Abstract
Basophils, which are normally confined to the circulation, can migrate to sites of allergic inflammation. Using the specific mAb, BB1, we detected basophil infiltration of the gastric mucosa of Helicobacter pylori-infected patients affected by moderate and severe gastritis. Basophils were not found in H. pylori-free individuals or in subjects with mild gastritis. The H. pylori-derived peptide, Hp(2–20), was a potent basophil chemoattractant in vitro, whereas the control peptide, Hp1, was ineffective. Basophils from peripheral blood of healthy volunteers expressed mRNA for the formyl peptide receptors, N-formyl-peptide receptor (FPR), FPR-like (FPRL)1, and FPRL2. Preincubation of basophils with FMLP or Hp(2–20) caused complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with a low concentration of FMLP, which binds with high affinity to FPR, but not to FPRL1 or FPRL2, did not affect the chemotactic response to Hp(2–20). In contrast, a high concentration of FMLP, which binds to FPRL1 and FPRL2, reduced the chemotactic response to Hp(2–20). The FPR antagonist, cyclosporin H, prevented chemotaxis induced by FMLP, but not by Hp(2–20). Hp(2–20) could be responsible, at least in part, for basophil infiltration of the gastric mucosa of H. pylori-infected patients presumably through the interaction with FPRL1 and FPRL2.
Helicobacter pylori is a Gram-negative bacterium, specialized in colonization of the human stomach (1). H. pylori infection is the major cause of gastroduodenal disease (2, 3) and entails an increased risk of gastric carcinoma (4, 5) and primary non-Hodgkin’s lymphoma of the stomach (6).
H. pylori is rarely found within the mucosa and mostly resides in the mucus layer overlaying the gastric epithelium (7). Thus, the factors that cause gastric inflammation presumably diffuse from the bacteria through the mucus and the gastric epithelium to cause chemotaxis of inflammatory cells such as neutrophils and monocytes/macrophages (8, 9). Many chemotactic factors secreted by H. pylori have been identified (10, 11, 12). Recently, a cecropin-like H. pylori peptide (Hp(2–20)) was found to be a monocyte chemoattractant (11). Cecropins are a group of peptides first discovered in the context of insect immunity and subsequently identified in bacteria (13). Cecropins are composed of two amphipathic α-helices joined by a hinge (14). H. pylori synthesizes cecropin-like amino-terminal peptide Hp(2–20) derived from the ribosomal protein, L1 (15). The receptors mediating monocyte activation caused by Hp(2–20) were identified as the formyl peptide receptor (FPR)3 homologues, FPR-like (FPRL)1 and FPRL2 (11).
Several natural N-formyl peptides, including the prototype FMLP, have been purified from bacterial supernatants, which suggests they are biologically relevant ligands for formyl peptide receptors. FMLP binds and activates G protein-coupled seven transmembrane (STM) receptors (16). Three STM receptors expressed by phagocytic leukocytes have been identified and cloned: neutrophils express the high affinity receptor (FPR) and its homologue FPRL1, whereas monocytes express FPR, FPRL1, and FPRL2 (17, 18). FPR is a high affinity receptor for FMLP, whereas FPRL1 has a much lower affinity (19, 20). FPRL1 is a promiscuous receptor activated by serum amyloid A (21), the prion peptide, PrP106–126 (22), lipoxin A4 (23), and various bacterial and synthetic peptides (11). In addition, we recently demonstrated that two HIV-1 gp41 peptides are chemoattractants for human basophils through interaction with FPRL1 (24). Cyclosporin H (CsH) and spinorphin are specific antagonists of the FMLP receptor subtype FPR (25, 26, 27).
Human basophils and mast cells (FcεRI+ cells) are the only cells expressing FcεRI and synthesizing histamine (28). There is increasing evidence that FcεRI+ cells are implicated in host defense against bacterial (29, 30) and viral infections (31, 32). Mast cells are present in the mucosa underlying the gastrointestinal tract, and mast cell degranulation has been reported in patients with peptic ulcer (33). The neutrophil-activating protein of H. pylori has been shown to induce activation of rodent mast cells (34). In addition, the vacuolating cytotoxin of H. pylori, VacA, induces chemotaxis and the release of cytokines from bone marrow-derived mast cells (35). Thus, H. pylori is involved in mast cell chemotaxis, and activation and release of proinflammatory mediators and cytokines. Although peripheral blood basophils may be increased in H. pylori-positive patients (36), studies of the in vitro effects of H. pylori on histamine release from basophils have yielded inconclusive results (37, 38, 39).
H. pylori-associated gastritis is characterized by infiltration of mononuclear and polymorphonuclear leukocytes into the gastric mucosa (40, 41). Basophil leukocytes, normally found only in the circulation, are able to migrate to sites of allergic inflammation (42, 43, 44, 45, 46). Basophils can be activated in vitro by several bacterial products (25, 47). Therefore, we investigated the presence of basophils in gastric mucosa of H. pylori-infected patients, and the in vitro effects of H. pylori-derived Hp(2–20) on basophils. In this study, we demonstrate basophil infiltration of gastric mucosa of H. pylori-infected patients affected by moderate and severe gastritis. We also demonstrate that Hp(2–20) is a potent chemoattractant for human basophils presumably through interaction with formyl peptide receptors, FPRL1 and FPRL2.
