Western blot analysis of monocyte chemotactic and activating factor or monocyte chemoattractant protein-1 (MCAF/MCP-1) from the culture supernatants of human nasal mucosa. Lane 1 represents culture supernatants of recombinant human MCAF/MCP-1 (positive control); lane 2, culture supernatants of sinus mucosa with chronic infective sinusitis; and lane 3, culture supernatants of nasal mucosa with allergy. The arrow indicates the band corresponding to approximately 14 kd, demonstrating the expression of MCAF/MCP-1 in these samples.
Histamine-releasing activity in supernatants of explant culture of human nasal mucosa (SCNM). Purified active fractions of SCNM with chronic sinusitis (Active Sup) induced significant histamine release compared with control fractions (Control Sup). After incubation with anti–monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1) antibody, a tendency toward inhibition was observed. The results of net histamine release are expressed as mean±SEM of 5 patients. The plus sign indicates that 10 to 250 µg/mL of anti-MCAF antibody was applied to the Active Sup.
Comparison of monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1)–positive cell counts in the nasal turbinate mucosa in nonallergic and allergic patients. The bar represents the median value.
Sequential 4-µm-thick sections of nasal mucosa from an allergic rhinitic patient stained with anti–monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1) (top) and Ki-M1p antibody (bottom). Almost all MCAF/MCP-1 immunoreactive cells were also stained with Ki-M1p, which recognizes macrophages and monocytes. The black arrows indicate cells that were stained positively with anti–MCAF/MCP-1 and Ki-M1p antibodies; white arrows, a cell that was stained positively with anti-MCAF/MCP-1 antibody only.
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Fujikura T, Otsuka H. Monocyte Chemotactic and Activating Factor/Monocyte Chemoattractant Protein-1–Mediated Histamine Release From Human Nasal Mucosa. Arch Otolaryngol Head Neck Surg. 1998;124(12):1331–1335. doi:10.1001/archotol.124.12.1331
MANY RECENT investigations1-3 focused on the mechanisms of allergen-induced late-phase response (LPR) in the airways. However, the pathogenesis of LPR is complex and is not completely understood. The recrudescence of symptoms in LPR is associated with an influx of eosinophils, neutrophils, mononuclear cells, and basophils.4,5 It seems likely that these cells contribute to the pathogenesis of LPR. The mast cell is the predominant source of mediators in the early-phase response. On the other hand, based on the pattern of mediator release observed in the LPR (ie, histamine and leukotriene but not prostaglandin D2), it has been speculated that basophils enter the nose during LPR and are responsible for the release of mediators.3 The stimulus for basophil activation in the LPR is not fully characterized. Several laboratories reported6-8 the existence of cytokinelike molecules—called histamine-releasing factors (HRFs)—that induce basophil or mast cell secretion of histamine in the absence of a particular antigen. Histamine-releasing factors are present in nasal washings of patients with allergic rhinitis.9 However, the molecular form of HRFs from nasal mucosa has not been revealed yet. Based on the results of a recent study,8 the major species of HRF are thought to belong to the chemokine family of cytokines. The activity of HRFs has been characterized primarily by using basophils as the target cells. Studies of mast cells have been limited in human beings but suggest a lesser responsiveness to chemokines.10 Monocyte chemotactic and activating factor or monocyte chemoattractant protein-1 (MCAF/MCP-1), which belongs to the CC chemokines, is known to be a potent HRF for basophils.11,12
The objective of this study is to investigate the existence of MCAF/MCP-1 in human nasal mucosa and to verify its role as an HRF. We detected MCAF/MCP-1 in the supernatants of explant culture of human nasal mucosa (SCNMs) using Western blot analysis and demonstrated the histamine-releasing activity of these supernatants.
