspension, the generation of C5a by C5 convertases on the surface of bacteria in fibrin gels is required to guide PMN to contact and ingest them.

Role of C5a in promoting PMN functions in vivo

Our findings suggest that C5a plays an essential role in PMN killing of S. epidermidis in fibrin-containing sites in vivo. They imply that in vivo C5a is important for guiding PMN to microbes enmeshed in tissues. This interpretation is consistent with the findings of Hopken et al. (1), Larsen et al. (36), and Ashman et al. (37), who showed that C5- or C5aR-deficient mice were unable to control the number of P. aeruginosa in the trachea or Candida albicans in CSF as compared with wild-type mice, despite a more intense infiltrate of PMN into the lungs or brains of C5- or C5aR-deficient mice. They also showed that PMN isolated from C5 or C5aR-deficient mice were as effective as PMN from wild-type mice in phagocytosis/killing of bacteria in vitro. Their studies showed that neither C5 nor C5aR is required for PMN to migrate out of the vasculature into mouse lung or brain (1, 36, 37). However, in C5- or C5aR-deficient mice, these extravasated PMN are ineffective in phagocytosis and killing of bacteria/yeast at these sites. This is consistent with what we have observed in fibrin gels (Table IIGo). These results suggest that in vivo the most important role of C5a is the guidance of extravasated PMN to microbes enmeshed in tissues, matrices, and/or mucous.

Our studies also indicate that C5a plays an essential role in PMN killing of bacteria in fibrin gels (Table IIGo and Fig. 3Go) but not in suspension (Table IIIGo). This suggests that bacterial killing by PMN in the bloodstream or in anatomical sites where bacteria are not entrapped in fibrin-containing matrices may occur independently of the presence of C5a. Indeed, C5 or C5aR deficiency in mice is not associated with increased susceptibility to bacterial infections. C5-deficient humans are generally healthy but do exhibit increased susceptibility to bacterial infection by neisserial organisms (38). C5aR knockout mice clear peritoneal infections with P. aeruginosa as effectively as wild-type mice (1). Therefore, PMN extravasation and migration at this and other sites must involve chemoattractants/chemokines other than C5a. Indeed, there is substantial evidence for this conclusion. For example, IL-8, not C5a, appears to play a dominant role in promoting PMN emigration from the vasculature into the CSF of rabbits infected intracisternally with S. pneumoniae (39, 40, 41).

Taken together, these studies lead us to make three additional observations about the roles of chemoattractants/chemokines in PMN-mediated host defense. First, they support our suggestion that the receptor for each chemoattractant/chemokine initiates a unique set of signals and that differences in PMN functions initiated by these signals become apparent only in the context of specific matrix proteins (6). Second, these findings support the hypothesis of Foxman et al. (42) that PMN migration and orientation in tissues is a multistep process in which the effects of different chemoattractants/chemokines predominate at each step along the way. Third, they call attention to the role of chemoattractants/chemokines other than C5a in body compartments and tissues that contain low concentrations of C5 (e.g., cerebrospinal space, alveoli, bronchi, and pleural and peritoneal cavities). Normal human plasma contains ~75 µg/ml C5 (43). As shown in Fig. 1Go, a serum concentration of at least 10% (~7.5 µg/ml C5) is required to induce a large number of PMN to migrate into S. epidermidis-containing fibrin gels. In body compartments containing low concentrations of C5, production of chemoattractants or chemokines by resident tissue macrophages may play an important role in recruiting and stimulating PMN migrate to sites of bacterial invasion.

LTB4 facilitates PMN chemotaxis and bacterial killing

In the presence of 40% NHS and of S. epidermidis, PMN crawled through fibrin gels in response to C5a at a rate of ~4 µm/min (Fig. 4Go). Addition of LTB4 to the compartment underlying these gels increased the rate of PMN chemotaxis to ~6 µm/min (Fig. 4Go). These results suggest that in tissues containing suboptimal C5 concentrations, such as those noted above, LTB4 generated by pioneer PMN, or by macrophages (44), speeds PMN migration to sites of bacterial invasion.

fMLP may be a bacterial virulence factor in the presence of fibrin

FMLP reduced PMN chemotaxis to ~2 µm/min, thereby slowing C5a-stimulated PMN chemotaxis in fibrin gels (Fig. 4Go) and PMN killing of S. epidermidis embedded in them (Figs. 5Go and 6Go). These results lend physiological relevance to our previous reports that fMLP inhibits LTB4-stimulated PMN chemotaxis through fibrin gels and plasma clots (6).

