Supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Ministry of Health, Labour and Welfare of Japan.
Human neutrophil peptide-1 aggravates dextran sulfate sodium-induced colitis†
Article first published online: 16 SEP 2011
Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.
Inflammatory Bowel Diseases
Volume 18, Issue 4, pages 667–675, April 2012
How to Cite
Hashimoto, S., Uto, H., Kanmura, S., Sakiyama, T., Oku, M., Iwashita, Y., Ibusuki, R., Sasaki, F., Ibusuki, K., Takami, Y., Moriuchi, A., Oketani, M., Ido, A. and Tsubouchi, H. (2012), Human neutrophil peptide-1 aggravates dextran sulfate sodium-induced colitis. Inflamm Bowel Dis, 18: 667–675. doi: 10.1002/ibd.21855
- Issue published online: 19 MAR 2012
- Article first published online: 16 SEP 2011
- Manuscript Accepted: 18 JUL 2011
- Manuscript Received: 14 JUL 2011
- human neutrophil peptide-1;
- ulcerative colitis;
- dextran sulfate sodium;
Human neutrophil peptide (HNP)-1, HNP-2, and HNP-3 (HNP-1–3) are useful biomarkers for ulcerative colitis (UC). The precise roles of these peptides in UC are poorly understood, however. The aim of this study was to determine whether HNP-1 affects disease activity in mice with experimental colitis.
Experimental colitis was induced in BALB/c or severe combined immunodeficiency (SCID) mice using dextran sulfate sodium (DSS). Mice were subsequently treated intraperitoneally with HNP-1 (100 μg/day) or phosphate-buffered saline (PBS) from day 4 to day 6. The severity of colitis was evaluated based on a disease activity index, histologic score, and cytokine expression.
Body weight and colon length significantly decreased and the disease activity index score, histologic score, and myeloperoxidase activity significantly increased in HNP-1-treated BALB/c mice compared with PBS-treated mice. Interferon-γ and tumor necrosis factor-α levels in colon culture supernatants-derived HNP-1-treated mice were also significantly higher, and interleukin (IL)-1β levels tended to increase in response to HNP-1. In addition, treating SCID mice with HNP-1 aggravated DSS-induced colitis and IL-1β levels in colon culture supernatants from these mice were significantly higher than in cultures obtained from control mice. Furthermore, in both BALB/c and SCID mice increased recruitment of F4/80-positive macrophages was observed in the inflamed colonic mucosa following HNP-1 injections.
High concentrations of HNP-1 aggravate DSS-induced colitis, including upregulated expression of such macrophage-derived cytokines as IL-1β. These results indicate that high concentrations of HNP-1–3 in patients with UC may exacerbate disease activity via increased cytokine production. (Inflamm Bowel Dis 2011;)
Inflammatory bowel disease (IBD) primarily manifests as one of two forms: ulcerative colitis (UC) or Crohn's disease (CD). Although the etiology of IBD is not clear, the mucosal immune system plays a central role in intestinal inflammation and injury and cytokines are critical modulators of inflammation. Of note, tumor necrosis factor (TNF)-α and interleukin (IL)-1β are thought to be key cytokines in IBD.1, 2
IBD is associated with the infiltration of a large number of leukocytes into the bowel mucosa and removing circulating leukocytes—including monocytes, granulocytes, and lymphocytes—is thought to be a promising approach for the treatment of IBD, particularly for UC in Japan.3, 4 The molecular mechanisms underlying the benefits of leukocyte aphaeresis have not been fully elucidated, however.
Defensins, such as the vertebrate α-defensins, are antimicrobial and cytotoxic peptides.5 α-Defensins include six subfamilies: four human neutrophil peptides (HNP-1 to HNP-4), human defensin (HD)-5, and HD-6.5, 6 HNPs are small cationic peptides that are predominantly expressed in normal bone marrow cells, neutrophils, natural killer cells, and T cells. HNPs are stored in azurophilic granules and are released in response to neutrophil activation.7–9 The amino acid sequences of HNP-1, HNP-2, and HNP-3 are similar structures, with the peptides consisting of 30, 29, and 30 amino acids, respectively. Twenty-nine of the amino acids are identical among the three peptides and we previously reported that neutrophils infiltrating the bowel mucosa in patients with active UC express HNP-1–3.10 In addition, a proteomic approach revealed that HNP-1–3 levels were elevated in sera from patients with UC compared with samples from patients with CD.10 Plasma concentrations of HNP-1–3 were also higher in patients with active UC than in healthy subjects or those with inactive UC, CD, or infectious enterocolitis. Furthermore, plasma concentrations of HNP-1–3 in patients with UC who responded to corticosteroid-based therapy decreased after treatment, whereas no change was observed in nonresponders.10 Interestingly, HNPs are reportedly antimicrobial and regulate IL-8 production and chemotaxis in neutrophils, lymphocytes, and macrophages.11, 12 The relationships between HNP-1–3 and intestinal inflammation or mucosal injury in patients with UC, however, have not been fully investigated. The aim of this study was to determine whether HNP-1 exacerbates disease activity in mice with experimental colitis.
