Neutrophil primary granule proteins HBP and HNP1–3 boost bacterial phagocytosis by human and murine macrophages (original) (raw)
PMN-sec enhances phagocytosis of bacteria in macrophages. PMN activation via β2 integrin cross-linking caused release of secretory vesicles and tertiary, secondary, and primary granules as shown by Western blot analysis for marker proteins in the PMN-sec (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI35740DS1). Human macrophages derived from monocytes were treated with PMN-sec for 24 hours followed by a 1-hour incubation period with Staphylococcus aureus or Escherichia coli that were IgG opsonized, complement opsonized, or nonopsonized. Treatment with PMN-sec caused a strong enhancement of phagocytosis of IgG-opsonized S. aureus or E. coli but not of complement-opsonized or nonopsonized bacteria (Figure 1, A and B). Treatment with PMN-sec also resulted in a comparable enhancement of phagocytosis of IgG-opsonized S. aureus by murine RAW264.7 cells and WEHI-3B cells (data not shown). Interestingly, treatment of human macrophages with PMN-sec not only increased the uptake of bacteria but also their capacity to intracellularly kill S. aureus and E. coli (Supplemental Figure 2). In further experiments, only IgG-opsonized bacteria were used in the phagocytosis assay.
PMN-sec products enhance phagocytosis in macrophages. (A and B) Human macrophages were treated with PMN_-sec_ or medium alone (ctrl) for 24 hours. After stimulation, fluorescent S. aureus (A) or E. coli (B) were injected into the medium. Bacteria were either opsonized with IgG or with complement or left nonopsonized. The number of incorporated bacteria per cell was quantified by fluorescence microscopy. For each analysis, 3–6 independent experiments were performed. *P < 0.05 versus respective control. (C) Comparison of phagocytic activity of peritoneal macrophages obtained from BALB/c or C57BL/6 mice. Macrophages were isolated from mice with intact WBC, neutropenic mice, and from neutropenic mice treated with PMN_-sec_. MFI as a measure of phagocytosed IgG-opsonized bacteria was read in a plate reader. For each analysis, 6 independent experiments were performed. †P < 0.05 versus intact WBC and PMN depletion plus PMN-sec of the respective strain.
PMN granule proteins stimulate bacterial phagocytosis in peritoneal macrophages in vivo. To further investigate the PMN-macrophage cross-talk in vivo a thioglycollate-induced peritonitis model, in which macrophages are exposed to PMN-sec products released into the peritoneum, was used. Subsequent incubation with bacteria and analysis of phagocytic capacity were done ex vivo. In BALB/c and C57BL/6 mice, we found that peritoneal macrophages obtained from neutropenic mice showed markedly reduced ability to phagocytose bacteria compared with mice with normal white blood cell count (WBC). The i.p. injection of human PMN-sec to neutropenic animals enhanced the phagocytic capacity of peritoneal macrophages (Figure 1C). To compare the quantity of PMN granule proteins in the PMN-sec with the conditions found in the peritoneal cavity in vivo, we analyzed the PMN-derived granule proteins myeloperoxidase (MPO) and MMP-9 in the PMN-sec as well as in the peritoneal lavage fluid. The activity of MPO and MMP-9 assessed in both specimens was found to be in a similar range (Supplemental Table 1). To exclude a direct effect of the PMN-depleting antibody on the phagocytic capacity, we treated peritoneal macrophages from mice with intact WBC with RB6-8C5 or control IgG. However, no differences regarding the phagocytic capacity could be detected (data not shown).
PMN-sec activates macrophages and enhances surface expression of FcγRs. In further experiments human macrophages were treated with PMN-sec over different times. Treatment for 24 hours was more effective in stimulating phagocytosis than treatment for 2 hours (Figure 2A). Moreover, we found that absence of PMN-sec during incubation with bacteria had no influence on the phagocytic capacity (Figure 2A), suggesting that PMN-sec acts rather on the macrophages than on the bacteria and that PMN-sec causes a long-standing activation of macrophages.