Materials and Methods
Human subjects and histologic diagnosis
Gastric biopsy specimens were obtained from 25 patients with H. pylori infection and 12 H. pylori-negative controls. Informed consent according to the guidelines of the University of Genoa Institutional Review Board for the Use of Humans in Research (Genoa, Italy) was obtained. Antral and corpus biopsies were fixed in 10% buffered formalin, paraffin-embedded, cut in serial 4-μm sections, and stained with H&E and Giemsa (48). Gastritis and H. pylori colonization were scored in accordance with the updated Sydney system (49). This required the assessment of four variables (mononuclear cell infiltration, activity of inflammation, mucosal atrophy, and H. pylori infection) on a four-point scale ranging from negative (0) to severe (3). The assessment was performed by two pathologists, independently. Each case was subsequently reassessed jointly and discordances were solved by consensus.
Reagents
A cecropin-like peptide, Hp(2–20), with a sequence (NH2-AKKVFKRLEKLFSKIQNDK-COOH) identical with that of the amino-terminal part of ribosomal protein L1 in H. pylori (50), was synthesized and purified by Innovagen (Lund, Sweden). The peptide, Hp1, with a sequence (NH2-AKKVFKRLELLFSKIQNDK-COOH), in which proline in position 10 had been substituted with lysine, and the hexapeptide Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm) was synthesized by Innovagen. The following were purchased: 60% HClO4 (Baker Chemical, Deventer, The Netherlands); human serum albumin (HSA), PIPES (Sigma-Aldrich, St. Louis, MO); HBSS, FCS, TRIzol, and Moloney murine leukemia virus transcriptase (Life Technologies, Grand Island, NY); FMLP (Calbiochem, La Jolla, CA); RPMI 1640 with 25 mM HEPES buffer, and Eagle’s MEM (Flow Laboratories, Irvine, U.K.); Dextran 70 and Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden). Fura 2-acetoxymethyl ester (fura 2-AM) was from Molecular Probes (Eugene, OR). CsH was obtained from Dr. D. Romer and Dr. E. Rissi (Novartis Pharmaceuticals, Basel, Switzerland).
Buffers
The PIPES buffer used in these experiments was made up of 25 mM PIPES, pH 7.4, 110 mM NaCl, and 5 mM KCl. The mixture is referred to as “P”. PCG contains, in addition to P, 5 mM CaCl2 and 1 g/L d-glucose (51). PACGM contains, in addition to P, HSA 3%, 1 mM CaCl2, 1 g/L dextrose, and 0.25 g/L MgCl2[chemps]6H2O, pH 7.4; PGMD contains 0.25 g/L MgCl2[chemps]6H2O, 10 mg/L DNase, and 1 g/L gelatin in addition to P, pH 7.4. PBS contains 8 g/L NaCl, 1.15 g/L Na2HPO4, 200 mg/L KCl, and 200 mg/L KH2PO4, pH 7.4.
Immunohistochemistry
The avidin-biotin-peroxidase technique was used for immunohistochemical staining of gastric biopsies (52). Briefly, deparaffinized sections were immersed in 3% H2O2 for 10 min to abolish endogenous peroxidase activity, washed in 0.05 M TBS (pH 7.4) for 15 min, and incubated with either BB1 mouse mAb or CD117/c-kit rabbit polyclonal Ab (DAKO, Glostrup, Denmark) for 1 h at 22°C. Slides were then rinsed three times in TBS, incubated for 20 min with Multilink Biotinylated anti-Ig (BioGenex Laboratories, San Ramon, CA) diluted 1/20, incubated for 20 min with streptavidin peroxidase (BioGenex Laboratories) diluted 1/20, and incubated for 3 min with 0.05% 3–3′-diaminobenzidine-tetrahydrochloride in 0.02% H2O2. Sections were counterstained with either Mayer’s hematoxylin or Giemsa, dehydrated, and mounted. BB1, a mAb recognizing a human basophil granular protein, was prepared as described elsewhere (53). This Ab does not react with lymphocytes, monocytes, neutrophils, platelets, eosinophils, mast cells, or any other cell type or tissue structure (54). Negative controls were obtained by omitting or substituting primary Ab with isotype-matched murine Abs. The density of BB1-immunoreactive cells was calculated in 5 high-power fields (×40 objective), corresponding to 361,000 μm2 by means of a computer-based imaging analysis system (Lucia, version 4.6; Nikon, Melville, NY).
Purification of peripheral blood basophils
Basophils were purified from peripheral blood cells of healthy volunteers seronegative for Abs to H. pylori, HIV-1, and HIV-2, aged from 21 to 38 years (mean 32.6 ± 5.8 years). “Buffy coat” cell packs from healthy volunteers were provided by the Immunohematology Service at the University of Naples Federico II (Naples, Italy). Informed consent according to the guidelines of the University of Naples Federico II Institutional Review Board for the Use of Humans in Research (Naples, Italy) was obtained. Cells were reconstituted in PBS containing 0.5 g/L HSA and 3.42 g/L sodium citrate, and loaded onto a countercurrent elutriator (model J2-21; Beckman Instruments, Fullerton, CA). Several fractions were collected, and fractions containing basophils in large numbers (>20 × 106 basophils) and of good purity (>15%) were enriched by discontinuous Percoll gradients (55). Basophils were further purified to near homogeneity (>98%) by depleting B cells, monocytes, NK cells, dendritic cells, erythrocytes, platelets, neutrophils, eosinophils, and T cells, using a mixture of hapten-conjugated CD3, CD7, CD14, CD15, CD16, CD36, CD45RA, and anti-HLA-DR Abs and MACS MicroBeads coupled to an anti-hapten mAb (Miltenyi Biotech, Bergisch Gladbach, Germany). The magnetically labeled cells were depleted by retaining them on a MACS column in the magnetic field of the MidiMACS (Miltenyi Biotech). Yields ranged from 3 to 10 × 106 basophils with purity above 98%, assessed by basophil staining with Alcian blue and counting in a Spiers-Levy eosinophil counter (51).
