Acta Oto-Laryngologica
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Retention Fluids of Chronic Sinusitis Induce Neutrophil Adherence to Microvascular Endothelial Cells Kazunori Itoh, Shoko Katahira, Tsutomu Matsuzaki, Masaru Ohyama & Katsunori Fukuda To cite this article: Kazunori Itoh, Shoko Katahira, Tsutomu Matsuzaki, Masaru Ohyama & Katsunori Fukuda (1992) Retention Fluids of Chronic Sinusitis Induce Neutrophil Adherence to Microvascular Endothelial Cells, Acta Oto-Laryngologica, 112:5, 882-889 To link to this article: http://dx.doi.org/10.3109/00016489209137487
Published online: 08 Jul 2009.
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Date: 25 March 2016, At: 20:36
Acta Otolaryngol (Stockh) 1992; 112: 882-889
Retention Fluids of Chronic Sinusitis Induce Neutrophil Adherence to Microvascular Endothelial Cells KAZUNORI ITOH, SHOKO KATAHIRA, TSUTOMU MATSUZAKI, MASARU OHYAMA and KATSUNORI FUKUDA From the Department of Otolaryngology, Faculty of Medicine, Kagoshima University, Kagoshima 890, Japan
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Itoh K, Katahira S, Matsuzaki T, Ohyama M, Fukuda K. Retention fluids of chronic sinusitis induce neutrophil adherence to microvascular endothelial cells. Acta Otolaryngol (Stockh) 1992; 112 882-889. The adherence of circulating leukocytes to the vascular endothelium is a critical step in the emigration of leukocytes through blood vessel walls to inflammatory lesions. The influence of nasal secretions on the adherence of neutrophils to the vascular endothelium was investigated using monolayers of human mucosal microvascular endothelial cells derived from the inferior turbinate. Preincubation of vascular endothelial cells with retention fluids from the maxillary sinus of the patients with chronic sinusitis showed increased neutrophil adherence. Recombinant IL-lB was also tested and found to induce adherence of neutrophils to human mucosal microvascular endothelial cells. However, no adhesive effect was observed with the nasal secretions of nasal allergy. An enzyme-linked immunosorbent assay detected considerable amounts of IL-lfi in the chronic sinusitis retention fluids, while the amounts of IL-la and TNF-a were very low. Thc increased adhesion of the neutrophils by the retention fluids of chronic sinusitis was also neutralized by the incubation with anti-IL-IP antibody in a dose dependent manner. These findings suggest that IL-lg in the paranasal secretion of chronic sinusitis induces the adherence of neutrophils to vascular endothelium and subsequent infiltration of neutrophils in the paranasal sinuses, thus contributing to the persistence of chronic sinusitis. Key words: interleukin- I, tumor necrosis factor, vascular endothelial cell, nasal
secretion.
INTRODUCTION Leukocyte infiltration is a four-step process: adherence to endothelial cells, penetration between adjacent endothelial cells, localization to the pcricyte basement membrane, and transverse across the pericyte basement membrane (1). The adherence of leukocytes to vascular endothelium is an essential event of the inflammatory cell infiltration process. Once leukocytes adhere to endothelial cells, they spontaneously cross the endothelial cell layer into the interstitial space (2). Several chemotactic factors of leukocytes such as C5a, formyl-methionyl-leucyl-phenylalanine (FMLP) ,leukotriene B4(LTB,), and bacterial endotoxins can increase neutrophil adhesion to endothelial cells (3-5). Several cytokines including interleukin-la (IL-la), interleukin-lb (IL-IB) and tumor necrosis factor o! (TNF-a) also can strongly promote neutrophil adherence to endothelial cells ( 5 , 6). Inflammatory cells infiltrates into parasinus mucosa, and nasal secretions are characteristic features of chronic sinusitis. Although the factors regulating ncutrophil emigration in inflammation have been studied extensively (7, 8), the chemotactic factors which originate in extravascular inflammatory foci in nasal and paranasal lesions and attract leukocytes from the vascular compartment are not fully understood. To define the role of neutrophil-endothelial cell interactions in chronic sinusitis, we have examined the effects of nasal and paranasal secretions on the neutrophil adherence to human mucosal microvascular endothelial cells
(HMMECs).