PMN-sec enhances activation of macrophages and expression of FcγRs. (A) Human macrophages were incubated with PMN_-sec_ for either 2 or 24 hours or with medium. In some wells, the PMN_-sec_ was washed off after the 2- or 24-hour incubation period and replaced by medium. Subsequently the phagocytic activity was quantified. For each analysis, 4 independent experiments were performed. *P < 0.05 versus control; **P < 0.05 versus control and the respective 2-hour treatment group. (B and C) Expression of activation markers (B) and phagocytic receptors (C) in macrophages in response to treatment with PMN_-sec_ for 24 hours. Expression is given as percentage change compared with basal expression. All values are isotype corrected. For each analysis, 6–8 independent experiments were performed. (D) Representative images of antibody staining for CD32 and CD64 in macrophages after treatment with PMN_-sec_ or medium. Scale bar: 10 μm. (E) Human macrophages were incubated with PMN_-sec_ or medium for 24 hours. Blocking antibodies toward CD64, CD32, or CD16 were added 30 minutes before incubation with IgG-opsonized S. aureus. The number of phagocytosed bacteria after 1 hour was quantified. For each analysis, 6 independent experiments were performed. †P < 0.05 versus group treated with PMN_-sec_.
To investigate whether this activation may involve altered activity of specific cell surface molecules, we measured the expression of activation markers (Figure 2B) and phagocytic receptors (Figure 2, C and D) on macrophages. While expression of CD36, CD80, and CD62L was not altered by treatment with PMN-sec, we found a clear increase in CD86, HLA class II, CD40, and CD25 expression, reflecting phenotypic changes seen in classically activated macrophages (16). Analysis of complement and FcγRs revealed a marked upregulation of CD32 and CD64 but not of CD16 or complement receptors (CD11b, CD11c, CD35). In control experiments the cell number was controlled for by counterstaining with the DNA-binding dye DAPI. In these experiments, we obtained identical results (Supplemental Figure 3). To investigate the role of the different FcγRs in enhanced phagocytosis, neutralizing antibodies to these molecules were applied in the phagocytosis assay. While antibodies to CD16 were without effects, blockade of CD64 or CD64 plus CD32 attenuated the enhanced phagocytosis significantly (Figure 2E).
The enhanced macrophage phagocytosis originates from proteins of PMN primary granules. To investigate the chemical nature of the secreted compounds that stimulate phagocytosis, the activity of the PMN-sec was tested after heat inactivation or pepsin treatment. Heat inactivation (data not shown) and treatment with pepsin completely abrogated the induction of phagocytosis in macrophages (Figure 3A), indicating that the PMNs release one or several proteins that are responsible for enhancing phagocytosis. To identify these proteins, we isolated the individual subsets of PMN granules using subcellular fractionation (17) and assayed the activity of the respective granule subset. To determine which fraction corresponded to which granule compartment, each individual fraction was analyzed for the presence of specific granule markers (Figure 3B). Fractions that contained only one type of granule were pooled and used to stimulate human macrophages. Using this approach, we found that the phagocytosis-enhancing activity resided predominantly in primary granules (Figure 3C).
Identification of active PMN granule components. (A) PMN_-sec_ was digested with pepsin and the remaining activity was tested. For each analysis, 4 independent experiments were performed. †P < 0.05 versus group treated with active PMN_-sec_. (B) Localization of neutrophil granules in fractions (x axis) obtained by subcellular fractionation of PMNs is shown by marker analysis. MPO activity was measured as a marker for primary granules. Western blots of the fractions probed with antibodies to lactoferrin, MMP-9, and CD35 indicate the localization of secondary and tertiary granules and of secretory vesicles, respectively. (C) Human macrophages were treated with PMN_-sec_, fractions of the indicated PMN granules, a mixture of the 4 different granule fractions (pooled), or medium and phagocytosis assay was performed. For each analysis, 6 independent experiments were performed. *P < 0.05 versus control. (D) Human macrophages were treated with proteins and peptides stored in human primary granules. Phagocytosis was compared with that obtained from treatment with PMN_-sec_ or medium. For each analysis, 6 independent experiments were performed. *P < 0.05 versus control.
