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Published ahead of print on April 17, 2003, doi:10.1164/rccm.200207-640OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 281-286, (2003)
© 2003 American Thoracic Society

Reduced Nitric Oxide in Sinus Epithelium of Patients with Radiologic Maxillary Sinusitis and Sepsis

Maria Deja, Thilo Busch, Sebastian Bachmann, Kerstin Riskowski, Valentina Câmpean, Brigitte Wiedmann, Michael Schwabe, Bertold Hell, Josef Pfeilschifter, Konrad J. Falke and Klaus Lewandowski

Departments of Anesthesiology and Intensive Care Medicine, Anatomy, and Maxillofacial Surgery, Charité, Campus Virchow-Klinikum; Department of Pathology, Charité, Campus Mitte, Humboldt University, Berlin; and Center of Pharmacology, Johann Wolfgang Goethe University, Frankfurt am Main, Germany

Correspondence and requests for reprints should be addressed to K. Lewandowski, M.D., Klinik für Anaesthesiologie und Operative Intensivmedizin, Charité, Campus Virchow-Klinikum, Humboldt-Universität zu Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany. E-mail: klaus.lewandowski{at}charite.de


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Radiologic maxillary sinusitis is an important risk factor for development of bronchopneumonia in mechanically ventilated patients. Nitric oxide produced within the paranasal sinuses is considered to provide an antibacterial environment and to modulate mucociliary clearance function. We hypothesized that a reduced formation of nitric oxide might contribute to the compromised local host defense in radiologic maxillary sinusitis and measured nitric oxide levels directly within maxillary sinuses of septic patients with radiologic maxillary sinusitis (n = 11), whose sinuses were fenestrated to eliminate a possible septic focus. Data were compared with those of patients without airway inflammation (n = 11, control subjects). Despite local inflammation and infection, we found considerably lower maxillary nitric oxide levels than in control subjects (31 ± 10 versus 2,554 ± 385 parts per billion, mean ± standard error of the mean, p < 0.001). Consistently, immunohistochemical and in situ hybridization investigations revealed strongly reduced expression of inducible nitric oxide synthase. By applying ultrastructural immunolocalization, we identified cilia and microvilli of the maxillary sinus epithelium as the major nitric oxide production site in control subjects. Our findings provide evidence of markedly reduced nitric oxide production in maxillary sinuses of patients with radiologic maxillary sinusitis and sepsis, implicating impaired local host defense and an increased risk for secondary infections.

Key Words: inducible nitric oxide synthase expression • nitric oxide • radiologic maxillary sinusitis

Radiologic maxillary sinusitis (RMS), defined as the presence of unilateral or bilateral opacification on a paranasal computerized tomography scan, reflecting air–fluid levels within the maxillary sinuses, is a frequent complication in critically ill patients (1). If contaminated with hospital-acquired pathogens, the stagnant secretions may cause the development of a nosocomial infectious sinusitis that is considered a possible focus of sepsis and as an important risk factor for ventilator-associated pneumonia (2). Nosocomial pneumonia significantly increases length of stay and mortality in the intensive care unit (3). The underlying mechanisms for fluid retention and compromised immune defense in RMS are not known. There is, however, experimental evidence that nitric oxide (NO) influences primary host defense. Thus, it has been demonstrated that the NO synthase antagonist NG-monomethyl-L-arginine reduced ciliary beat frequency in prestimulated bovine bronchial epithelial cells by 40% (4). In similar fashion, NG-monomethyl-L-arginine blocked the increase in ciliary beat frequency evoked by the stimulation of muscarinic receptors in ciliated epithelial cells of human tonsils (5). Consequently, inhalation of the nebulized NO donor nitroprusside in human volunteers at a dose of 3 mg increased nasal mucociliary activity in vivo by 57% (6). Apart from its effects on ciliary function, NO is considered to take part in nonspecific immune defense mechanisms by its ability to inhibit the growth of microorganisms (7–9). Furthermore, it is an endogenous inhibitor of both basal and neurogenic mucus secretion in rodents (10).