Acta Otolaryngol (Stockh) 112
Neutrophil adherence to endothelial cells 883
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MATERIAL AND METHODS Cell culture HMMECs were isolated from human nasal mucosa during turbinotomy in patients with hypertrophic rhinitis or nasal allergy as previously reported (9). Briefly, the surgically-removed nasal mucosa was washed with Dulbecco's phosphate buffered saline (DPBS). After removal of the epithelial layer and periosteum, the mucosal tissues were cut into 4-mm square sections and incubated for 1 h at 37°C in DPBS containing 0.05% collagenase (type 11) and 1 YOethylenediaminetetraacetic acid (EDTA). After washing with DPBS, the sections were pressed by using a mucosa elevator from the center of each tissue section toward the periphery to release endothelial cells. The cells were collected by centrifugation and placed onto a 6-mm plastic culture dish coated with type I collagen prepared from human placenta. After incubation for 1 h, the plate was thoroughly washed to remove floating cells. The attached cells were covered with MCDB 107 containing 10% FBS and 75pg/ml crude endothelial cell growth factors (ECGF). After the cells were confluent, they were harvested with 0.05% trypsin, 0.02% EDTA in DPBS, and cells were split at a ratio of 1:4 for inoculation into new culture dishes. Each harvest and split was referred to as one passage and was performed once a week. HMMECs at passage 3 to 5 were used in these experiments. Purifeation and "Cr-labeling of neutrophils Six percent dextran-saline-diluted anticoagulated blood from human volunteers was obtained following venipuncture. Neutrophils were isolated after centrifugation over Percoll gradients and hypotonic lysis according to Schleimer et al. (5). Both purity and viability of neutrophils consistently exceeded 95%. Cells were labeled with 3.7 MBq sodium 51Cr-chromate by incubation for 30 min at 37°C in culture media. Labeled cells were then washed three times with culture media at room temperature, and resuspended in culture media at 2.5 x lo6 cells/ml for use in the neutrophil-HMMEC adherence assay. Preparation of nasal and paranasal secretions Chronic sinusitis was defined by typical X-ray findings and clinical examination showing the persistent mucoid or mucropurulent nasal discharge lasting more than 3 months. Nasal allergy was defined by typical clinical symptoms, a positive skin test, and nasal provocation by using a house dust-disk. Sinus retention fluids were obtained from 14 adult patients with chronic sinusitis by antral puncture or during antrostomy. The presence of increased numbers of neutrophils and mononuclear cells in the parasinus retention fluids was confirmed by Gimusa staining. Nasal secretions were collected from 6 adult patients with nasal allergy by aspiration from the nasal cavity following antigen provocation. All the following procedures were camed out below 4°C unless otherwise indicated. Nasal and paranasal secretion were added to 3 volumes of DPBS, stirred for 30 s and incubated for 20 min. After centrifugation at 400 x G for 10 min in order to remove cell fractions, the supernatant was again centrifugated at 25 000 x G for 1 h. An aliquot of the supernatant was used in quantiative analysis of IL-la, /3 and TNF-a. To remove the mucinous fractions, the remaining supernatant was further centrifugated at 400 000 x G for 4 h and the supernatant was used in the neutrophil-HMMEC adherence assay. Samples were stored at -4OT until subsequent use. Neutrophil-HMMEC adherence assay The neutrophil-HMMEC adherence assay was performed using 24-well plates of confluent HMMECs monolayers. The plates were incubated in 0.25ml of samples from nasal and
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paranasal secretions and 0.25 ml of MCDB 107 containing 1% FBS in a CO, incubator for 6 h. After washing the wells thrice with MCDB 107, 0.5 ml of MCDB 107 and 0.1 ml of "Cr-labeled neutrophils suspended in the same medium (2.5 x 106/ml)were added to each well. After incubation for 20 min at 37 C, nonadherent cells were removed by aspiration and rinsing three times. Adherent cells were dissolved with 1 ml of 1 M ammonium hydroxide and radioactivities were quantitated using a gamma counter (10). Results were indicated as a percentage of the total added counts bound to the HMMECs monolayers. Replicates had a coefficient of variation of less than 10%. Meosuremenr of I L - l z , j and TNF-z
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Quantitative analysis of IL-lr,B and TNF-I in each sample from nasal and paranasal secretions was performed by enzyme-linked immunosorbent assay (ELISA). RESULTS Previously, we have reported that HMMECs isolated from the mucosa of the inferior turbinate had a short life span in culture compared with human umbilical vein endothelial cells. In the present investigations, we found that HMMECs can be maintained for up to 30 passages over 10 months, using MCDB 107 medium supplemented with 10% FBS and 75 pm/ml of crude ECGF. In the present studies, HMMECs at passages 3 to 5 were used. These cells appeared as polygonal monolayer at confluency (Fig. 1A) and expressed Factor-VIII-related antigen (Fig. 1 B), findings which are characteristic of endothelial cells. Neutrophil adherence to HMMECs occurred in vitro by the stimulation of these cells with pooled samples of chronic sinusitis retention fluids (Fig. 2) or human rIL-Ij (Fig. 2, 3) in a dose- and time-dependent fashion. Phase contrast micrographs indicated that the neutrophils adhered to the HMMECs in the presence of retention fluid from chronic sinusitis (Fig. 4) and IL-I (Fig. 4), but not control medium (Fig. 4) or in the presence of nasal secretion from nasal allergy (Fig. 4). These micrographs also showed that neutrophils adhered to HMMECs and not to the exposed collagen or plastic (Fig. 4). The neutrophil adherence-promoting activities of nasal and paranasal secretions from the patients with chronic sinusitis and nasal allergy were estimated by the incubation with HMMEC monolayers. The adhesion of neutrophils to HMMECs was induced approximately 3-fold by paranasal secretion from chronic sinusitis (Fig. 5). However, nasal secretion from nasal allergy patients showed activities as low as control.
(4
(B) Fig. I . Human mucosal microvascular endothelial cells in culture at passage 5 . (A) Phase contrast micrographs of monolayer of HMMECs ( x IOO), ( B ) Indirect immunofluorescence staining for Factor
VIII-related antigen (
x 400).
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0
I
I
1
I
I
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2
4
6
=-
IL-1
m--m
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Fig. 2. Effect of pooled sample of the secretions from chronic sinusitis and human rILlb on adherence of neutrophils to HMMECs. HMMECs monolayers were incubated with pooled sample (100 pl/well; closed square) or human rlL-1b (100 units/we& open square) for indicated time and tested for adherence of neutrophils.
Time (h)
/
30
8 m
e L
20
U
6
/
f dr
Fig. 3. Human rlL-Ib induces neutrophils adherence to HMMECs. HMMECs monolayen were incubated with indicated concentrations of human rlL-1fi for 4 h at 37°C and tested for adherence of neutrophils.