Purification of human neutrophils, monocytes, and dendritic cells
Neutrophils were purified from normal subjects, as previously described (25). The resulting cell preparations contained ∼99% polymorphonuclear neutrophils; platelet contamination was always <1%, as determined by direct counting. Cell preparations consisted of >96% viable neutrophils as revealed by trypan blue dye exclusion. Human PBMC were isolated from buffy coat cell packs by routine Ficoll-Hypaque density gradient centrifugation. Monocytes were purified from human PBMC with the use of the magnetic cell sorter CD14 monocyte isolation kit (Miltenyi Biotec), according to the manufacturer’s recommendation. The purity of monocytes was checked by FACScan analysis. Monocyte preparations with a purity above 95% were used. The generation of dendritic cells from monocytes was conducted as follows: monocytes were differentiated to immature dendritic cells by incubating at 1 × 106/ml in RPMI 1640 medium (RPMI 1640 plus 10% FBS, 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, and 100 μg/ml streptomycin) in the presence of recombinant human (rh) GM-CSF (50 ng/ml) and rhIL-4 (50 ng/ml) at 37°C in a humidified CO2 (5%) incubator for 7 days. To induce dendritic cell maturation, immature dendritic cells were cultured in the same cytokine mixtures supplemented with rhTNF-α (50 ng/ml) for 48 h at 37°C in a humidified CO2 (5%) incubator.
Chemotaxis assay
Basophil chemotaxis was performed using a modified Boyden chamber technique as described elsewhere (55, 56). Briefly, 25 μl of PACGM buffer or various concentrations of the chemoattractants in the same buffer were placed in triplicate in the lower compartment of a 48-well microchemotaxis chamber (NeuroProbe, Cabin John, MD). The lower compartments were covered with 5-μm-pore polycarbonate membranes (Nucleopore, Pleasanton, CA). Fifty microliters of the cell suspensions (5 × 104/well) resuspended in PACGM were pipetted into the upper compartments. The chemotactic chamber was then incubated for 1 h at 37°C in a humidified incubator with 5% CO2 (Automatic CO2 Incubator, model 160 IR; ICN Flow, Milan, Italy). At the end of incubation, the membrane was removed; the upper side was washed with PBS, and the filter was fixed, stained with May-Grunwald-Giemsa, and mounted on a microscope slide with Cytoseal (Stephens Scientific, Springfield, NJ). Basophil chemotaxis was quantitated microscopically by counting the number of cells attached to the surface of the 5-μm cellulose nitrate filter. In each experiment, 10 fields per triplicate filter were measured at a ×40 magnification. The results were compared with buffer controls. Checkboard analyses were performed to discriminate between chemotaxis and nondirected migration (chemokinesis) of basophils. In these experiments, basophils were placed in the upper chemotactic chambers and various concentrations of Hp(2–20), FMLP, or PACGM buffer were added to the upper or lower wells or to both. Spontaneous migration (chemokinesis) was determined in the absence of chemoattractant or when stimuli were added to either the lower or upper chambers. The basophil migratory response to chemotactic stimuli was largely due to chemotaxis and not to chemokinesis. Indeed, a checkboard analysis, in which chemoattractants above and below the filters varied, resulted in significant migration only when there was a gradient of the factor below the filters.
Calcium fluxes
Intracellular Ca2+ concentration ([Ca2+]i) changes were measured using the intracellular fluorescent indicator, fura 2-AM (57). Cells were loaded with 3 μM fura 2-AM for 30 min at 37°C. Fluorescence was measured with a Varian Cary Eclipse (Varian, Leini, Italy) fluorescence spectrophotometer (excitation and emission wavelengths, 340 and 509 nm, respectively) equipped with magnetic stirring and temperature control. To minimize leakage of trapped fura 2-AM, 200 μM sulfinpyrazone was included in the medium (57). The results were not affected by sulfinpyrazone. At the end of each incubation, digitonin (50 μg/ml) and EGTA (20 mM) were added to measure maximal (Fmax) and minimal (Fmin) fluorescence values. Values of [Ca2+]i were calculated according to the formula: [Ca2+]i = Kd[(F − Fmin)/(Fmax − F)] (57).
Histamine release
Basophils (∼6 × 104 basophils per tube) were resuspended in PCG, and 0.1 ml of the cell suspension was placed in 12 × 75 mm polyethylene tubes (Sarstedt, Princeton, NJ) and warmed to 37°C; 0.1 ml of each prewarmed releasing stimulus was added, and incubation was continued at 37°C for 45 min (51). At the end of this step, the reactions were stopped by centrifugation (1000 × g, 22°C, 2 min), and the cell-free supernatants were assayed for histamine content with an automated fluorometric technique (58). Total histamine content was assessed by lysis induced by incubating the cells with 2% HClO4 before centrifugation. To calculate histamine release as a percentage of total cellular histamine, the spontaneous release of histamine from mast cells (2 to 6% of the total cellular histamine) was subtracted from both the numerator and denominator (56). All values are based on the means of duplicate or triplicate determinations. Replicates differed in histamine content by <10%.