Nitric oxide is produced endogenously within the respiratory tract and is detectable in exhaled gas of humans and other mammals (11, 12). After the discovery that more than 80% of the total exhaled quantity is released into the upper airways (13, 14), the epithelial cells in the paranasal sinuses were identified as the major source of NO release into the respiratory tract (15). Nitric oxide concentrations of several thousand parts per billion (ppb) were measured directly within the maxillary sinuses of healthy adult volunteers. Molecular biochemical investigations suggested a constitutively expressed enzyme sharing messenger ribonucleic acid sequence homology with human hepatocyte inducible nitric oxide synthase (iNOS) to be almost exclusively responsible for paranasal NO production in healthy adults (15).

We hypothesized that reduced maxillary NO synthesis might contribute to the impaired local host defense in RMS. In this study, we investigated maxillary NO release and ultrastructural localization of iNOS in patients with RMS and sepsis. Data were compared with those from patients who underwent maxillofacial surgery to correct for dysgnathia. These patients were free of systemic and airway inflammation and served as control subjects.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Population
The study protocol was approved by the local ethics committee. With written informed consent given by themselves or by their next of kin, 22 intubated and mechanically ventilated adult patients (11 male, 11 female; body weight, 77 ± 5 kg; age, 39 ± 3 years) were included in the study. Eleven were critically ill patients fulfilling criteria for RMS (1): total opacification or presence of an air–fluid level within one or both maxillary sinuses in CT scans (see the online supplement for details concerning the duration of mechanical ventilation, tubing routes, and positional maneuvers). All patients with RMS fulfilled the criteria of sepsis (sepsis, n = 1; severe sepsis, n = 3; septic shock, n = 7) according to the current definition (16) and underwent fenestration of the maxillary sinuses to eliminate a possible septic focus. Six patients with septic shock received low-dose hydrocortisone (240 mg/day Hydrocortison, Aventis Pharma, Bad Soden, Germany) for immunomodulatory therapy (17). Five patients with RMS fulfilled criteria for acute respiratory distress syndrome (18). Eleven control patients, free of systemic and airway inflammation, were voluntarily fenestrated during surgical osteotomy to correct dysgnathia under general anesthesia. All control subjects received preoperative corticosteroids (1 g of Solu-Decortin; Merck, Darmstadt, Germany) intravenously for antiedematous therapy.

Gas Sampling and NO Measurements
For gas sampling, either a steel needle (Yale spinal; inner diameter, 2 mm; Becton Dickinson, Fraga, Spain) (in control subjects) or a blunt, curved cannula (in patients with RMS) was advanced into the maxillary sinuses and was connected to a glass syringe, which was itself linked with an occlusive, extremely inert and gas-tight Tedlar bag (Supelco Sigma-Aldrich, Deisenhofen, Germany). We collected 1,000 ml of gas in aliquots of 50 ml with a continuous aspiration rate of 0.1 L/minute. Patients with RMS were investigated in addition, if feasible, between days 2 and 3 and between days 4 and 27 of their intensive care unit stay. Nitric oxide and nitrogen dioxide concentrations were measured immediately by chemiluminescence analysis (19) (CLD700AL analyzer; Eco-Physics, Duernten, Switzerland). Net NO excretion rate was calculated by multiplying sampling flow rate by the NO concentration difference between sampled gas and ambient air (see online data supplement for details).