10
0
0.01
0.1
1
10
1M)
loo0
L-13 concentration (uniWweU)
Concentrations of IL-la, IL-1s and TNF-a, cytokines which are candidates for inducing neutrophil adherence, were measured in the nasal and paranasal secretions by ELISA. In most cases of chronic sinusitis, the paranasal secretions contained a considerable amount of IL-IF (Fig. 6), whereas this cytokine could not be detected in nasal secretions of nasal allergy. The amounts of IL-la were also low, and TNF-z could not be detected in either nasal or paranasal secretions of chronic sinusitis or nasal allergy. To confirm that IL-Is does indeed act as a stimulus inducing neutrophil adherence to HMMECs, an anti-IL-lj antibody was tested in the neutrophil adherence assay. The adherence-inducing activity of pooled samples of paranasal secretions from the patients of chronic sinusitis was neutralized by an anti-IL-ls antibody in a dose-dependent manner (Fig. 7). Furthermore, these activities were also inactivated by heat-treatment at 56 C for 30min (data not shown), which could exclude the potential contaminant of endotoxin (10).
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Fig. 4. Phase contrast micrographs show retention fluid of chronic sinusitis and human rIL-I/3 induce neutrophils adherence to HMMECs. HMMECs monolayers were coincubated with neutrophils after preincubation for 100min with either medium alone (A), maxillary sinus retention fluid of chronic sinusitis (B),medium containing 2 units/ml of human IL-I (C) or nasal secretion of nasal allergy (D).
Fig. 5. Effect of the nasal or paranasal secretions on adherence of neutrophils to HMMECs monolayers. S'Cr-labeled neutrophils (2.5 x lo5) were added to confluent HMMECs monolayers preincubated with either medium alone ( - ), maxillary sinus retention fluids of chronic sinusitis (C.S.), nasal secretions of nasal allergy (N.A.). *p < 0.005.
DISCUSSION With the development of antibiotic drugs the prevalence of chronic sinusitis has decreased but remains a common clinical problem. Part of the pathogenesis of chronic sinusitis is the retention within the paranasal sinuses of secretions which contain numerous leukocytes. Most leukocytes in these secretions are neutrophils, but some are mononuclear leukocytes. It has been proposed that migrated leukocytes release various proteases which may cause
Neutrophil adherence to endothelial cells
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TNF
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IL-la
,
O-
C.S.
N.A.
C.S.
II
N.A.
0
!
Fig. 6. Quantitative analysis of ILla,@and TNFa in the nasal fluids of chronic sinusitis and nasal allergy. These were estimated in the maxillary sinus retention fluids of chronic sinusitis (C.S.) and nasal secretions of nasal allergy (N.A.) by ELISA
IL-lP
0 SINUSITIS 0
I
50 -
0
I T
,
,
,
,
0
0.1
1
10
Fig. 7. Neutralizing effect of anti-human IL-I@ antibody on neutrophilHMMECs adherence. HMMECs monolayers were preincubated with pooled samples of chronic sinusitis (250 pllweli; closed circle) or human rIL-I@(50 units/well; open circle) in the presence of indicated concentration of anti-human IL-I@ antibody for 6 hours, and tested for adherence of neutrophils.
100
anti 11-1J Ab concentration (;rg/wdD
mucosal damage (1 1, 12). The mechanism of the continuous migration of leukocytes into paranasal and nasal cavities is not well understood. Thus, the influx of circulating leukocytes is a critical step of the inflammatory reaction and is initiated by the interaction of leukocytes with the vascular endothelium (1). These interactions in vitro have been studied using long-term cultivation of human umbilical vein endothelial cells (HUVECs) (3-6). In pathological conditions such as chemotactic-fragment-inducedlung inflammation, leukocytes migrate to the alveolar space from the capillaries and small venules by adherence to the vascular endothelium ( 13). In the present study, we used HMMECs rather than HUVECs to study the role of endothelial cells in the pathogenesis of chronic sinusitis, since HMMECs are derived from the organ involved in the pathogenesis. Using HMMECs, we have demonstrated that the paranasal secretions of chronic sinusitis induce the adherence of HMMECs for isolated neutrophils. Although leukocyte-endothelial cell interactions are important for leukocytes infiltration in acute inflammation (2-6), neutrophil-HMMEC adherence promoted by the
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secretions from chronic sinusitis may also be significant in the prolongation of chronic inflammation in nasal and paranasal regions. Our results indicate that the pathological secretions consisting of migrated leukocytes trigger the further migration of leukocytes into the nasal and paranasal cavities. In other words, this vicious cycle may be an important part of the pathogenesis of chronic sinusitis, though both the origin and the nature of the leukocyte-migration inducing factors are still unknown. Several factors modulate neutrophil migration into nasal and paranasal cavities. Many of these factors, including chemotactic peptide (FMLP), complement fragment (CSa) (3), lipoxygenase products (LTB,) (4), bacterjal endotoxin ( 5 ) and some cytokines (5, 6). have been reported to affect the adherence of neutrophils to cultured HUVECs. IL-1, TNF-a and endotoxin play an important role in acute inflammation since these cytokines stimulate the expression of adhesion molecules such as intercellular adhesion molecule