Hence, we used purified or recombinant forms of proteins and peptides that are stored in primary granules and analyzed their activity in the phagocytosis assay. The selection was based upon their abundance in primary granules and their reported capability to activate macrophages. The concentration at which each individual protein was used was adjusted to what has earlier been found to activate monocytes or macrophages (8, 18–20). The panel of proteins consisted of elastase, human neutrophil peptides 1–3 (HNP1–3, α-defensins), proteinase-3, HBP (CAP37/azurocidin), lysozyme, MPO, and cathepsin G. Notably, the concentrations of the proteins used in our assay were similar to the amounts we found in the PMN-sec (HBP, 3.4 μg/ml; HNP1–3, 2.5 μg/ml; cathepsin G, 2.1 μg/ml; MPO, 1.3 U/ml). Among the proteins tested, only recombinant HBP and HNP1–3 purified from human PMNs were found to enhance the phagocytosis of S. aureus by human macrophages (Figure 3D).
Chemical identification of HBP and HNP1–3 as enhancers of bacterial uptake in macrophages. Complementary to the previous approach, the PMN-sec was separated by reversed-phase HPLC, and the fractions were tested for their biological activity. Only pooled fractions 45–47 and 60–62 caused a marked enhancement of phagocytic activity (Figure 4A). Screening of HPLC fractions for the presence of HBP and HNP1–3 revealed positive signals for HBP in fractions 60–62 (Figure 4B). For HNP1–3, strong immunoreactivity was found in fraction 46 (Figure 4C) and weak staining in fraction 47 (data not shown). Western blot analysis of pooled fractions 60–62 revealed a strong immunoreactive band with a molecular weight corresponding to that of HBP (Figure 4B). After Coomassie staining of the remaining protein in the gel, a band of 27 kDa was visualized. The protein in this gel plug was trypsinized, and the masses of the generated fragments were determined with MALDI–mass spectrometry (MALDI-MS). Further MS-Fit search on these mass values indicated 33.8% coverage and 6 matched fragments corresponding to HBP, supporting the presence of HBP in fractions 60–62.
Identification of HBP and HNP1–3 as enhancers of bacterial phagocytosis in macrophages. (A) Fractionation of PMN_-sec_ by reversed-phase HPLC. PMN_-sec_ was loaded onto a C18 column. Proteins (right y axis, solid curve) were eluted with a gradient of ACN with 0.1% TFA (right y axis, dotted line). Gray bars indicate the phagocytic activity resulting from stimulation with material of 3 consecutive fractions (left y axis). Basal phagocytosis and phagocytosis in response to PMN_-sec_ are indicated by the lower and upper dashed line, respectively. Arrows indicate active fractions (45–47 and 60–62). For each analysis, 4 independent experiments were performed. (B) Immunological detection of HBP in fraction 60–62 using dot blot (left panel). These fractions were pooled and further analyzed with Western blot analysis (right panel). As positive control for both Western and dot blot, 40 ng recombinant HBP was used. (C) HPLC fractions were screened for the presence of HNP1–3 by dot blot analysis. Positive staining was detected in fractions 46 (insert). Inserts of dot blots in B and C were run on the same gel but were noncontiguous. As positive control 20 ng HNP1 was used. Determination of the mass values of material in fraction 46 with MALDI-MS gave molecular weights close to the theoretical values of HNP1–3.
To verify the presence of HNP1–3 in fraction 46, the material was analyzed with mass spectrometry (Figure 4C) and N-terminal sequence analysis. The obtained mass values were 3,442.30, 3,371.1, and 3,486.4 Da, corresponding to HNP1 (3,442.0 Da), HNP2 (3,371.0 Da), and HNP3 (3,486.1 Da). N-terminal sequence analysis of the material in fraction 46 yielded 3 sequences comprising 20 amino acids residues, which are identical to the N-terminal residues of HNP1–3 (ACYCRIPACIAGERRYGTCI, CYCRIPACIAGERRYGTCIY, and DCYCRIPACIAGERRYGTCI).
Enhanced phagocytosis in response to PMN granule proteins is mediated by HBP and HNP1–3. To investigate the contribution of HBP and HNP1–3 to enhanced phagocytosis, we immunodepleted the PMN-sec of these polypeptides. HBP removal caused a reduction of the enhanced phagocytosis by almost 70%, while HNP1–3–depletion damped the effect by approximately 25% (Figure 5A). Combined depletion of HBP and HNP1–3 resulted in almost complete elimination (83%) of the enhanced phagocytosis, attributing a virtually exclusive role to HBP and HNP1–3.