MATERIALS AND METHODS
Synthetic HNP-1 was purchased from the Peptide Institute (Osaka, Japan) and dissolved in phosphate-buffered saline (PBS). McCoy's 5A medium, RPMI 1640 medium, fetal bovine serum, penicillin-streptomycin, PBS, L-glutamine, and HEPES buffer were obtained from Invitrogen (La Jolla, CA). TetraColor One was obtained from Seikagaku Biobusiness (Tokyo, Japan). Colorimetric immunoassays to quantify cell proliferation based on bromodeoxyuridine (BrdU) incorporation during DNA synthesis were obtained from Roche Diagnostics (Mannheim, Germany). Annexin V-FITC apoptosis detection kits and tetramethylbenzidine/H2O2 substrate solution were obtained from BD Biosciences (Princeton, NJ). Dextran sodium sulfate (DSS; mean molecular weight, 5,000 Da) and cetyltrimethylammonium chloride were obtained from Wako Pure Chemical Industries (Osaka, Japan). Citrate buffer solution was obtained from Sigma-Aldrich Japan (Tokyo, Japan). Interferon (IFN)-γ, IL-1β, and TNF-α ELISA kits were obtained from R&D Systems (Minneapolis, MN).
Specific pathogen-free male BALB/c mice (7 weeks of age, weighing 25–29 g) were obtained from Kyudo (Saga, Japan). Male severe combined immunodeficiency (SCID) mice (C.B-17/lcr-scid/scid Jcl, 7 weeks of age, weighing 20–24 g) were obtained from CLEA Japan (Tokyo, Japan). Mice were maintained in standard wire cages and allowed free access to food and water for 1 week before entering the study. This study was approved by the Animal Experiment Ethics Committee of the Kagoshima University Graduate School of Medical and Dental Sciences.
Induction of Colitis
Experimental colitis was induced in BALB/c or SCID mice by administering 4.0% or 3.0% DSS in drinking water, respectively, for 7 days. Mice were intraperitoneally injected with HNP-1 (100 μg/day) or PBS from day 4 to day 6. Serum HNP-1 levels were measured using a human HNP 1–3 enzyme-linked immunosorbent assay (ELISA) kit (Hycult Biotechnology, Ude, Netherlands) according to the manufacturer's instructions. Mice were sacrificed and the severity of colitis was evaluated based on body weight, colon length, clinical scoring, and histologic scoring of colitis. Clinical scoring was used to determine values for a disease activity index (DAI), which was based on weight loss, stool consistency, and bleeding, as described previously by Murthy et al.13 Each clinical parameter was scored on a scale of 0–4 and the parameter values were added together. For histopathologic examination, the cecum and rectum were dissected longitudinally and the length of the entire colon was measured. Sections of colon were fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Histologic scoring was based on the method described previously by Cooper et al.14 Colon damage was categorized into five groups: grade 0, normal mucosa; grade 1, loss of one-third of the crypts; grade 2, loss of two-thirds of the crypts; grade 3, lamina propria covered with a single epithelial layer with mild inflammatory cell infiltration; grade 4, erosions and marked inflammatory cell infiltration. All scores were obtained in a blinded fashion by two investigators. Colonic tissue myeloperoxidase (MPO) activity, an indicator of the extent of neutrophil infiltration, was assessed as described previously by Qualls et al.15
For macrophage staining, F4/80 antigen was detected using methods similar to those described previously.16 To assess proliferation of colonic epithelial cells, Ki-67 was stained using methods similar to those described previously.17 In brief, after DSS was administered to BALB/c or SCID mice with or without HNP-1 for 7 days, deparaffinized sections of colon tissues were incubated with F4/80 monoclonal rat antimouse antibodies (AbD Serotec, Raleigh, NC) or anti-Ki-67 monoclonal antibodies (MIB-5; Dako Cytomation, Copenhagen, Denmark), followed by incubation with Histofine Simple Stain MAX PO (Rat; Nichirei, Japan). Following visualization of F4/80 or Ki-67 antigen, F4/80- or Ki-67-positive cells were counted based on the average obtained from five areas under a microscope at 400× magnification.