Microbiological, Histologic, Immunohistochemical, and In Situ Hybridization Investigations
Swabs for bacterial cultures and open mucosal biopsies of maxillary sinus epithelium were taken from n = 6 patients and n = 6 control subjects. Histopathological examinations were based on hematoxylin–eosin staining. Cryostat sections of 5 µm were treated with a primary anti-iNOS antibody (20), diluted 1:200. For signal detection, a secondary horseradish peroxidase-conjugated swine anti-rabbit antibody (Dako, Copenhagen, Denmark), diluted 1:100, was applied. Fine structural immunoperoxidase labeling was accomplished according to an established protocol (21). Briefly, 30-µm-thick cryostat sections were incubated overnight with the primary anti-iNOS antibody, diluted 1:25, followed by treatment with the secondary peroxidase-labeled antibody, diluted 1:25. Semithin (1 µm) and ultrathin (60 nm) sections were viewed by light and electron microscopy, respectively. An iNOS riboprobe was generated from deoxyribonucleic acid complementary to base pairs 3180 to 3853. Hybridization on 5-µm cryostat sections was performed, as described elsewhere (22), overnight at 50°C with 30 ng of probe in 40 µl of buffer. Sections were then incubated for 2 hours with an antidigoxigenin antibody (Dako) diluted 1:50, washed, and analyzed by high-resolution interference-contrast microscopy (see online supplement for details about the techniques and quantification of histologies and immunostaining).

Statistical Analysis
Differences between groups were explored with the Mann–Whitney U test. Intragroup comparisons were performed by Friedman analysis of variance followed by post hoc tests according to Wilcoxon with Bonferroni's correction. Correlation analysis was performed with the Spearman rank correlation coefficient (SPSS statistical software, version 10.0.7; SPSS, Chicago, IL). Statistical significance was accepted for p < 0.05. Data are presented as means ± standard error of the mean.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In patients with RMS and sepsis (n = 11), mean NO concentrations measured in the maxillary sinuses within 24 hours of fenestration were significantly and markedly lower than in patients with healthy paranasal sinuses (n = 11) (31 ± 10 versus 2,554 ± 385 ppb, p < 0.001; see Table 1) . These values corresponded to net NO excretion rates of 2 ± 1 and 254 ± 39 nl/minute, respectively (p < 0.001, patients with RMS and sepsis versus control subjects). Nitric oxide levels within the maxillary sinuses exceeded those measured in ambient air in all cases (p < 0.01 both for control subjects and patients with RMS and sepsis), demonstrating endogenous NO production. In control subjects, maxillary nitrogen dioxide concentration was significantly increased compared with ambient air (p < 0.01), but remained below 10% of the corresponding NO levels, indicating no substantial NO/nitrogen dioxide conversion by reaction with oxygen. Data from NO measurements conducted on various days after fenestration in n = 8 patients with RMS are displayed in Figure 1 . Within 3 days, NO concentration remained in the same range (day 1, 21 ± 9 ppb; days 2–3, 48 ± 17 ppb; NS). Additional determinations, conducted between days 4 and 27, revealed an increase in NO concentration to 554 ± 5 ppb (p < 0.05 versus day 1, and p < 0.05 versus days 2–3). Only in one patient was a "normal" value of 1,780 ppb detected. At the time of measurement this patient was still receiving corticosteroids (hydrocortisone at 250 mg/day, intravenous).


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  		TABLE 1. Results of nitric oxide and nitrogen dioxide measurements in gas sampled from the maxillary sinus

 


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  		Figure 1. Time course of maxillary sinus nitric oxide (NO) concentration in patients with RMS and sepsis (n = 8). Within 3 days of fenestration, maxillary NO levels remained low. Nitric oxide levels increased significantly in consecutive measurements between days 4 and 27, but only one patient reached a value comparable to those of control subjects. *p < 0.05.

 
We did not detect a relationship between either the number of sinus cavities affected, the presence of acute respiratory distress syndrome, or the use of corticosteroids, and NO release into the maxillary sinuses in patients with RMS and sepsis. There was no correlation between the number of diseased sinuses and the maxillary NO concentration (r = –0.06; p = 0.86, NS), or the net NO excretion rate (r = 0.06; p = 0.86, NS). Nitric oxide levels in the maxillary sinuses were comparable in patients with and without acute respiratory distress syndrome (32 ± 14 ppb, and 30 ± 14 ppb, respectively; p = 0.86, NS). The range of values for the net NO excretion rate was not significantly different among RMS patients who received low-dose hydrocortisone (1–6 nl/minute) and RMS patients without administration of corticosteroids (1–9 nl/minute) (p = 0.27, NS).