I (ICAM-1) (14), vascular cell adhesion molecule 1 (VCAM-I) (15) and endothelial leukocyte adhesion molecules (ELAM-I) ( 16). Furthermore, these cytokines stimulate HUVECs to promote trans-endothelial neutrophils ( 17). Since IL-l#i is present at a high concentration in the secretion of patients with chronic sinusitis and we have demonstrated the inhibition of neutrophils-HMMECs adhesion by anti-IL-I#i antibody, we believe that IL-1fl is an important factor inducing neutrophil migration in chronic sinusitis. Additional experiments, however, to prove that IL-l#i is present in nasal secrtion will be required (10). IL-I is a pleiotropic cytokine and interacts with many types of cells including neutrophils, lymphocytes, fibroblasts and vascular endothelial cells (18). This cytokine has also been known as the key mediator not only of the various acute phases of inflammatory reactions, but also of some chronic inflammations like rheumatoid arthritis (18). To confirm the activity of IL-lfl in chronic sinusitis, the expression and distribution of IL-I@ and adhesion molecules (14-16) should be investigated by employing the immunohistological and molecular biological techniques. The incomplete inhibition of neutrophil-HMMECs adherence by the anti-IG1 fl antibody in these experiments also suggests that neutrophil migration into the paranasal sinuses is not solely due to IL-I@ but may include other factors such as LTB4, C5a, and bacterial products. Current therapy of chronic sinusitis includes antibiotics and repeated drainage or aspiration of retention fluids from paranasal sinuses. The results of the present investigation provide a pathophysiological basis for removal of retained secretions, as the reduction in leukocyte should diminish cytokine release and thereby retard further leukocyte migration. Furthermore, a human IL-I receptor antagonist was recently cloned and characterized (19, 20), and may be beneficial in chronic sinusitis. ACKNOWLEDGEMENTS We are indebted to Dr Jeffrey B. Kopp and Dr Peter D. Burbelo, Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes of Health, USA, for reviewing the manuscript and for critical comments.
REFERENCES 1. Harlan JM. Leukocyte-endothelial interactions. Blood 1985; 65: 513-25. 2. Oda T, Nagai K, Katori M. Time course of migration of polymorphonuclear leukocytes through venular endothelium in hamster cheek pouch. In: Tsuchiya M et al, eds. Microcirculation: An update. Vol. 1. Amsterdam: Elsevier Science Publ., 1987: 727-8. 3. Tonnesen MG, Smedly LA, Henson PM. Neutrophil-endothelial cell interaction modulation of neutrophil adhesivenessinduced by complement fragments C5a and C5a des arg phenylalain in vitro. J Clin Invest 1984; 74 1581-92.
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4. Hoover RL, Karnovsky MJ, Austen KF et al. Leukotriene B4 action on endothelium mediates augmented neutrophil/endothelial adhesion. Proc Natl Acad Sci USA 1984; 81: 2191 -3. 5. Schleimer RP, Rutledge BK. Cultured human vascular endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin 1, endotoxin, and tumor-promoting phorbol diesters. J Immunol 1986; 136 649-54. 6. Bevilacqua MP, Pober JS, Wheeler ME et al. Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J Clin Invest 1985; 76: 2003-11. 7. van Epp DE, Garcia ML. Enhancement of neutrophil function as a result of prior exposure to chemotactic factor. J Clin Invest 1980; 66: 167-75. 8. Ginsburg I, Quie PG. Modulation of polymorphonuclear leukocyte chemotaxis by leukocyte extracts, bacterial products, inflammatory exdates, and polyelectrolytes. Inflammation 1980; 4 301-11. 9. Fukuda K, lmamura Y, Koshihara Y et al. Establishment of human mucosal microvascular endothelial cells from inferior turbinate in culture. Am J Otolaryngol 1989; 10: 85-91. 10. Bochener BS, Landy SD, Plaut M et al. Interleukin 1 production by human lung tissue. J Immunol 1987; 139: 2297-302. 1 1. Ohlsson K, Ohlsson I. The neutral proteases of human granulocyte: Isolation and partial characterization of granulocyte elastase. Eur J Biochem 1974; 4 2 519-27. 12. Fukuda K, Itoh K, Ooyama T. A protease inhibitor from human allergic nasal secretions. Acta Otolaryngol (Stockh) 1987; 104: 539-44. 13. Shaw JO. Leukocytes in chemotactic fragment-induced lung inflammation. Vascular emigration and alveolar surface migration. Am J Pathol 1980; 101a; 283-302. 14. Stauton DE, Marlin SD, Stratowa C et al. Primary structure of ICAM-I demonstrates interaction between members of immunoglobu.in and integrin supergene families. Cell 1988; 52: 925-33. 15. Osborn L, Hession C, Tizard R et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 1989; 59: 1203-11. 16. Bevilacqua MP, Stengelin S, Gimbrone Jr MA et al. Endothelial leukocyte adhesion molecule 1: An inducible receptor for neutrophil related to complement regulatory proteins and lectins. Science 1989; 243: 1160-5. 17. Moser R, Schleiffenbaum B, Groscutrh P et al. Interleukin 1 and tumor necrosis Factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage. J Clin Invest 1989; 83: 444-55. 18. Maury CPJ. Interleukin-1 and the pathogenesis of inflammatory diseases. Acta Med Scand 1986; 220: 291-4. 19. Eisenberg SP, Evans RJ, Arend WP et al. Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist. Nature 1990, 343: 341 -6. 20. Carter DB, Deibel Jr MR, Dunn CJ et al. Purification, cloning, expression and biological characterization of an interleukin-1 receptor antagonist protein. Nature 1990; 344: 633-7.