Colon Organ Cultures
The colon organ culture conditions have been described previously by Azuma et al.18 On day 7 after DSS administration the transverse colon was obtained from each mouse. A segment of distal colon was removed from each animal, cut open longitudinally, and washed in PBS containing penicillin and streptomycin. Segments 1 cm in length were placed in 24-well flat-bottom culture plates (Asahi Glass, Tokyo, Japan), containing 1 mL of fresh RPMI 1640 medium supplemented with penicillin and streptomycin, and incubated at 37°C for 24 hours. This experiment was performed using 13 mice from each group. Culture supernatants were stored at −30°C until analysis. Concentrations of IFN-γ, IL-1β, and TNF-α in the supernatants were measured using cytokine ELISA kits according to the manufacturer's protocol and analyzed in duplicate using a microplate reader (Bio-Rad Laboratories, Hercules, CA). Cytokine concentrations in the samples were calculated using standard curves. Protein concentrations in each sample were determined using an RC DC protein assay (Bio-Rad Laboratories).
Human HT-29 colon cancer cell line (European Collection of Cell Cultures, EC910722201) were obtained from Dainippon Sumitomo Pharma Biomedical (Osaka, Japan) and maintained at 37°C in 5% CO2 and McCoy's 5A medium supplemented with 10% heat-inactivated fetal bovine serum, streptomycin, penicillin, and L-glutamine.
Stimulation of Cells with HNP-1 and Cell Proliferation
The viability of HT-29 cells was assessed using the TetraColor ONE assay, which examines mitochondrial activity. DNA synthesis was assessed in the cells using a BrdU incorporation assay according to the manufacturer's instructions. Before the experiments cells were trypsinized and washed twice with PBS. Cells were seeded at a concentration of 3 × 104 cells/mL (TetraColor ONE assay) or 1 × 105 cells/mL (BrdU incorporation assay) in 96-well plates containing 90 μL of serum-free medium and incubated for 24 hours. HNP-1 dissolved in PBS or PBS alone were added to the 96-well plates and the cells were incubated in a total volume of 100 μL medium/well with various concentrations of HNP-1 for 12, 24, 48, or 72 hours (n = 6–12 for each condition). For the TetraColor ONE assay, 10 μL of TetraColor ONE solution was added to each well and the cells were incubated at 37°C for 2 hours. Each well was analyzed using a microplate reader (Bio-Rad Laboratories) at 450 nm (reference wavelength, 620 nm). The proliferation rate and BrdU incorporation in HNP-1-treated cells were assessed and absorbance values obtained with untreated cells were defined as 100%.
Annexin V Staining and FACS
An Annexin V-FITC apoptosis detection kit was used to determine the percentage of cells undergoing apoptosis. HT-29 cells were seeded in 12-well plates and grown to ≈80% confluence. Before the experiments, cells were washed twice with PBS and serum-free medium was added. HNP-1 dissolved in PBS or PBS alone was added and the cells were incubated for 6, 12, 18, or 24 hours. The cells were then trypsinized and washed with PBS. The cells were resuspended in 1× binding buffer at a concentration of 1 × 106 cells/mL and Annexin V-FITC (5 μL) and propidium iodide (PI; 10 μL) were added. After incubation in the dark for 15 minutes the samples were analyzed using a FACScan instrument (BD Biosciences). Dead cells were defined as those that were positive for both Annexin V and PI. Data were analyzed using Win MDI software and are shown as means ± SD from three separate experiments.
Results are expressed as means ± SD or dot plots. Data were analyzed for statistical differences using Mann–Whitney U-tests or Fisher's exact tests. Statistical significance was defined as P < 0.05.
Serum Concentrations of HNP-1–3 in Control Mice After HNP-1 Administration
Three BALB/c mice were intraperitoneally injected with single doses of 100 μg of HNP-1 and serum HNP-1 levels were measured using ELISA. After administration of HNP-1, levels of the peptide were 177.33 ± 5.04 ng/mL, 292.40 ± 43.61 ng/mL, 148.27 ± 18.46, and 16.89 ± 14.86 ng/mL at 1, 3, 12, and 24 hours, respectively.