To study the pathophysiological mechanisms for the reduced NO production, we investigated the expression of iNOS protein and iNOS-specific messenger ribonucleic acid in ciliated epithelial cells of the maxillary sinuses. On the basis of immunohistochemical peroxidase-labeled staining of iNOS in control subjects and patients with RMS and sepsis (each, n = 6 biopsies) we found substantial differences between these groups. In all control subjects, iNOS immunoreactivity produced a distinct apical staining of the epithelial cells. In contrast, in patients with RMS and sepsis, iNOS immunoreactivity was faint or absent (Figures 2A and B) . A quantitative analysis of the immunostaining was performed in n = 5 control subjects and in n = 5 patients with RMS and sepsis. Total optical density as calculated by summing up optical densities over all pixel areas with signals due to immunostaining was significantly different between groups (control subjects, 22.9 ± 2.4; RMS group, 11.4 ± 1.6; p < 0.01). Accordingly, there was a 10-fold difference in total pixel areas with signal between control subjects and patients with RMS (control subjects, 27,000 ± 8,100 pixel areas; RMS group, 2,700 ± 300 pixel areas; p < 0.01).



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  		Figure 2. Immunoreactive sinus inducible nitric oxide synthase distribution in biopsy samples from maxillary sinus epithelium. Samples from control subjects (A) reveal strong immunoreactivity in the apical part of the ciliated epithelial cells near the surface. A much weaker signal is obtained in ciliated epithelium of patients with RMS and sepsis (B). Original magnification: x900.

 
Detailed analysis of iNOS levels in our control patients showed that the immunoreactivity was located within the cilia. The signal was strongest in the apical zone of the epithelium directly beneath the lumenal cell membrane, and in the basal part of cilia (Figure 3) . Immunoelectron microscopy of the same material showed iNOS immunoreactivity predominantly associated with the ciliary matrix, with microvilli interspersed between the cilia, and with the apical plasma membrane (Figure 4A) . This signal was absent in patients with RMS and sepsis (Figure 4B). In situ hybridization with an iNOS-specific complementary ribonucleic acid probe revealed that substantial amounts of iNOS message were consistently labeled in a major subapical portion of the epithelium in all control samples (Figure 5A) . Samples from patients with RMS and sepsis exhibited a much weaker signal, indicating substantially diminished expression of iNOS messenger ribonucleic acid (Figure 5B).



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  		Figure 3. Preembedding immunohistochemistry of maxillary sinus epithelium from a control biopsy. At high-resolution light microscopy, we demonstrate a distinct signal for inducible nitric oxide synthase protein in the apical cell border and cilia. Original magnification: x1,800.

 


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  		Figure 4. Preembedding immunoelectron microscopy of a control biopsy sample. Maxillary sinus epithelium shows inducible nitric oxide synthase immunoreactivity within cilia and microvilli (A). The signal is absent in biopsy material of a patient with RMS and sepsis (B). Original magnification: x7,000.

 


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  		Figure 5. In situ hybridization on maxillary sinus epithelium. The inducible nitric oxide synthase messenger ribonucleic acid signal is concentrated in the upper part of the epithelium as well as in the basal cell layer from a control biopsy (A). In biopsies from patients with RMS and sepsis, no labeling was detected (B). Original magnification: x900.