Twenty-one patients with mite allergy (10 male and 11 female; age range, 15-45 years; mean±SEM age, 27±2 years), 7 nonallergic patients (5 male and 2 female; age range, 29-48 years; mean±SEM age, 35±2 years), and 5 patients with chronic infective sinusitis (2 male and 3 female; age range, 34-66 years; mean±SEM age, 50±6 years) participated in the study. Written informed consent was obtained from all patients before starting the study. All allergic patients had positive test results for mite nasal allergy detected by a clinical history, a rhinoscopic examination, a nasal smear for eosinophilia, an intradermal test, a nasal provocation test, and determination of specific mite IgE antibodies by a radioallergosorbent test. Although 4 patients with mite allergy and 1 patient with chronic infective sinusitis had a positive reaction to a radioallergosorbent test specific for Japanese cedar pollen, the studies were performed out of season. All other patients with mite allergy and chronic infective sinusitis had a negative reaction to radioallergosorbent tests for other common aeroallergens, such as molds or grasses. Nonallergic patients had negative results in all tests. Twelve allergic patients were treated with immunotherapy. They were given subcutaneous injections of an aqueous extract of house dust (TORII, Torii Co, Tokyo, Japan) at a 1:100 to 1:1000 dilution for longer than 6 months.
Antihuman MCAF/MCP-1 was purchased from Pepro Tech Inc (Rocky Hill, NJ), and goat anti-rabbit IgG, F(ab‘)2was purchased from Immunotech SA (Marseille, France). Ki-M1p antihuman monocyte and macrophage antibody were purchased from Seikagaku Kogyo Co (Tokyo), and mouse IgG1 (MOPC21) was purchased from Sigma Chemical Co (St Louis, Mo). The staining kit for the streptavidin-biotin method of immunohistochemistry was purchased from Nichirei Co (Tokyo).
During surgery, we took pieces of nasal mucosa (approximate weight, 1-3 g) from the nasal turbinate or the maxillary sinus and cut them into 1- to 2-mm cubic fragments. About 20 fragments were put into 8 mL of RPMI 1640 tissue culture medium containing HEPES, 25 mmol; penicillin, 100 U/mL; streptomycin sulfate, 100 µg/mL; and gentamicin sulfate, 50 µg/mL; and were incubated with 5% carbon dioxide at 37°C for 48 hours. After collecting SCNM samples of 500 mL each from 5 patients with nasal allergy or chronic sinusitis, the pooled samples were concentrated 200-fold by ultrafiltration and placed in low molecular weight (3500 d) cutoff dialysis tubing. Dialysis was performed for 48 hours at 4°C. After centrifugation again at 40,000g for 20 minutes, we got 2.5-mL samples, of which protein concentration was about 0.5 g/mL.
Each SCNM sample (1.0 mL) of nasal turbinate mucosa and sinus mucosa, was diluted 1:50 with 0.01 mol/L phosphate-buffered saline solution (pH, 7.2). Sonic treatment with (sonic oscillator UD201, Tomy Seiko, Tokyo, Japan) was done for 5 minutes. After boiling at 100°C for 5 minutes with the same volume of sample buffer, containing 2-mercaptoethanol, electrophoretic separation of this supernatant was performed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with a 12% polyacrylamide separating gel according to the procedure of Laemmli.13 Peptide fragments resolved by sodium doLaemmli.13 Peptide fragments resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were transformed into a nitrocellulose membrane with a constant current (1 A) for 1 hour according to the method of Towbin et al.14 Nonspecific membrane protein binding sites were blocked and then membranes were probed with rabbit antihuman MCAF polyclonal antibody. After incubation for 24 hours, membranes were reacted with alkaline phosphatase–labeled goat anti-rabbit IgG, F(ab‘)2 for 2 hours.
Purification of culture supernatants was performed according to the following procedure. After concentration (200-fold) and dialysis, the crude supernatants were applied to a column and eluted with 0.01-mol/L Tris hydrochloride (pH 7.4). According to the pattern of filtration, we divided the supernatants into several fractions. From the results of a preliminary study (T.F. and H.O., unpublished data, November 9, 1993), we chose 1 fraction in which histamine-releasing activity could be found, and then this fraction was concentrated 10-fold. Leukocytes were prepared by dextran sedimentation (6% dextran and 0.85% sodium chloride). Purified SCNM, 30 µL, was incubated with dextran-sedimented leukocytes, 100 µL, containing basophils, 1.5 × 106/mL, at 37°C for 30 minutes. This suspension was centrifuged at 140g for 3 minutes and separated into supernatants and sediment, then histamine was determined by radioimmunoassay. The percentage of histamine release was calculated according to the following formula: histamine in the supernatant divided by histamine in the supernatant plus histamine in the sediment; the value of spontaneous histamine release was subtracted from this percentage. Spontaneous histamine release was assessed by incubating cells in HEPES-buffered saline solution (pH 7.4) alone.