Contribution of HBP and HNP1–3 to enhanced phagocytosis. (A) Human macrophages were treated with medium, PMN_-sec_, or secretion depleted of HBP, HNP1–3, or both. Phagocytic activity is expressed in percentage of enhanced phagocytosis above control in response to PMN_-sec_, which was set to 100%. For each analysis, 4 independent experiments were performed. *P < 0.05 versus group treated with PMN_-sec_. (B) Comparison of phagocytic activity of peritoneal macrophages from mice with intact WBC or neutropenic mice. Neutropenic mice were also injected i.p. with HBP (10 μg/mouse), HNP1–3 (2 μg/mouse), or both. MFI, as a measure of phagocytosed IgG-opsonized bacteria, was read in a plate reader. For each analysis, 5 independent experiments were performed. †P < 0.05 versus fluorescence intensity in macrophages obtained from PMN-depleted mice. (C) Thioglycollate-treated neutropenic mice received PMN-sec, PMN-sec depleted of HBP, or PMN-sec depleted of both HBP and HNP1–3. *P < 0.05 versus group treated with PMN-sec. For each analysis, 5 independent experiments were performed. (D) A polyclonal antibody to HBP or control IgG were injected i.p. into thioglycollate-treated mice. The phagocytic capacity of peritoneal macrophages was tested ex vivo 4 days after initiating peritonitis. ‡P < 0.05 versus isotype control. For each analysis, 4 independent experiments were performed.
In our peritonitis model, we tested the effect of HBP and HNP1–3 on the phagocytic capacity of peritoneal macrophages from neutropenic BALB/c mice. Injection of HNP1–3 tended to increase the ability to phagocytose, whereas HBP significantly enhanced the phagocytic capacity in peritoneal macrophages (Figure 5B). Interestingly, combination of HBP and HNP1–3 almost completely restored the phagocytic competence of peritoneal macrophages. In further experiments, we injected PMN-sec, PMN-sec depleted of HBP, and PMN-sec depleted of both polypeptides. Depletion of HBP from the PMN-sec almost completely abolished its phagocytosis-enhancing effect (Figure 5C). In yet another approach, we injected 50 μg polyclonal anti-HBP i.p. together with the thioglycollate broth at day 0 and every 24 hours after until the macrophages were harvested. Application of the anti-HBP antibody resulted in a significant reduction of the phagocytic capacity of macrophages harvested from the peritoneum as compared with mice treated with irrelevant IgG (Figure 5D). To investigate if the recruitment of PMNs to the peritoneal cavity was compromised by this treatment, we analyzed the number of PMNs by FACS. Importantly, we found no difference in the recruitment of PMNs (CD45+Gr1+F4/80–) to the peritoneal cavity (data not shown). Also the number of macrophages (CD45+Gr1–F4/80+) was not affected by the HBP antibody (data not shown).
Although murine macrophages respond to human HNP1–3 in this study and elsewhere (21), it is well established that murine PMNs are devoid of HNPs. Therefore, we wanted to confirm our findings in a different species known to have HNPs (22). In a rat peritonitis model, we could demonstrate that PMN depletion reduced the phagocytic function in peritoneal macrophages (Supplemental Figure 4). Injection of HBP or HNP1–3 partly restored phagocytic activity in these animals with predominance for HBP.