PMN express on their plasma membranes a neutral endopeptidase, CD10, for which fMLP is an excellent substrate (45). Our finding that carboxypeptidase Y blocks the inhibitory effects of fMLP on PMN bactericidal activity suggests that CD10 may facilitate PMN migration into sites containing bacteria and fibrin by degrading N-formylated peptides released by the bacteria.

N-formylated peptides are generated by a wide variety of bacteria (18, 46). The lumen of the human intestine contains ~10-7 M N-formylated peptides (18). Regardless of whether fMLP was placed in the bottom compartment of chemotaxis chambers containing fibrin gels or in the fibrin gels themselves, 10-6 M fMLP was required to fully block PMN migration and bacterial killing in response to the gradients of C5a generated in these experiments (Figs. 4Go and 5Go), and to block PMN chemotaxis in response to 10-7 M LTB4 in previous experiments (6). However, the presence of 10-7 M or 10-9 M fMLP within fibrin gels was sufficient to block bacterial killing by ~80 or ~30%, respectively (Fig. 6Go). Thus, concentrations of fMLP similar to those found in the normal colon in vivo (18) are more than sufficient to exert a strong inhibitory effect on PMN killing of bacterial pathogens in fibrin-containing matrices. Therefore, we suggest that in the presence of fibrin fMLP is a bacterial virulence factor.

Mechanisms by which LTB4 promotes and fMLP inhibits C5a-stimulated PMN chemotaxis through fibrin gels

Our studies show that fMLP and LTB4 exert qualitatively different effects on chemotaxis of PMN through fibrin gels (6), activation of PMN integrins (47), and production of H2O2 by PMN (48). FMLP and LTB4 signal via heptahelical receptors coupled to G{alpha}i-containing heterotrimeric G proteins. Pertussis toxin, which inactivates G{alpha}i, blocks all PMN functions stimulated by fMLP and LTB4 (48). Given their apparent similarities in signaling mechanisms, how might these receptors activate qualitatively distinct PMN effector functions?

One possibility is that while fMLP and LTB4 receptors use the same G{alpha}i subunits, they might activate different G{beta}{gamma} subunits. Five different G{beta} subunits and 12 different G{gamma} subunits have been identified so far (49). Studies ongoing in our laboratory show that PMN contain several isoforms of G{beta} subunits. Different heptahelical receptors have been shown to activate different G{beta} or G{gamma} subunits. For example, both muscarinic M4 and somatostatin receptors couple to G{alpha}0 but to different G{beta}{gamma} subunits (50, 51). Azpiazu et al. (52) have shown that molecular contacts between G{gamma} subunits and heptahelical receptors are necessary for G protein activation, indicating that heptahelical receptors could specifically bind particular G{gamma} subunits. There is also evidence that specific G{beta}{gamma} subunits are responsible for different effector functions and signaling mechanisms. For example, a G{beta}3 mutant enhances chemotaxis of human PMN in response to IL-8 and fMLP (53, 54) but has no effect on superoxide production stimulated by either ligand. Activation of phosphoinositide 3-kinase and phospholipase C has been shown to be mediated by different G{beta}{gamma} subunits (55).

A second possibility derives from the observation that heptahelical receptors can directly couple to cytoplasmic effector proteins other than heterotrimeric G proteins. For example, Luttrell et al. (56) and McDonald et al. (57) have shown that following G protein activation, specific {beta}-arrestins bind to {beta}-adrenergic receptors, thereby activating Src and mitogen-activated protein kinases; and Neptune et al. (58) have reported that it is the heptahelical receptor, and not the G{alpha} subunit, which determines the cellular function that is stimulated when the receptor is activated by its cognate ligand. Thus, at least two mechanisms have been described by which different heptahelical receptors that activate the same G{alpha} subunits could nonetheless stimulate qualitatively distinct signal cascades and cellular functions.


   Acknowledgments
 
We thank Dr. Steven Greenberg for critical reading of this paper and Miles Burger for helpful discussions about signaling mechanisms of heptahelical receptors.


   Footnotes
 
1 This work was supported by National Institute of Allergy and Infectious Diseases Grant AI-20418 (to S.C.S.), and by a predoctoral fellowship from National Institute of Allergy and Infectious Diseases (training grant 5T32AI-07525 to Y.L.). Back

2 Address correspondence and reprint requests to Dr. Samuel C. Silverstein, Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032. E-mail address: scs3{at}columbia.edu Back

3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; C5a, a fragment of cleaved C5; SCPA, streptococcal C5a peptidase; LTB4, leukotriene B4; NHS, normal human serum; HIS, heat-inactivated human serum; ZAS, zymosan-activated serum; CSF, cerebrospinal fluid; TSB, trypticase soy broth. Back

Received for publication July 16, 2001. Accepted for publication November 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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