To demonstrate the effect of anti–MCAF/MCP-1 antibody on histamine release induced by SCNMs, the purified SCNM sample was preincubated with rabbit antihuman MCAF polyclonal antibody at concentrations of 10, 100, or 250 µg/mL at 37°C for 1 hour and centrifuged at 10,000g for 10 minutes. After these treatments, assay of histamine-releasing activity in the supernatants was performed.
Nasal biopsy samples were taken from turbinates during surgical treatment of nasal obstruction. Specimens (approximately 5 × 20 mm) were placed in Carnoy solution and fixed for 2 days. Each specimen was embedded in paraffin and cut with a microtome into 4-µm-thick sections. After deparaffinization, immunohistochemical staining was carried out by treatment with the streptavidin-biotin method. As primary antibody, 100 µg/mL of mouse antihuman MCAF/MCP-1 monoclonal antibody was applied for 24 hours at 4°C. Control slides were similarly treated, but mouse IgG1 was used instead of the primary antibody. Using a light microscope (magnification ×200), we chose 50 areas (0.06 mm2 per area) at random in the lamina propria and counted the number of MCAF/MCP-1–positive cells in the area. Staining of macrophages and monocytes was carried out using the same system, but Ki-M1p was used as the primary antibody.
The percentage of histamine release is shown as mean±SEM, and statistical analyses were performed with the paired Wilcoxon signed rank test using a statistical computer program (FISHER, StatSoft Inc, Tulsa, Okla). The number of MCAF/MCP-1–positive cells was compared between 2 groups—allergy and nonallergy—using the Mann-Whitney U test. Differences were considered statistically significant at P<.05.
In Western blot analysis of SCNM with allergy or chronic sinusitis, bands corresponding to approximately 30 kd (before sonic treatment) and 13 to 15 kd (after sonic treatment) were observed (Figure 1). As a positive control, recombinant MCAF/MCP-1 migrated at 8 to 9 kd as a single band. The molecular weight of MCAF/MCP-1 is 8.7 kd, but when purified materials containing MCAF/MCP-1 are electrophoresed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, at least 2 bands corresponding to approximately 14 and 15 kd are observed.15 Some of the bands above 30 kd were considered its polymer or a nonspecific reaction with albumin.
From the results of a preliminary study (T.F. and H.O., unpublished data, October 5, 1993), statistically significant histamine release was not observed in both culture supernatants (data not shown), but there was a tendency for our samples from sinus mucosa to contain more MCAF/MCP-1 than nasal mucosa with allergy. Thus, we purified and concentrated the SCNM sample with chronic sinusitis and reacted it with leukocytes again. Basophil-rich fractions of dextran-sedimented leukocytes from 5 individuals with nasal allergy were challenged with this SCNM. Net histamine release induced by 1 active fraction was 4.6%±12.1%, and this histamine release was statistically significant compared with that of controls (P<.05). The fraction that contained no proteins was used as a control, and the value of its histamine release was −0.2%±0.1%.
We also assessed the inhibition of histamine release by anti–MCAF/MCP-1 polyclonal antibody. Net histamine release values induced by active fraction of SCNM after preincubation with anti–MCAF/MCP-1 were 2.4%±0.7% (with 10 µg/mL of anti–MCAF/MCP-1), 0.7%±0.3% (anti–MCAF/MCP-1, 100 µg/mL), and 1.4%±0.3% (anti–MCAF/MCP-1, 250 µg/mL). The tendency toward inhibition (P=.06) was observed (Figure 2).
In nasal mucosa of 11 patients with allergic rhinitis, several MCAF/MCP-1–producing cells (97.6±21.1 cells/mm2; range, 40.1-290.9 cells/mm2) were observed (P<.01; Figure 3). Cells positive for MCAF/MCP-1 were observed in the lamina propria, but not in the epithelial layer. The number of these cells in the 5 patients without nasal allergy was 25.1±3.7 cells/mm2 (range, 22.4-36.2 cells/mm2).
Sequential 4-µm-thick sections of nasal mucosa from an allergic patient were stained with anti–MCAF/MCP-1 monoclonal antibody and Ki-M1p antibody recognizing monocytes and macrophages. Almost all MCAF/MCP-1 immunoreactive cells were also stained with Ki-M1p (Figure 4).