HNP-1 Aggravated DSS-induced Colitis in BALB/c Mice
In BALB/c mice, body weights of HNP-1-treated mice were significantly lower than those of control animals on day 7 (19.58 ± 0.96 g vs. 20.62 ± 1.12 g, P = 0.04; Fig. 1A), and the colons of HNP-1-treated mice were significantly shorter than those in the PBS-treated group (8.29 ± 0.64 cm vs. 9.26 ± 0.76 cm, P = 0.02; Fig. 1B). In addition, DAI scores were significantly higher in the HNP-1-treated mice compared with those in the PBS-treated group (9.33 ± 2.65 vs. 6.20 ± 2.74, P = 0.02; percentage of DAI scores greater than 6: 87.5% vs. 22.2%, P = 0.01; Fig. 1C).
Histologic examination of the distal colons in the HNP-1-treated mice revealed marked erosion of the mucosal layer, an absence of glandular epithelium, and inflammatory cell infiltration in the submucosal area (Fig. 2A). Histologic scores were significantly higher in the HNP-1-treated group than in the PBS-treated group (3.89 ± 0.33 g vs. 2.90 ± 0.99 g, P = 0.03; percentage of scores greater than 3: 87.5% vs. 33.3%, P < 0.05; Fig. 2B). Colonic MPO activity, an indicator of neutrophil infiltration, was significantly higher in HNP-1-treated mice compared with control mice (207.03 ± 52.35 U/mg tissue vs. 160.20 ± 43.11 U/mg tissue, P < 0.05, n = 7 for each group; Fig. 2C). In addition, F4/80-positive macrophages were detected in colonic mucosa that was inflamed in response to DSS with or without HNP-1 administration (Fig. 3A). More F4/80-positive macrophages were detected in sections from HNP-treated mice than in samples from the PBS-treated group (Fig. 3B). In contrast, Ki-67-positive colonic epithelial cells were detected in both the HNP- and PBS-treated mice (Fig. 3C), with no statistical differences between the groups in the number of Ki-67-positive cells (Fig. 3D).
IFN-γ, TNF-α, and IL-1β Levels in Colon Culture Supernatants Derived from BALB/c Mice Treated with DSS and HNP-1
IFN-γ and TNF-α concentrations in colon culture supernatants were significantly higher in samples obtained from HNP-1-treated BALB/c mice compared with samples derived from control mice (Fig. 4). In addition, concentrations of IL-1β tended to be higher in cultures obtained from the HNP-1-treated group compared with those derived from control mice, although the result was not statistically significant.
Aggravation of DSS-induced Colitis by HNP-1 in SCID Mice
To determine whether HNP-1-mediated aggravation of DSS-induced colitis depends on T-helper 1 CD4+ T cells, we investigated the effects of HNP-1 on DSS-induced colitis in SCID mice. Although body weights and colon lengths were not significantly affected by HNP-1 (Fig. 5A,B), DAI scores for DSS-induced colitis in HNP-1-treated SCID mice were significantly higher compared with PBS-treated mice (5.63 ± 2.83 vs. 2.08 ± 2.02, P < 0.01; percentage of scores greater than 4: 62.5% vs. 8.3%, P = 0.02; Fig. 5C). HNP-1 injection resulted in higher histologic scores for DSS-induced colitis in SCID mice (2.38 ± 1.30 vs. 1.25 ± 0.45, P =0.04; percentage of scores greater than 2: 50.0% vs. 0%, P = 0.01; Fig. 6A). Colonic MPO activity did not differ in the two groups (Fig. 6B). In addition, more F4/80-positive macrophages were detected in the HNP-1-treated group than in the PBS-treated group (Fig. 6C), whereas no significant difference was observed in the number of Ki-67-positive cells (Fig. 6D). Furthermore, IL-1β concentrations in colon culture supernatants were significantly higher for samples obtained from HNP-1-treated SCID mice with DSS-induced colitis than in samples derived from mice that did not receive HNP-1 (Fig. 6E). IFN-γ and TNF-α were not detected in colon cultures obtained from either HNP-1- or PBS-treated SCID mice.