Isolation of cellular mRNA and RT-PCR analysis
Total RNA was isolated from purified blood basophils, monocytes, neutrophils, and monocyte-derived dendritic cells using the SV96 total RNA isolation system (Promega, Madison, WI) according to the manufacturer’s instructions, and treated with RNase-free DNase I. Reverse transcription was performed with 5 mM MgCl2, oligo(dt)16 primer, and murine leukemia virus reverse transcriptase according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA) on a thermocycler (GeneAmp PCR System 2400; Applied Biosystems). cDNA was then titrated for β-actin message and equivalent templates of cDNAs were amplified for FPR, FPRL1, and FPRL2 using specific primers designed according to the published sequences (GenBank accession nos. NM_002029 for FPR; NM_001462 for FPRL1; and NM_002030 for FPRL2). The primers for FPR were 5′-ACCTGCTGTATCTCTTGGCT-3′ (forward) and 5′-TGACCATGAAGAATGGCAAA-3′ (reverse), which resulted in a fragment of 215 bp. The primers for FPRL1 were 5′-TTGGTTTCCCTTTCAACTGG-3′ (forward) and 5′-ACTTAAAGCATGGGGTTGAG-3′ (reverse) which resulted in a fragment of 158 bp. The primers for FPRL2 were 5′-ATTAAATCCAGCCGTCCCTT-3′ (forward) and 5′-ACTAGTGGGCAAAGAGCGAA-3′ (reverse), which results in a fragment of 273 bp. The amplification protocol consisted in 40 cycles as follows: denaturation, 30 s at 95°C; annealing, 30 s at 60°C and 2 min primer extension at 68°C. A final extension at 68°C for 10 min was performed. PCR products, together with a DNA ladder as a size standard, were separated on a 2.5% agarose gel, stained with ethidium bromide, and photographed. Real-time quantitative PCR was performed on the iCycler (Bio-Rad, Hercules, CA) using the PE Applied Biosystems SYBR Green PCR kit and the FPR, FPRL1, and FPRL2 primers described above.
IL-4 and IL-13 ELISA
47).
Lactate dehydrogenase (LDH) assay
LDH release at the end of the incubations served as an index of cytotoxicity. It was measured in cell-free supernatants using a commercially available kit (Sigma-Aldrich, St. Louis, MO) (24).
Statistical analysis
The results are expressed as mean ± SEM. Statistical significance was analyzed by one-way ANOVA and when the F value was significant, by Duncan’s multiple range test (59). Differences were considered significant when p < 0.05.
Results
Identification of basophils in gastric mucosa of H. pylori-infected patients
Sixty-three biopsy specimens were taken during upper endoscopy in the gastric body (n=30) and antrum (n=33) from 25 patients with H. pylori infection and 12 H. pylori-negative controls. They were examined by immunohistochemistry using the mAb BB1 that specifically recognizes human basophils also in tissue (45, 53). In the 12 H. pylori-negative controls, no basophils were detected in 9 antral biopsies and 12 corpus biopsies. The amount of basophils was very variable in H. pylori-infected individuals. No BB1-positive cells were detected in the 11 H. pylori-positive cases with no evidence of inflammation or with mild activity inflammation. In contrast, five of the eight patients with moderate activity, and four of the six patients with severe activity harbored BB1-positive basophils. In H. pylori-positive cases basophils were detected in the antral mucosa and in the oxyntic mucosa. Basophils, as well as other granulocytes and mononuclear cells, were observed beneath the surface and foveolar epithelium (Fig. 1⇓A). Basophils were scattered in the lamina propria (Fig. 1⇓B), and in 30% of the cases with basophil infiltration, the BB1-positive cells also aggregated in clusters (Fig. 1⇓, C and D). Occasionally, basophils were found in the lumen of blood vessels (Fig. 1⇓E). The number of BB1-positive cells, assessed by imaging analysis, ranged from 3 to 12 per 5 high-power fields in sections with scattered cells and from 25 to 79 in sections with clusters. The percentage of basophils compared with other inflammatory cells (mononuclear cells, range: 89–91%; neutrophils, range: 1.2–2.5%; eosinophils, range: 1.6–3.6%; and mast cells, range: 2.2–3.2%) ranged from 1.1 to 3%. An interesting feature was the close spatial association between H. pylori in the mucus layer and on the apical surface of the epithelial lining and basophil infiltration in the subepithelial layer (Fig. 2⇓). However, basophils were also found in the basal part of the lamina propria. The examination of serial sections showed no overlap between BB1-positive and CD117/c-kit-immunoreactive cells (data not shown). There was no labeling when BB1 Ab was replaced by a nonrelevant murine Ab (data not shown).
Gastric mucosa from patients with H. pylori chronic active gastritis immunostained with BB1 mAb. The arrow in each panel indicates immunoreactive basophils. A, BB1-immunoreactive basophils are closely associated with surface epithelium; B, an isolated basophil beneath the foveolar epithelium; C and D, clusters of basophils around the foveolar and gland epithelia; E, intramucosal small blood vessel containing a single basophil (immunoperoxidase with light hematoxylin counterstain).