HBP and HNP1–3 stimulate enhanced phagocytosis via distinct signaling pathways. Above we show that the enhanced phagocytosis stimulated by PMN granule proteins critically depends on FcγRs and on the presence of HBP and HNP1–3. In further experiments, we attempted to tie these observations together. Treatment of macrophages with HBP or HNP1–3 caused a strong increase in expression of CD32 and CD64 (Figure 6, A and C). HBP has previously been shown to function as a soluble ligand to β2 integrins (23) and to activate monocytes through binding this receptor (24). In our experiments, incubation of macrophages with HBP caused an immediate Ca2+ mobilization, which was inhibited in the presence of the β2 integrin blocking mAb IB4 (Figure 6B). Blockade of β2 integrins not only prevented activation of macrophages but also attenuated expression of CD32 and CD64 by treatment with HBP (Supplemental Table 2). Similar observations were made in a murine system. There, enhanced phagocytosis of bacteria by murine RAW264.7 macrophages in response to HBP was abolished in the presence of the CD18 blocking antibody GAME46 (data not shown). In peritoneal macrophages from neutropenic CD18-deficient mice, stimulation with HBP did not induce a significant increase in bacterial uptake, which is in contrast to macrophages from neutropenic wild-type mice (Figure 7A). In a further step, we injected HBP into the peritoneum of neutropenic wild-type and CD18-deficient mice. While testing the phagocytic capacity ex vivo we found that HBP significantly enhanced phagocytosis in wild-type mice but not in CD18-deficient mice (Figure 7B). Moreover, we used isolated peritoneal macrophages from neutropenic MyD88- and IRF3-deficient mice to investigate the potential involvement of TLR signaling. In contrast to macrophages from CD18-deficient mice, HBP induced a significant increase in bacterial phagocytosis in macrophages obtained from neutropenic MyD88- and IRF3-deficient mice (Figure 7A), indicating that (a) enhanced phagocytosis in response to HBP does not involve TLR signaling and (b) that the effect of HBP is not due to LPS contamination. The same relationships were found when HBP was injected i.p. into neutropenic MyD88- and IRF3-deficient mice (Figure 7B). To gain insight into which intracellular pathway HBP activates in macrophages after binding to β2 integrins, we used inhibitors of various signaling molecules. Of the substances tested, inhibitors of tyrosine kinases and of MAP kinases greatly reduced the HBP-induced expression of CD32 and CD64, while inhibition of phospholipase C, protein kinase C, and phosphatidylinositol-3-kinase were without effects (Supplemental Table 2).
Characterization of the enhanced phagocytosis by HBP and HNP1–3. (A and C) Human macrophages were treated with increasing concentrations of (A) HBP or (C) HNP1–3 for 24 hours, and the expression of CD32 and CD64 was assessed by immunofluorescence. Data are expressed as a percentage of basal expression, which was set to 100%. For each analysis, 5 independent experiments were performed. *P < 0.05 versus control. (B and D) Intracellular Ca2+-mobilization in macrophages labeled with the Ca2+-sensitive dye fluo4/AM in response to stimulation with (B) HBP (1 μg/ml) and (D) HNP1–3 (1 μg/ml). After labeling and washing, fluorescence was read in a plate reader every 30 seconds, from 60 seconds before stimulation until 300 seconds after stimulation. Data are expressed as percent of fluorescence intensity at time 0. IB4 (10 μg/ml) or suramin (100 μM) were added to analyze the involvement of β2 integrins and P2Y6 receptors in the respective activation. For each analysis, 4 independent experiments were performed. *P < 0.05 versus control treatment. (E) Western blot for CD64 of whole cell lysate of human macrophages treated with HBP (1 μg/ml, 24 hours) in the presence or absence of siRNA to CD64.
Enhanced phagocytosis mediated by HBP depends on CD18 but not TLR signaling, while enhanced phagocytosis induced by HNP1–3 is independent of CD18 and TLR signaling. (A) Peritoneal macrophages were isolated from neutropenic mice of various strains and treated ex vivo with medium, HBP (1 μg/ml), or HNP1–3 (1 μg/ml) for 24 hours. Thereafter, the phagocytic capacity was assessed using IgG-opsonized S. aureus. For each analysis, 4 independent experiments were performed. *P < 0.05 versus respective control. (B) Comparison of phagocytic capacity of peritoneal macrophages from different strains. Mice were rendered neutropenic and then injected i.p. with either PBS or HBP. For each analysis, 3–5 independent experiments were performed. †P < 0.05 versus neutropenic mice of the respective strain.