High Concentrations of HNP-1 Significantly Inhibited HT-29 Cell Viability In Vitro
To investigate the effects of HNP-1 on colon epithelial cell viability, HT-29 cells were treated with a dilution series of HNP-1 from 5 μg/mL to 200 μg/mL for 24 hours. A TetraColor One cell proliferation assay revealed that HT-29 cell proliferation was induced by 5 μg/mL or 10 μg/mL HNP-1, whereas a cytotoxic effect was observed in HT-29 cells at HNP-1 concentrations greater than 100 μg/mL (Fig. 7A). In addition, 100 μg/mL HNP-1 was time-dependently cytotoxic for HT-29 cells (Fig. 7B), and BrdU incorporation in HT-29 cells decreased significantly at HNP-1 concentrations greater than 100 μg/mL (Fig. 7C).
To evaluate the effects of HNP-1 on cytotoxicity and apoptosis in colon epithelial cells, HT-29 cells were incubated with 100 μg/mL HNP-1 and stained for Annexin-V and PI. Dead cells were defined as those that were positive for both markers (Fig. 7D). The percentage of cells that were dead in the HT-29 cultures treated with 100 μg/mL HNP-1 significantly increased at 18 hours and 24 hours compared with untreated cultures (Fig. 7E). In contrast, marked numbers of apoptotic cells—defined as those positive for Annexin-V but negative for PI—were not observed in this experiment. This result indicates that HNP-1 caused nonapoptotic cell death.
We previously reported that plasma HNP-1–3 are biomarkers for active UC and predictors of treatment efficacy in affected patients.10 In vitro, HNP-1–3 increase inflammatory cell migration12, 19 and cause these populations to secrete inflammatory cytokines.20, 21 HNP-1 is also cytotoxic to various eukaryotic and tumor cells.22–25 No studies, however, have addressed whether HNP modulate disease activity and cytokine expression in experimental colitis in vivo or colon epithelial cells in vitro. Here we show that HNP-1 aggravates DSS-induced colitis, an experimental model of UC, in both BALB/c and SCID mice. The enhanced disease was likely mediated, at least in part, by HNP-1-induced cytokine production in macrophages. In contrast, although HNP-1 also inhibited the proliferation of a colon cancer cell line, high concentrations of HNP-1 were required. Thus, HNP-1 appears to modulate DSS-induced colitis primarily via a cytokine-mediated, T-cell-independent mechanism.
Compared with healthy individuals, patients with UC have more mucosal bacteria,26 which invade the colonic mucosa.27 These patients have higher neutrophil numbers in the colon mucosa.28 Interestingly, HNP-1–3 are expressed by infiltrating neutrophils and colon epithelium,10, 29 and higher levels of these peptides have been detected in patients with active UC than in patients in which the disease is inactive.10, 30 One study showed that HNP-1–3 concentrations in the stool of patients with IBD were ≈14 times higher than in samples obtained from healthy individuals.31 In addition, HNP-1–3 concentrations are higher at sites of inflammation than in plasma.32, 33 We have shown that HNP-1 exacerbated DSS-induced colitis. Together, these results indicate that neutrophils contribute to the pathogenesis of UC; high concentrations of HNP-1–3 in response to infiltrating bacteria exacerbate the mucosal disorder at inflamed sites in patients with UC.
Although disease markers, including DAI and histologic scores, significantly differed between HNP-1-treated and control mice, the differences may not be physiologically relevant (Figs. 1, 2, 5, 6). The intraperitoneal dosage of HNP-1 was 100 μg, and serum HNP-1 levels were greater than 100 ng/mL during the first 12 hours following a single injection. These serum HNP-1 levels are similar to the plasma HNP-1–3 levels observed in patients with active UC (mean levels: 203.1 ng/mL),10 although the concentrations were not sustained in our experimental model. Thus, higher and/or more sustained HNP-1 concentrations may induce more severe colitis and larger differences between HNP-1-treated and control groups. Of note, no enterocolitis was detected in BALB/c mice after a single HNP-1 injection without DSS (data not shown). DSS-induced colitis was also primarily observed in the mid to distal colon, with little disease detected in the proximal colon. Histologic scores for the proximal colon were not significantly different between the HNP-1-treated and control groups (data not shown). These results indicate that HNP-1 exacerbates, but does not cause, colitis, and this defensin peptide may be a critical aggravator of colitis in patients with UC.