Manuscript received Nouember 5, 1991; accepted January 22, 1992 Address for correspondence: Kazunori Itoh, Department of Otolaryngology, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890, Japan
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Retention Fluids of Chronic Sinusitis Induce Neutrophil Adherence to Microvascular Endothelial Cells Kazunori Itoh, Shoko Katahira, Tsutomu Matsuzaki, Masaru Ohyama & Katsunori Fukuda To cite this article: Kazunori Itoh, Shoko Katahira, Tsutomu Matsuzaki, Masaru Ohyama & Katsunori Fukuda (1992) Retention Fluids of Chronic Sinusitis Induce Neutrophil Adherence to Microvascular Endothelial Cells, Acta Oto-Laryngologica, 112:5, 882-889 To link to this article: http://dx.doi.org/10.3109/00016489209137487
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [RMIT University Library]
Date: 25 March 2016, At: 20:36
Acta Otolaryngol (Stockh) 1992; 112: 882-889
Retention Fluids of Chronic Sinusitis Induce Neutrophil Adherence to Microvascular Endothelial Cells KAZUNORI ITOH, SHOKO KATAHIRA, TSUTOMU MATSUZAKI, MASARU OHYAMA and KATSUNORI FUKUDA From the Department of Otolaryngology, Faculty of Medicine, Kagoshima University, Kagoshima 890, Japan
Downloaded by [RMIT University Library] at 20:36 25 March 2016
Itoh K, Katahira S, Matsuzaki T, Ohyama M, Fukuda K. Retention fluids of chronic sinusitis induce neutrophil adherence to microvascular endothelial cells. Acta Otolaryngol (Stockh) 1992; 112 882-889. The adherence of circulating leukocytes to the vascular endothelium is a critical step in the emigration of leukocytes through blood vessel walls to inflammatory lesions. The influence of nasal secretions on the adherence of neutrophils to the vascular endothelium was investigated using monolayers of human mucosal microvascular endothelial cells derived from the inferior turbinate. Preincubation of vascular endothelial cells with retention fluids from the maxillary sinus of the patients with chronic sinusitis showed increased neutrophil adherence. Recombinant IL-lB was also tested and found to induce adherence of neutrophils to human mucosal microvascular endothelial cells. However, no adhesive effect was observed with the nasal secretions of nasal allergy. An enzyme-linked immunosorbent assay detected considerable amounts of IL-lfi in the chronic sinusitis retention fluids, while the amounts of IL-la and TNF-a were very low. Thc increased adhesion of the neutrophils by the retention fluids of chronic sinusitis was also neutralized by the incubation with anti-IL-IP antibody in a dose dependent manner. These findings suggest that IL-lg in the paranasal secretion of chronic sinusitis induces the adherence of neutrophils to vascular endothelium and subsequent infiltration of neutrophils in the paranasal sinuses, thus contributing to the persistence of chronic sinusitis. Key words: interleukin- I, tumor necrosis factor, vascular endothelial cell, nasal
secretion.
INTRODUCTION Leukocyte infiltration is a four-step process: adherence to endothelial cells, penetration between adjacent endothelial cells, localization to the pcricyte basement membrane, and transverse across the pericyte basement membrane (1). The adherence of leukocytes to vascular endothelium is an essential event of the inflammatory cell infiltration process. Once leukocytes adhere to endothelial cells, they spontaneously cross the endothelial cell layer into the interstitial space (2). Several chemotactic factors of leukocytes such as C5a, formyl-methionyl-leucyl-phenylalanine (FMLP) ,leukotriene B4(LTB,), and bacterial endotoxins can increase neutrophil adhesion to endothelial cells (3-5). Several cytokines including interleukin-la (IL-la), interleukin-lb (IL-IB) and tumor necrosis factor o! (TNF-a) also can strongly promote neutrophil adherence to endothelial cells ( 5 , 6). Inflammatory cells infiltrates into parasinus mucosa, and nasal secretions are characteristic features of chronic sinusitis. Although the factors regulating ncutrophil emigration in inflammation have been studied extensively (7, 8), the chemotactic factors which originate in extravascular inflammatory foci in nasal and paranasal lesions and attract leukocytes from the vascular compartment are not fully understood. To define the role of neutrophil-endothelial cell interactions in chronic sinusitis, we have examined the effects of nasal and paranasal secretions on the neutrophil adherence to human mucosal microvascular endothelial cells
(HMMECs).