In contrast to what was found for stimulation with HBP, IB4 treatment did not prevent HNP1–3–induced macrophage activation (Supplemental Table 3). In addition, none of the inhibitors used to block intracellular signaling pathways reduced the HNP1–3–induced expression of CD32 and CD64 (Supplemental Table 3). Recently, Kinee et al. demonstrated that PMN-derived HNPs mediate their effect on epithelial cells via binding to P2Y6 receptors (25). Therefore, it was of interest to investigate the influence of the P2Y-specific inhibitor suramin on the activation of macrophages by HNP1–3. HNP1–3 clearly activated macrophages as indicated by intracellular Ca2+ mobilization (Figure 6D). However, suramin failed to block HNP1–3–mediated activation and FcγR expression (data not shown). Similarly, treatment with Pertussis toxin (PTx) did not have any influence on the HNP1–3–induced macrophages activation (Supplemental Table 3). The efficiency of HNP1–3 treatment was also not blocked in macrophages deficient in CD18, MyD88, or IRF3 (Figure 7A). Taken together, these data indicate that HNP1–3 act via a pathway independent of TLR, CD18, and adenosine receptor signaling.
To reveal if the enhanced expression of CD32 and CD64 induced by HBP/HNP1–3 was due to de novo expression, we used emetine and anisomycin. Both compounds abrogated the HBP- and HNP1–3–mediated expression of CD32 and CD64 (Supplemental Tables 2 and 3). Moreover, whole-cell lysates of HBP-treated macrophages exhibited higher amounts of CD64 compared with control cells (Figure 6E). siRNA to CD64 resulted in a decreased expression of this receptor in control cells and a markedly reduced responsiveness to HBP (Figure 6E), suggesting that HBP stimulates de novo synthesis of CD32 and CD64.
The effects of HBP and HNP1–3 on the macrophage phenotype depend on autocrine stimulation via TNF-α and IFN-γ. The classic activation of macrophages is initiated by TNF-α and IFN-γ (16). We therefore investigated whether treatment with HBP or HNP1–3 induces release of these cytokines from macrophages. Both HBP and HNP1–3 stimulated release of TNF-α and, to a smaller extent, IFN-γ (Figure 8, A and D). Blocking antibodies to TNF-α significantly reduced the effect of HBP and HNPs in the phagocytosis assay, whereas antibodies to IFN-γ had minor effects (Figure 8, B and E). Reduced phagocytosis in response to anti–TNF-α was related to a reduced CD32 and CD64 expression, while anti–IFN-γ exerted effects on the CD64 expression only (Figure 8, C and F). In control experiments, we counterstained the cells with the DNA-binding dye DAPI to adjust for the cell number. Here we reached identical results (Supplemental Figure 5). Antibodies to TNF-α also reduced the HBP-mediated enhancement of HLA class II, CD40, and CD86 expression (Supplemental Figure 6, A and B). As for the expression of CD32 and CD64, we investigated the involvement of specific signaling pathways in the release of TNF-α and IFN-γ upon stimulation with HBP or HNP1–3. The HBP-induced cytokine release was clearly prevented in the presence of the CD18 antibody IB4 and inhibitors to tyrosine kinases and MAP kinases, while none of these treatments reduced the HNP1–3–mediated release of TNF-α or IFN-γ (data not shown). Furthermore, treatment with siRNA to CD64 blocked the enhanced expression of this receptor in response to HBP, while levels of TNF-α and IFN-γ remained elevated (data not shown).
Involvement of TNF-α and IFN-γ in the HBP- and HNP1–3–mediated enhanced phagocytosis. (A and D) Human macrophages were treated with (A) HBP or (D) HNP1–3 and the concentration of TNF-α and IFN-γ was determined by ELISA at different time points. For each analysis, 4 independent experiments were performed. †P < 0.05 indicates significant effect of treatment. (B and E) The contribution of TNF-α and IFN-γ was assessed in the phagocytosis assay after stimulation of human macrophages with (B) HBP or (E) HNP1–3 by addition of neutralizing antibodies to TNF-α (5 μg/ml) or IFN-γ (10 μg/ml). For each analysis, 4 independent experiments were performed. *P < 0.05 versus HBP or HNP1–3 treatment groups. (C and F) Importance of TNF-α and IFN-γ for the upregulation of CD32 and CD64 on human macrophages in response to (C) HBP or (F) HNP1–3 evaluated through use of neutralizing antibodies. Receptor expression is assessed by immunofluorescence staining and displayed as percent change compared with control. For each analysis, 5 independent experiments were performed. *P < 0.05 versus HBP or HNP1–3 treatment groups.