Chemokines induce a variety of proinflammatory effects, including influx of inflammatory cells, and activation of basophils and eosinophils.16,17 There is some evidence that CC chemokines may be implicated in the pathogenesis of allergic inflammation.16-19 Monocyte chemotactic and activating factor/monocyte chemoattractant protein-1, which belongs to the CC chemokine family of cytokines, is known to activate basophils and is responsible in large part for histamine-releasing activity.8,11,12 We could not directly show the histamine-releasing activity using nasal mucosa with allergy, but the results of our study demonstrate the existence of MCAF/MCP-1 in SCNM with chronic inflammation, which possibly acts as an HRF. In our study, 4 of 5 patients with allergy selected for preparation of SCNM samples, were treated with immunotherapy. Immunotherapy reduces the production of HRF in patients with asthma.20 Therefore, MCAF/MCP-1 production from SCNM might not be enough to induce a high degree of histamine release.
In the present study, MCAF/MCP-1 immunoreactivity was observed in the nasal submucosa of allergic patients but not in the epithelial layer. The main symptom of LPR in the nasal allergy is nasal obstruction, not sneezing or rhinorrhea. The basophils that migrated from peripheral blood to the nasal submucosa may be activated by MCAF/MCP-1, produced also in the submucosa, and may release histamine. This reaction causes nasal congestion, but because the reaction occurs only in the submucosa, it is difficult to stimulate nerve endings in or just beneath the epithelial layer. Thus, it may not be enough to cause sneezing or nasal secretion.
We speculate that MCAF/MCP-1 is constantly produced and is stored in nasal mucosa with allergy. The degree of basophil infiltration may also regulate the pathogenesis of LPR. Although the results of several studies4,21 demonstrate the presence of basophils in nasal allergic inflammatory sites, the mechanisms of basophil influx into the nose remain unclear. Among CC chemokines, MCAF/MCP-1 has only moderate chemotactic activity for basophils, but RANTES and monocyte chemoattractant protein-3 (MCP-3) are more effective as chemoattractants for basophils.16,19 The activity of CC chemokines as chemoattractants for inflammatory cells in nasal mucosa should be investigated.
The cell sources of MCAF/MCP-1 are macrophages, monocytes, fibroblasts, endothelial cells, lymphocytes, and some types of tumor cells.22 Recently, Becker and coworkers23 reported the presence of messenger RNA for MCAF/MCP-1 in culture nasal epithelial cells, but, based on the results of our immunohistochemical study, most MCAF/MCP-1–producing cells in nasal mucosa were monocytes, macrophages, and some endothelial cells. Human airway epithelial cells may have potential activity to produce MCAF/MCP-1, but in human nasal mucosa with allergy, the significance of their MCAF/MCP-1 production has not been established yet.
Production of MCAF/MCP-1 occurs only in the states with some stimulation from inflammatory cells or tumor cells.22 Viral or bacterial infection has been known to induce chemokine production,19,24 and the results of our study reveal that chronic infection induces MCAF/MCP-1 production from sinus mucosa. The role of MCAF/MCP-1 in infection is not fully understood. Human airways are continuously exposed to infections and irritants. Therefore, chemokines are useful for local regulation of inflammatory response and injury of the airways. On the other hand, chemokines induced by viral or bacterial infection may participate in the protraction of allergic inflammation. In nasal allergy, the factors that stimulate the production of MCAF/MCP-1 remain unknown. Tumor necrosis factor α is one of the cytokines that induce MCAF/MCP-1 production from macrophages.22,23 Under the accumulated exposure of antigen, mast cells and macrophages may be activated, and tumor necrosis factor α released from these cells may induce MCAF/MCP-1 production. Macrophages in nasal mucosa possibly provide an autocrine pathway to maintain migration and effect function of those cells.
In perennial allergic rhinitis, nasal mucosa is constantly exposed to antigen, and early-phase response and LPR may overlap. Although in a clinical condition LPR of nasal allergy is not clearly observed, stored MCAF/MCP-1 in nasal submucosa may act as an HRF and may contribute not only to the LPR but also to the protraction of allergic inflammation in nasal mucosa.
Accepted for publication July 8, 1998.
We thank Hisashi Okuni, MD, and Yuko Todome, PhD, for their technical advice and Minoru Okuda, MD, for his helpful review of the manuscript.
Reprints: Terumichi Fujikura, MD, Department of Otorhinolaryngology–Head and Neck Surgery, Nippon Medical School, Dai 2 Hospital, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-0063 Japan.