Acta Otolaryngol (Stockh) 112
Neutrophil adherence to endothelial cells 883
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MATERIAL AND METHODS Cell culture HMMECs were isolated from human nasal mucosa during turbinotomy in patients with hypertrophic rhinitis or nasal allergy as previously reported (9). Briefly, the surgically-removed nasal mucosa was washed with Dulbecco's phosphate buffered saline (DPBS). After removal of the epithelial layer and periosteum, the mucosal tissues were cut into 4-mm square sections and incubated for 1 h at 37°C in DPBS containing 0.05% collagenase (type 11) and 1 YOethylenediaminetetraacetic acid (EDTA). After washing with DPBS, the sections were pressed by using a mucosa elevator from the center of each tissue section toward the periphery to release endothelial cells. The cells were collected by centrifugation and placed onto a 6-mm plastic culture dish coated with type I collagen prepared from human placenta. After incubation for 1 h, the plate was thoroughly washed to remove floating cells. The attached cells were covered with MCDB 107 containing 10% FBS and 75pg/ml crude endothelial cell growth factors (ECGF). After the cells were confluent, they were harvested with 0.05% trypsin, 0.02% EDTA in DPBS, and cells were split at a ratio of 1:4 for inoculation into new culture dishes. Each harvest and split was referred to as one passage and was performed once a week. HMMECs at passage 3 to 5 were used in these experiments. Purifeation and "Cr-labeling of neutrophils Six percent dextran-saline-diluted anticoagulated blood from human volunteers was obtained following venipuncture. Neutrophils were isolated after centrifugation over Percoll gradients and hypotonic lysis according to Schleimer et al. (5). Both purity and viability of neutrophils consistently exceeded 95%. Cells were labeled with 3.7 MBq sodium 51Cr-chromate by incubation for 30 min at 37°C in culture media. Labeled cells were then washed three times with culture media at room temperature, and resuspended in culture media at 2.5 x lo6 cells/ml for use in the neutrophil-HMMEC adherence assay. Preparation of nasal and paranasal secretions Chronic sinusitis was defined by typical X-ray findings and clinical examination showing the persistent mucoid or mucropurulent nasal discharge lasting more than 3 months. Nasal allergy was defined by typical clinical symptoms, a positive skin test, and nasal provocation by using a house dust-disk. Sinus retention fluids were obtained from 14 adult patients with chronic sinusitis by antral puncture or during antrostomy. The presence of increased numbers of neutrophils and mononuclear cells in the parasinus retention fluids was confirmed by Gimusa staining. Nasal secretions were collected from 6 adult patients with nasal allergy by aspiration from the nasal cavity following antigen provocation. All the following procedures were camed out below 4°C unless otherwise indicated. Nasal and paranasal secretion were added to 3 volumes of DPBS, stirred for 30 s and incubated for 20 min. After centrifugation at 400 x G for 10 min in order to remove cell fractions, the supernatant was again centrifugated at 25 000 x G for 1 h. An aliquot of the supernatant was used in quantiative analysis of IL-la, /3 and TNF-a. To remove the mucinous fractions, the remaining supernatant was further centrifugated at 400 000 x G for 4 h and the supernatant was used in the neutrophil-HMMEC adherence assay. Samples were stored at -4OT until subsequent use. Neutrophil-HMMEC adherence assay The neutrophil-HMMEC adherence assay was performed using 24-well plates of confluent HMMECs monolayers. The plates were incubated in 0.25ml of samples from nasal and
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Acta OIolaryngol (SIockh) I12
paranasal secretions and 0.25 ml of MCDB 107 containing 1% FBS in a CO, incubator for 6 h. After washing the wells thrice with MCDB 107, 0.5 ml of MCDB 107 and 0.1 ml of "Cr-labeled neutrophils suspended in the same medium (2.5 x 106/ml)were added to each well. After incubation for 20 min at 37 C, nonadherent cells were removed by aspiration and rinsing three times. Adherent cells were dissolved with 1 ml of 1 M ammonium hydroxide and radioactivities were quantitated using a gamma counter (10). Results were indicated as a percentage of the total added counts bound to the HMMECs monolayers. Replicates had a coefficient of variation of less than 10%. Meosuremenr of I L - l z , j and TNF-z
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Quantitative analysis of IL-lr,B and TNF-I in each sample from nasal and paranasal secretions was performed by enzyme-linked immunosorbent assay (ELISA). RESULTS Previously, we have reported that HMMECs isolated from the mucosa of the inferior turbinate had a short life span in culture compared with human umbilical vein endothelial cells. In the present investigations, we found that HMMECs can be maintained for up to 30 passages over 10 months, using MCDB 107 medium supplemented with 10% FBS and 75 pm/ml of crude ECGF. In the present studies, HMMECs at passages 3 to 5 were used. These cells appeared as polygonal monolayer at confluency (Fig. 1A) and expressed Factor-VIII-related antigen (Fig. 1 B), findings which are characteristic of endothelial cells. Neutrophil adherence to HMMECs occurred in vitro by the stimulation of these cells with pooled samples of chronic sinusitis retention fluids (Fig. 2) or human rIL-Ij (Fig. 2, 3) in a dose- and time-dependent fashion. Phase contrast micrographs indicated that the neutrophils adhered to the HMMECs in the presence of retention fluid from chronic sinusitis (Fig. 4) and IL-I (Fig. 4), but not control medium (Fig. 4) or in the presence of nasal secretion from nasal allergy (Fig. 4). These micrographs also showed that neutrophils adhered to HMMECs and not to the exposed collagen or plastic (Fig. 4). The neutrophil adherence-promoting activities of nasal and paranasal secretions from the patients with chronic sinusitis and nasal allergy were estimated by the incubation with HMMEC monolayers. The adhesion of neutrophils to HMMECs was induced approximately 3-fold by paranasal secretion from chronic sinusitis (Fig. 5). However, nasal secretion from nasal allergy patients showed activities as low as control.
(4
(B) Fig. I . Human mucosal microvascular endothelial cells in culture at passage 5 . (A) Phase contrast micrographs of monolayer of HMMECs ( x IOO), ( B ) Indirect immunofluorescence staining for Factor
VIII-related antigen (
x 400).
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0
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Fig. 2. Effect of pooled sample of the secretions from chronic sinusitis and human rILlb on adherence of neutrophils to HMMECs. HMMECs monolayers were incubated with pooled sample (100 pl/well; closed square) or human rlL-1b (100 units/we& open square) for indicated time and tested for adherence of neutrophils.