Two BB1-immunoreactive basophils (arrows) in the upper part of the lamina propria. The overlaying surface epithelium is heavily colonized by H. pylori (immunoperoxidase with Giemsa counterstain).
Effects of peptides Hp(2–20) and Hp1 on chemotaxis of human basophils
We evaluated the in vitro effects of a wide range of concentrations (10−9 to 3 × 10−5 M) of Hp(2–20) on chemotaxis of basophils purified (>98%) from peripheral blood from healthy individuals seronegative for Abs to H. pylori, HIV-1, and HIV-2. Fig. 3⇓ shows the results of six experiments that demonstrate that nanomolar concentrations of Hp(2–20) caused basophil chemotaxis, which plateaued at 3 × 10−7 M. To determine whether Hp(2–20)-induced migration of basophils resulted from chemotaxis or chemokinesis, checkerboard analysis was performed, and showed that Hp(2–20) dose-dependently induced the migration of basophils when added to the lower wells of the chemotaxis chamber. Increasing concentrations of Hp(2–20) added with the cells to the upper wells did not induce directional basophil migration (data not shown). Thus, Hp(2–20)-induced migration of basophils resulted from chemotaxis rather than from chemokinesis. Hp(2–20) contains a perfect amphipathic α-helical structure, similar to that found in cecropins, which can be interrupted by replacing key amino acids (13, 15). Replacement of the proline residue in position 10 of Hp(2–20) with lysine, an amino acid that lacks a polar side chain, disrupts the helical structure (15). No basophil chemotaxis was induced with a wide range of concentrations (10−9 to 10−4 M) of the K→L-substituted control peptide, Hp1 (Fig. 3⇓). In these experiments, low concentrations of the hexapeptide, WKYMVm (10−10–10−7 M), which activates FPR, FPRL1, and FPRL2 (60), proved to be a very potent chemotactic stimulus plateauing at 10−8 M.
Effects of Hp(2–20), Hp1, and WKYMVm on human basophil chemotaxis. Basophils obtained from peripheral blood of donors negative for H. pylori, HIV-1 and HIV-2 Abs were allowed to migrate with the indicated concentrations of peptides for 1 h at 37°C in a humidified (5% CO2) incubator. Values are the mean ± SEM of six experiments with different basophil preparations. Error bars are not shown when graphically too small.
Induction of Ca2+ mobilization in human basophils by Hp(2–20)
Because an increase in free cytoplasmic Ca2+ is a prerequisite for intracellular transduction of chemotactic signals (61), we analyzed whether Hp(2–20) induces Ca2+ influx in human basophils. Fig. 4⇓ shows the results of a typical experiment of four in which the addition of Hp(2–20) (50 μM) to basophils loaded with the fluorescent Ca2+ indicator fura 2-AM produced a rapid and transient increase in intracellular Ca2+ concentration. The pattern of Ca2+ mobilization caused by Hp(2–20) is typical of FPR agonists (62). No Ca2+ mobilization was obtained with the K→L-substituted control peptide Hp1 (50 μM). A low concentration of the hexapeptide WKYMVm (5 × 10−7 M), which activates FPR, FPRL1, and FPRL2 (60), caused a rapid and transient increase in intracellular Ca2+ concentration.
Effects of peptides Hp(2–20), Hp1, and WKYMVm on Ca2+ mobilization in human basophils purified from normal donors negative for H. pylori, HIV-1 and HIV-2 Abs. Peptide Hp(2–20) (50 μM) and WKYMVm (5 × 10−7 M) induced rapid and transient Ca2+ mobilization in human basophils, whereas the K→L-substituted control peptide Hp1 (50 μM) caused no Ca2+ mobilization.
Effect of peptides Hp(2–20), Hp1, FMLP, WKYMVm, and gp41 2019 on histamine release from human basophils
We evaluated the effects of increasing concentrations of several peptides on histamine release from basophils purified (>98%) from peripheral blood from healthy individuals. The results of the six experiments illustrated in Fig. 5⇓ demonstrate that Hp(2–20) and Hp1 did not cause the release of histamine from basophils. Also gp41 2019, which presumably acts through the activation of FPRL1 and FPRL2 (24) did not induce histamine secretion from basophils. The concentrations of Hp(2–20) and Hp1 peptides used did not induce spontaneous LDH release from basophils (data not shown). In these experiments, we used, as a control, FMLP, which activates a STM receptor on basophils independent of IgE receptor (51, 63). As previously shown (64), FMLP (10−8-10−6 M) induced histamine release from basophils from normal donors. In these experiments, low concentrations of WKYMVm (10−9-10−7 M) were the most potent stimuli for the release of histamine from basophils. In three experiments, neither Hp(2–20) nor Hp1 induced IL-4 or IL-13 release from basophils. In these experiments anti-IgE induced the release of both IL-4 and IL-13 from basophils (data not shown). Hp(2–20) did not induce histamine release from basophils purified from peripheral blood of H. pylori-infected individuals (data not shown).
Effects of WKYMVm, FMLP, Hp(2–20), Hp1, and gp41 2019 on histamine release from human basophils purified from normal donors negative for H. pylori, HIV-1 and HIV-2 Abs. Basophils were incubated with the indicated concentrations of stimuli for 45 min at 37°C. Values are the mean ± SEM obtained from six experiments with different basophil preparations. Error bars are not shown when graphically too small.