HNP-1, a 3,442-Da member of the α-defensin family, is an antibiotic peptide containing 30 amino acid residues and three intramolecular disulfide bonds. HNP-1 is produced by human neutrophils, stored in azurophilic granules (30%–50% of the total protein content of these granules), and released in response to inflammation.7, 8 HNP-1 affected the expression of such inflammatory cytokines as TNF-α, IFN-γ, and IL-1β, which aggravated DSS-induced colitis in mice, although the effects on IL-1β levels were not significant (Fig. 4). In addition, IL-1β mRNA expression levels were about 2.5 times higher in colon tissue obtained from BALB/c mice with DSS-induced colitis and treated with HNP-1 compared with those that did not receive HNP-1 (data not shown), although the cell types in which IL-1β was expressed were not clear; candidate populations include lamina propria CD11b macrophages and intestinal epithelial cells. HNP-1 also affected IL-1β expression in SCID mice with DSS-induced colitis, although TNF-α and IFN-γ were not detected in ex vivo experiments. Furthermore, the cells recruited to the inflamed colonic mucosa in mice with DSS-induced colitis were mainly macrophages, which increased in number following HNP-1 treatment of BALB/c mice. Similar results were observed in SCID mice, which lack lymphocytes. HNP-1–3 increase inflammatory cell migration12, 19 and cause macrophages to secrete TNF-α and IFN-γ.20 HNP-1 reportedly upregulated expression levels of TNF-α and IL-1β in monocytes activated by Staphylococcus aureus or phorbol myristate acetate (PMA).21 A porcine neutrophil antimicrobial peptide similar to defensins and HNP-1 has been shown to modify IL-1 release.34 In addition, Granz et al35 reported that direct cellular contact with stimulated T cells induces the expression/activation of proinflammatory factors and pathways in human monocytes. Thus, HNP-1 may primarily affect IL-1β secretion by macrophages in SCID mice and expression of TNF-α and IFN-γ may require T-cell binding in BALB/c mice; this effect may require stimulatory conditions, such as DSS-induced colitis.
Our in vitro studies indicate that HNP-1 has both inhibitory and promoting effects on colon epithelial cell proliferation, although it should be noted that we used cancer cells (Fig. 6). HNP-1 at low concentrations was shown to enhance cell proliferation, whereas high HNP-1 concentrations reduced the proliferation of respiratory epithelial cells, blood corpuscle origin cells, and oral squamous carcinoma cells.22–25 In contrast, the effects of HNP-1 on Ki-67 immunostaining in colon epithelial cells from mice with DSS-induced colitis were similar among BALB/c and SCID mice treated with HNP-1 or PBS (Figs. 3D, 6D). Although HNP-1 levels may not reach 100 μg/mL under normal physiologic conditions (Fig. 7A), HNP-1–3 levels in patients with bacterial infections can certainly exceed this concentration.36 Furthermore, local concentrations of HNP-1–3 at the site of inflammation are likely higher than plasma levels.33 Although in vitro assays may not reveal all of the in vivo effects of HNP-1, we have described a possible mechanism by which HNP-1–3 aggravate UC. Furthermore, although the effects of HNP-1 on cell proliferation are likely not specific to colon epithelial cells,33 the cytotoxicity of HNP-1 in colon epithelial cells, together with dysregulated inflammatory cytokine expression, may contribute to colitis.37 Further examinations are needed to build on our results—for example, in vitro assays using normal colon epithelial cells with relatively low concentrations of HNP-1 and assays of regional HNP-1–3 concentrations in patients with UC.
HNP-1–3 are antimicrobial and help to prevent bacterial infections that can contribute to IBD.38 Moreover, neutrophils were recently shown to be an important source of the potent immunosuppressive cytokine IL-10 at the site of infection during sepsis.39 On the other hand, leukocytapheresis is an effective method to treat UC. TNF-α and IL-1β levels are reduced by leukocytapheresis,3, 4 and removing activated leukocytes may reduce HNP-1–3 levels, resulting in lower inflammatory cytokine concentrations and reduced colonic inflammation. Thus, although HNP-1–3 may produce both beneficial and pathologic effects, patients with UC are likely to benefit from reducing high expression levels of HNP-1–3.
In conclusion, HNP-1 may aggravate UC, in part, by elevating levels of inflammatory cytokines, including TNF-α, IFN-γ, and IL-1β, potentially via a T-cell-independent pathway.
We thank Ms. Yuko Morinaga for technical assistance.