Time (h)
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8 m
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Fig. 3. Human rlL-Ib induces neutrophils adherence to HMMECs. HMMECs monolayen were incubated with indicated concentrations of human rlL-1fi for 4 h at 37°C and tested for adherence of neutrophils.
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1M)
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Concentrations of IL-la, IL-1s and TNF-a, cytokines which are candidates for inducing neutrophil adherence, were measured in the nasal and paranasal secretions by ELISA. In most cases of chronic sinusitis, the paranasal secretions contained a considerable amount of IL-IF (Fig. 6), whereas this cytokine could not be detected in nasal secretions of nasal allergy. The amounts of IL-la were also low, and TNF-z could not be detected in either nasal or paranasal secretions of chronic sinusitis or nasal allergy. To confirm that IL-Is does indeed act as a stimulus inducing neutrophil adherence to HMMECs, an anti-IL-lj antibody was tested in the neutrophil adherence assay. The adherence-inducing activity of pooled samples of paranasal secretions from the patients of chronic sinusitis was neutralized by an anti-IL-ls antibody in a dose-dependent manner (Fig. 7). Furthermore, these activities were also inactivated by heat-treatment at 56 C for 30min (data not shown), which could exclude the potential contaminant of endotoxin (10).
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Fig. 4. Phase contrast micrographs show retention fluid of chronic sinusitis and human rIL-I/3 induce neutrophils adherence to HMMECs. HMMECs monolayers were coincubated with neutrophils after preincubation for 100min with either medium alone (A), maxillary sinus retention fluid of chronic sinusitis (B),medium containing 2 units/ml of human IL-I (C) or nasal secretion of nasal allergy (D).
Fig. 5. Effect of the nasal or paranasal secretions on adherence of neutrophils to HMMECs monolayers. S'Cr-labeled neutrophils (2.5 x lo5) were added to confluent HMMECs monolayers preincubated with either medium alone ( - ), maxillary sinus retention fluids of chronic sinusitis (C.S.), nasal secretions of nasal allergy (N.A.). *p < 0.005.
DISCUSSION With the development of antibiotic drugs the prevalence of chronic sinusitis has decreased but remains a common clinical problem. Part of the pathogenesis of chronic sinusitis is the retention within the paranasal sinuses of secretions which contain numerous leukocytes. Most leukocytes in these secretions are neutrophils, but some are mononuclear leukocytes. It has been proposed that migrated leukocytes release various proteases which may cause
Neutrophil adherence to endothelial cells
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,
O-
C.S.
N.A.
C.S.
II
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!
Fig. 6. Quantitative analysis of ILla,@and TNFa in the nasal fluids of chronic sinusitis and nasal allergy. These were estimated in the maxillary sinus retention fluids of chronic sinusitis (C.S.) and nasal secretions of nasal allergy (N.A.) by ELISA
IL-lP
0 SINUSITIS 0
I
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I T
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,
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Fig. 7. Neutralizing effect of anti-human IL-I@ antibody on neutrophilHMMECs adherence. HMMECs monolayers were preincubated with pooled samples of chronic sinusitis (250 pllweli; closed circle) or human rIL-I@(50 units/well; open circle) in the presence of indicated concentration of anti-human IL-I@ antibody for 6 hours, and tested for adherence of neutrophils.
100
anti 11-1J Ab concentration (;rg/wdD
mucosal damage (1 1, 12). The mechanism of the continuous migration of leukocytes into paranasal and nasal cavities is not well understood. Thus, the influx of circulating leukocytes is a critical step of the inflammatory reaction and is initiated by the interaction of leukocytes with the vascular endothelium (1). These interactions in vitro have been studied using long-term cultivation of human umbilical vein endothelial cells (HUVECs) (3-6). In pathological conditions such as chemotactic-fragment-inducedlung inflammation, leukocytes migrate to the alveolar space from the capillaries and small venules by adherence to the vascular endothelium ( 13). In the present study, we used HMMECs rather than HUVECs to study the role of endothelial cells in the pathogenesis of chronic sinusitis, since HMMECs are derived from the organ involved in the pathogenesis. Using HMMECs, we have demonstrated that the paranasal secretions of chronic sinusitis induce the adherence of HMMECs for isolated neutrophils. Although leukocyte-endothelial cell interactions are important for leukocytes infiltration in acute inflammation (2-6), neutrophil-HMMEC adherence promoted by the
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secretions from chronic sinusitis may also be significant in the prolongation of chronic inflammation in nasal and paranasal regions. Our results indicate that the pathological secretions consisting of migrated leukocytes trigger the further migration of leukocytes into the nasal and paranasal cavities. In other words, this vicious cycle may be an important part of the pathogenesis of chronic sinusitis, though both the origin and the nature of the leukocyte-migration inducing factors are still unknown. Several factors modulate neutrophil migration into nasal and paranasal cavities. Many of these factors, including chemotactic peptide (FMLP), complement fragment (CSa) (3), lipoxygenase products (LTB,) (4), bacterjal endotoxin ( 5 ) and some cytokines (5, 6). have been reported to affect the adherence of neutrophils to cultured HUVECs. IL-1, TNF-a and endotoxin play an