Effects of CsH on basophil chemotaxis induced by Hp(2–20) and FMLP
We previously demonstrated that CsH is a specific inhibitor of the release of preformed and de novo-synthesized mediators induced by FMLP from basophils (25). More recent results have demonstrated that CsH is a specific FPR antagonist (18, 26). In three experiments, we examined the effects of CsH (800 nM) on basophil chemotaxis induced by FMLP (5 × 10−7 M) and by Hp(2–20) (500 nM). Preincubation (15 min at 37°C) of basophils with CsH significantly inhibited (>70%) basophil chemotaxis induced by FMLP. In none of these experiments did CsH inhibit basophil chemotaxis induced by Hp(2–20) (Fig. 6⇓). These results are compatible with the hypothesis that FMLP induced basophil chemotaxis through the activation of FPR, whereas Hp(2–20) acted through different subtypes of FMLP receptors.
Effects of CsH on human basophil chemotaxis induced by FMLP or Hp(2–20). Basophils isolated from normal donors negative for H. pylori, HIV-1 and HIV-2 Abs were preincubated (5 min at 37°C) with CsH (800 nM) or buffer. Basophils were allowed to migrate toward FMLP (5 × 10−7 M) or Hp(2–20) (5 × 10−7 M) for 1 h at 37°C in a humidified (5% CO2) incubator. Values are the mean ± SEM obtained from three experiments with different basophil preparations. ∗, p < 0.01 when compared with FMLP.
FPR, FPRL1, and FPRL2 mRNA in human basophils
We analyzed the expression of FPR, FPRL1, and FPRL2 transcripts in human basophils, neutrophils, and monocytes by RT-PCR. Fig. 7⇓A shows the results of a representative experiment from among three different donors. We found that FPR mRNA and FPRL1 mRNA were expressed in basophils, neutrophils, and monocytes isolated from the peripheral blood of healthy volunteers. We also found that FPRL2 mRNA was expressed in basophils and monocyte-derived dendritic cells, as previously reported (16, 60) (Fig. 7⇓B). The quantitative expression of FPR, FPRL1, and FPRL2 mRNA in human basophils, neutrophils, and monocyte-derived dendritic cells was assayed by a real-time PCR technique that provides a sensitive and quantitative enumeration of sequences over at least seven orders of magnitude of starting numbers. Fig. 8⇓ shows the expression of FMLP receptors (FPR, FPRL1, and FPRL2) relative to β-actin in human basophils, neutrophils, and monocyte-derived dendritic cells. These results confirm that basophils express mRNA for FPRL1 and FPRL2, in addition to FPR. As a control, FPRL1 was detected in human neutrophils and FPRL2 was found in dendritic cells.
A, FPR and FPRL1 mRNA expression in human basophils, neutrophils, and monocytes purified from healthy volunteers negative for H. pylori, HIV-1 and HIV-2 Abs. RNA transcripts for FPR and FPRL1 were analyzed by RT-PCR. B, FPRL2 mRNA expression in human basophils and monocyte-derived dendritic cells from healthy volunteers negative for H. pylori, HIV-1 and HIV-2 Abs. RNA transcript for FPRL2 was analyzed by RT-PCR.
Real-time quantitative PCR analysis of the expression of FPR, FPRL1, and FPRL2 in relation to β-actin in human basophils, neutrophils, monocytes, and monocyte-derived dendritic cells. The figure shows the results of a representative experiment from among three different donors.
Cross-desensitization between Hp(2–20) and FMLP
The relationship between peptides Hp(2–20) and FMLP was further examined by evaluating the effects of cross-desensitization between these stimuli on basophil chemotaxis. Purified basophils were treated with buffer or with a low (5 × 10−7 M) or high (10−4 M) concentration of FMLP or with Hp(2–20) (5 × 10−7 M) in P-EDTA for 30 min at 37°C. At the end of incubation, cells were washed and suspended in PACGM. Fig. 9⇓ shows the results of five experiments in which the response to Hp(2–20) was abolished by preincubation with the homologous stimulus. When basophils were desensitized by preincubation with a low concentration (5 × 10−7 M) of FMLP, the response to heterologous stimulus Hp(2–20) was not significantly affected. Differently, when basophils were desensitized by exposure to a high concentration (10−4 M) of FMLP, which binds also to FPRL1 (65), the response to Hp(2–20) was significantly desensitized.
Effects of cross-desensitization between low (5 × 10−7 M) or high (10−4 M) concentrations of FMLP and Hp(2–20) (5 × 10−7 M) on basophil chemotaxis. Basophils isolated from normal donors negative for H. pylori, HIV-1 and HIV-2 Abs were incubated in PIPES buffer containing EDTA (4 mM), FMLP (5 × 10−7 M), FMLP (10−4 M), or Hp(2–20) (5 × 10−7 M) for 30 min at 37°C. At the end of incubation, cells were washed (two times) and resuspended in PACGM and challenged with the chemotactic stimuli (Hp(2–20), 5 × 10−7 M). Basophils were allowed to migrate for 1 h at 37°C in a humidified incubator with 5% CO2. Values are the mean ± SEM from five experiments. ∗, p < 0.01 when compared with cells preincubated with buffer and stimulated with Hp(2–20).