important role in acute inflammation since these cytokines stimulate the expression of adhesion molecules such as intercellular adhesion molecule I (ICAM-1) (14), vascular cell adhesion molecule 1 (VCAM-I) (15) and endothelial leukocyte adhesion molecules (ELAM-I) ( 16). Furthermore, these cytokines stimulate HUVECs to promote trans-endothelial neutrophils ( 17). Since IL-l#i is present at a high concentration in the secretion of patients with chronic sinusitis and we have demonstrated the inhibition of neutrophils-HMMECs adhesion by anti-IL-I#i antibody, we believe that IL-1fl is an important factor inducing neutrophil migration in chronic sinusitis. Additional experiments, however, to prove that IL-l#i is present in nasal secrtion will be required (10). IL-I is a pleiotropic cytokine and interacts with many types of cells including neutrophils, lymphocytes, fibroblasts and vascular endothelial cells (18). This cytokine has also been known as the key mediator not only of the various acute phases of inflammatory reactions, but also of some chronic inflammations like rheumatoid arthritis (18). To confirm the activity of IL-lfl in chronic sinusitis, the expression and distribution of IL-I@ and adhesion molecules (14-16) should be investigated by employing the immunohistological and molecular biological techniques. The incomplete inhibition of neutrophil-HMMECs adherence by the anti-IG1 fl antibody in these experiments also suggests that neutrophil migration into the paranasal sinuses is not solely due to IL-I@ but may include other factors such as LTB4, C5a, and bacterial products. Current therapy of chronic sinusitis includes antibiotics and repeated drainage or aspiration of retention fluids from paranasal sinuses. The results of the present investigation provide a pathophysiological basis for removal of retained secretions, as the reduction in leukocyte should diminish cytokine release and thereby retard further leukocyte migration. Furthermore, a human IL-I receptor antagonist was recently cloned and characterized (19, 20), and may be beneficial in chronic sinusitis. ACKNOWLEDGEMENTS We are indebted to Dr Jeffrey B. Kopp and Dr Peter D. Burbelo, Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes of Health, USA, for reviewing the manuscript and for critical comments.
REFERENCES 1. Harlan JM. Leukocyte-endothelial interactions. Blood 1985; 65: 513-25. 2. Oda T, Nagai K, Katori M. Time course of migration of polymorphonuclear leukocytes through venular endothelium in hamster cheek pouch. In: Tsuchiya M et al, eds. Microcirculation: An update. Vol. 1. Amsterdam: Elsevier Science Publ., 1987: 727-8. 3. Tonnesen MG, Smedly LA, Henson PM. Neutrophil-endothelial cell interaction modulation of neutrophil adhesivenessinduced by complement fragments C5a and C5a des arg phenylalain in vitro. J Clin Invest 1984; 74 1581-92.
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4. Hoover RL, Karnovsky MJ, Austen KF et al. Leukotriene B4 action on endothelium mediates augmented neutrophil/endothelial adhesion. Proc Natl Acad Sci USA 1984; 81: 2191 -3. 5. Schleimer RP, Rutledge BK. Cultured human vascular endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin 1, endotoxin, and tumor-promoting phorbol diesters. J Immunol 1986; 136 649-54. 6. Bevilacqua MP, Pober JS, Wheeler ME et al. Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J Clin Invest 1985; 76: 2003-11. 7. van Epp DE, Garcia ML. Enhancement of neutrophil function as a result of prior exposure to chemotactic factor. J Clin Invest 1980; 66: 167-75. 8. Ginsburg I, Quie PG. Modulation of polymorphonuclear leukocyte chemotaxis by leukocyte extracts, bacterial products, inflammatory exdates, and polyelectrolytes. Inflammation 1980; 4 301-11. 9. Fukuda K, lmamura Y, Koshihara Y et al. Establishment of human mucosal microvascular endothelial cells from inferior turbinate in culture. Am J Otolaryngol 1989; 10: 85-91. 10. Bochener BS, Landy SD, Plaut M et al. Interleukin 1 production by human lung tissue. J Immunol 1987; 139: 2297-302. 1 1. Ohlsson K, Ohlsson I. The neutral proteases of human granulocyte: Isolation and partial characterization of granulocyte elastase. Eur J Biochem 1974; 4 2 519-27. 12. Fukuda K, Itoh K, Ooyama T. A protease inhibitor from human allergic nasal secretions. Acta Otolaryngol (Stockh) 1987; 104: 539-44. 13. Shaw JO. Leukocytes in chemotactic fragment-induced lung inflammation. Vascular emigration and alveolar surface migration. Am J Pathol 1980; 101a; 283-302. 14. Stauton DE, Marlin SD, Stratowa C et al. Primary structure of ICAM-I demonstrates interaction between members of immunoglobu.in and integrin supergene families. Cell 1988; 52: 925-33. 15. Osborn L, Hession C, Tizard R et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 1989; 59: 1203-11. 16. Bevilacqua MP, Stengelin S, Gimbrone Jr MA et al. Endothelial leukocyte adhesion molecule 1: An inducible receptor for neutrophil related to complement regulatory proteins and lectins. Science 1989; 243: 1160-5. 17. Moser R, Schleiffenbaum B, Groscutrh P et al. Interleukin 1 and tumor necrosis Factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage. J Clin Invest 1989; 83: 444-55. 18. Maury CPJ. Interleukin-1 and the pathogenesis of inflammatory diseases. Acta Med Scand 1986; 220: 291-4. 19. Eisenberg SP, Evans RJ, Arend WP et al. Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist. Nature 1990, 343: 341 -6. 20. Carter DB, Deibel Jr MR, Dunn CJ et al. Purification, cloning, expression and biological characterization of an interleukin-1 receptor antagonist protein. Nature 1990; 344: 633-7.
Manuscript received Nouember 5, 1991; accepted January 22, 1992 Address for correspondence: Kazunori Itoh, Department of Otolaryngology, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890, Japan