Discussion
In this study, we show that basophils can be a component of the inflammatory infiltrate in the gastric mucosa of patients with active inflammation caused by H. pylori. In addition, we demonstrate that a H. pylori-derived peptide, Hp(2–20), is a potent chemoattractant for basophils presumably by interacting with FPRL1 and FPRL2 receptors.
We used BB1, a basophil granule-specific mAb that recognizes a protein of ∼124 kDa and does not cross-react with mast cells, eosinophils, neutrophils, lymphocytes or macrophages (54, 66). Although it is well documented that basophils infiltrate the site of allergic inflammation (42, 43, 44, 45, 46), we show that basophil infiltration can occur at the site of bacterial-induced inflammation. Using immunohistochemistry, basophils were undetectable in gastritis-free, H. pylori-negative individuals, and in H. pylori-positive subjects with no or a low grade of gastric inflammation. Conversely, BB1-positive basophils were identified in >60% of gastric biopsies of H. pylori-infected patients with moderate and severe gastritis. We do not yet know why basophils were not detected in some patients with active gastritis caused by H. pylori. One may speculate that this finding reflects: 1) biological and clinical diversity of patients with H. pylori-induced gastritis; 2) different strains of H. pylori inducing distinct patterns of infiltrating inflammatory cells, as shown experimentally (67); 3) host genetic factors; or finally, 4) technical aspects. In fact, in relation to the latter, we found that individual cells showed some variation in intensity of staining, which likely reflects either different levels of cell sectioning or different stages of basophil degranulation. We cannot exclude the possibility that partial or complete basophil degranulation may result in some underestimation of the number of basophils infiltrating the gastric mucosa (42). In fact, it has been demonstrated that basogranulin, the basophil granule protein recognized by the mAb BB1, can be released after stimulation of basophils (68). Nevertheless, the basophil specificity of BB1 clearly indicates that basophils infiltrated the gastric mucosa in most patients with moderate and severe inflammation caused by H. pylori.
Mast cells and basophils are the main effector cells in IgE-mediated allergic responses, but they also play important roles in innate immune responses against bacteria by releasing proinflammatory mediators and cytokines (29, 30). Human FcεRI+ cells are involved in bacterial infections in a variety of ways (25, 47, 69, 70). In vitro studies have demonstrated that several H. pylori-derived peptides induce and/or potentiate mediator release from FcεRI+ cells. For instance, H. pylori extracts potentiate histamine release from rat mast cells (71). In addition, the neutrophil-activating protein of H. pylori induces the release of preformed mediators and of IL-6 from peritoneal mast cells (34). Finally, VacA activates mast cells so leading to the migration and production of proinflammatory cytokines (35). Moreover, oral treatment of mice with VacA caused acute inflammation of gastric mucosa and mast cell accumulation (35). Thus, several H. pylori proteins activate rodent mast cells, presumably through different immunologic mechanisms.
Human basophils, but not mast cells, express receptors for FMLP that induce their chemotaxis and the release of proinflammatory mediators (25). Three N-formyl peptide receptors have been identified on different cells (17, 18). These receptor subtypes belong to the STM, G protein-coupled rhodopsin superfamily (72, 73). The FPR has a high affinity for FMLP and is activated by nanomolar concentrations of FMLP. FPRL1 is a promiscuous receptor activated in response to higher concentrations of FMLP (16), the prion peptide, PrP106–126 (22), lipoxin A4 (23), Hp(2–20) (11), serum amyloid A (21), and various synthetic peptides (18). Interestingly, the Hp(2–20) peptide does not share any sequence homologies with any of the other agonists, in agreement with the promiscuous feature of FPRL1. Human neutrophils and monocytes express FPRL2, and Hp(2–20) exerts its chemotactic effect also through activation of this receptor subtype (18). Our results provide the first demonstration that human basophils express FPR, FPRL1, and FPRL2 receptors.
CsH blocks FPR-evoked responses (18, 25, 26). Accordingly, CsH blocked the chemotactic activity of FMLP on basophils, but had no effect on the response evoked by peptide Hp(2–20). We performed cross-desensitization experiments to verify the specificity of the activation route for peptide Hp(2–20) and FMLP. Upon binding of FMLP to its FPR, the occupied receptor is phosphorylated (74), thus cells are desensitized and unable to generate signals through the same receptor. We found that basophils preincubated with FMLP in the absence of Ca2+ did not generate a chemotactic response when rechallenged with the same agonist (homologous desensitization). Similarly, basophils preincubated with peptide Hp(2–20) were desensitized to a subsequent challenge with the homologous stimulus. Heterologous desensitization yielded interesting results. When basophils were preincubated with a low concentration (5 × 10−7 M) of FMLP, which binds with high affinity only to FPR, but not to FPRL1 (72), the chemotactic response to the heterologous stimulus (peptide Hp(2–20)) was not affected. In contrast, when basophils were exposed to a high concentration (10−4 M) of FMLP, which binds also to FPRL1 (72), the chemotactic response to peptide Hp(2–20) was significantly reduced. The results of these two groups of experiments are consistent with the hypothesis that peptide Hp(2–20) and FMLP act through different N-formyl peptide receptor subtypes to induce chemotaxis of human basophils: FMLP acts essentially through the interaction with FPR, whereas peptide Hp(2–20) presumably acts through FPRL1 and FPRL2. This hypothesis is supported by the different pharmacologic effects of CsH on basophil chemotaxis induced by FMLP and peptide Hp(2–20). Until receptor-specific agonists/antagonists become available it is not possible to determine the relative contribution of FPRL1 and FPRL2 for the signals transduced by Hp(2–20). We cannot exclude the possibility that peptide Hp(2–20), besides activating FPRL1 and FPRL2, might activate other, as yet unknown, receptor(s) on basophils.
The observation that the synthetic hexapeptide, WKYMVm, is a very potent stimulus for both histamine release and chemotaxis of human basophils is intriguing. The presence of a d-methionine at its terminus is critical for WKYMVm’s biological activity (75). WKYMVm, in addition to being a high affinity ligand for FPR, is an agonist also for FPRL1 and FPRL2 (18, 60). The possibility exists that WKYMVm induces mediator release through activation of FPR, and activates basophil chemotaxis through FPRL1 and FPRL2. Why basophils recognize d-methionine-containing peptides is not as obvious as why they recognize bacterial-derived N-formylated peptides. Natural proteins normally include only l-amino acids. However, there are a few exceptions: the cell wall of Gram-negative bacteria and a number of oligopeptides produced as antibiotic agents by a variety of bacteria contain d-amino acids (76, 77). The WKYMVm receptor may thus have evolved to enable human basophils to recognize microbial agents. This hypothesis is consistent with the observation that human FcεRI+ cells are involved in natural immunity against several microbial agents (25, 47).
Granulocyte and mononuclear cell infiltration is characteristic of bacterial infections. By contrast, to our knowledge, basophils have never been detected at sites of bacterial infections. The interactions between basophils and microbial agents are exceedingly complex, reflecting long periods of coevolution (78, 79). There is compelling evidence that basophils and their mediators participate in host defense against parasites (79) and viruses (24, 31, 32, 64). It has been shown that peripheral blood basophils may be increased in H. pylori-positive patients (36). In this study, we provide the first direct evidence that basophils can contribute to H. pylori-induced mucosal inflammation and that a bacterial infection is associated with basophil infiltration. Although we found that Hp(2–20) is chemotactic for human basophils, this peptide was not found to induce the release of mediators from basophils purified from either H. pylori-positive or -negative individuals. However, several stimuli can locally activate infiltrating basophils. For example, a mechanism by which basophils may be activated in H. pylori gastritis could implicate IL-8, which is secreted from inflamed mucosa (80) and is able to induce histamine release from basophils (81). In addition, it cannot be excluded that basophils from H. pylori-positive individuals can be activated through an IgE-mediated mechanism (37, 39).
Our study suggests that the Hp(2–20) peptide contributes to the recruitment of basophils to the inflammatory component of H. pylori-infected gastric tissue. Betten et al. (11) demonstrated that Hp(2–20) activates human monocytes to induce lymphocyte dysfunction and apoptosis. Taken together, these observations suggest that Hp(2–20) may contribute through various mechanisms to the dysregulation of the immune system in H. pylori infection.
The basophil infiltration of gastric mucosa in H. pylori-infected individuals was unexpected and unprecedented. The role(s) of basophils and their mediators in the inflammatory response to H. pylori remains to be determined. It is possible that platelet-activating factor, histamine, cysteinyl leukotriene C4, and other proinflammatory mediators released by basophils contribute to H. pylori-associated gastric damage. Alternatively, we cannot exclude the hypothesis that TH2-like cytokines (IL-4 and IL-13) synthesized by basophils (47, 64) modulate TH1-mediated mucosal inflammation (82).
In conclusion, we provide the first evidence that basophils infiltrate the gastric mucosa of patients with moderate and severe chronic active gastritis caused by H. pylori. We also demonstrate that a H. pylori-derived peptide (Hp(2–20)) is a potent chemoattractant for basophils through interaction with FPRL1 and FPRL2. The biological and clinical significance in host defense and immune responses in H. pylori infection of our findings requires additional investigations.
Acknowledgments
We are indebted to Dr. Giuseppe Spadaro (Division of Clinical Immunology and Allergy, University of Naples Federico II, Naples, Italy) for providing access to patients with H. pylori infection. We dedicate this paper to Rita Levi Montalcini.
Footnotes
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↵1 This work was supported by grants from the Ministero dell’ Instruzione, Università e Ricerca National Project “Helicobacter pylori infection: host-pathogen interactions”, the Istituto Superiore Sanità (AIDS Project 40C.60 and 40D.57), and Ministero della Salute “Alzheimer Project”, (Rome, Italy). G.M. is the recipient of the Esculapio Award 2002 (Accademia Tiberina, Rome, Italy).
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↵2 Address correspondence and reprint requests to Dr. Gianni Marone, Department of Clinical Immunology and Allergy, University of Naples Federico II, Via S. Pansini 5, 80131 Napoli, Italy. E-mail address: marone{at}unina.it
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↵3 Abbreviations used in this paper: FPR, formyl peptide receptor; FPRL, FPR-like; STM, seven transmembrane; HSA, human serum albumin; fura 2-AM, fura 2-acetoxymethyl ester; CsH, cyclosporin H; LDH, lactate dehydrogenase; [Ca2+]i, intracellular Ca2+ concentration; rh, recombinant human.
- Received November 6, 2003.
- Accepted April 1, 2004.
- Copyright © 2004 by The American Association of Immunologists
References
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