 
Histopathological and microbiological analysis of biopsy specimen of control subjects revealed no abnormalities. Examination of maxillary sinus biopsies from patients with RMS and sepsis documented a dispersed edematous stroma in otherwise basically regular ciliated epithelium without indication of erosion. Furthermore, and importantly, mixed cellular infiltrates with lymphocytes (28 ± 7 cells per high-power field) and plasma cells (6 ± 1 cells per high-power field) indicated nonspecific inflammation in all investigated cases. In three patients additional infiltration of neutrophilic leukocytes was present, suggesting histologic evidence of infection. In two of the latter, positive bacterial cultures (Staphylococcus aureus and Propionibacterium acnes; Pseudomonas aeruginosa, respectively) were detectable on microbiological analysis and contributed to the diagnosis of an infectious sinusitis. Analysis of the immunostaining signal revealed no difference in the range of values between RMS patients with and without infection (8.6–14.6 and 8.4–16.2, respectively). The ranges of values for maxillary NO concentration also did not depend on the presence or absence of infection (RMS with infection, 15 ± 48 ppb; RMS without infection, 10 ± 74 ppb). The extent of the structural impairment, however, was slightly more pronounced in patients with infection with regard to edema formation and ciliary thinning out. In five of the investigated patients with RMS we found none, and in one patient with RMS only a clinically insignificant number of eosinophilic leukocytes (two cells per high-power field). Therefore, an allergic etiology of RMS was excluded in all patients (see Figures E2 and E3 in the online supplement).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To investigate whether RMS is associated with locally decreased NO production, we measured NO levels directly within the sinus lumen and investigated iNOS expression in the maxillary sinus epithelium. We found that patients with RMS and sepsis, in contrast to control subjects without airway inflammation, had a significant and sustained decrease in NO release into the maxillary sinuses. Using immunohistochemical and in situ hybridization methods, we demonstrated that this was related to a markedly attenuated abundance of iNOS protein and iNOS messenger ribonucleic acid in the surface lining epithelium. Our results with control subjects confirm the previously reported finding of Lundberg and coworkers (15) that the abundance of iNOS in apical regions of the maxillary sinus epithelium is responsible for the high NO levels within the sinus lumen of healthy adult volunteers. Further, we provide the first evidence that maxillary iNOS in human subjects free of airway inflammation is localized mainly within cilia and microvilli. The different distribution of iNOS messenger ribonucleic acid over large parts of the epithelial cells reflects an effective targeting mechanism of the protein from its site of translation to its final destination site. The resulting spatially restricted localization of iNOS supports NO release into the sinus lumen and simultaneously reduces adverse effects on the cell nucleus. It is well known that microorganisms invading cilia may cause ultrastructural changes in the microtubuli leading to mucociliary dysfunction (23). Work from several authors reveals that the NO concentrations we found in the maxillary sinuses of control subjects suffice to block the growth of cultured bacteria and viruses (7–9). This provides an efficient mechanism for nonspecific host defense.

In addition, NO production within the cilia may favor modulation of ciliary beat frequency by direct action on dynein arms. Endogenous NO leads to formation of cyclic guanosine monophosphate, which in turn activates protein kinase G. The latter, in the end, evokes the effects of the signal transduction pathway via phosphorylation (24). Experimental studies indicate two molecular biological mechanisms. First, protein kinase G may induce a calcium influx (25) subsequently leading to an upregulation of dynein adenosine triphosphatase (26). Second, G-kinase phosphorylates some of the dynein-associated proteins at the axons of the cilia (27, 28). In cytoplasmic dynein, such subunits influence dynein–adenosine triphosphatase activity (29, 30). Accordingly, previous studies showed a ciliary beat frequency of 17 Hz in epithelial cells from human maxillary sinus (31) versus a ciliary beat frequency of 11 Hz in human epithelial cells taken from the concha nasalis inferior (32), where only weak iNOS activity has been reported (15). We hypothesize that the high NO production within the paranasal sinuses contributes to maintain ciliary beat frequency at a level sufficient for optimal mucociliary clearing function. Because the sinus ostia are not located at the anatomically lowest level, a continuous, highly efficient active transport is required to avoid mucous plugging within the maxillary sinuses.

In general, proinflammatory cytokines and lipopolysaccharides have the potential to induce iNOS expression. Consequently, several inflammatory diseases that affect the interstitium and airway epithelium are associated with increased NO release into the airways. This explains increased levels of exhaled NO in patients with asthma (33), fibrosing alveolitis (34), bronchiectasis (35), lymphocytic bronchiolitis (36), and allergic rhinitis (37). Various studies provided indirect evidence of increased iNOS expression as the primary reason for impaired vascular reactivity in the acute phase of sepsis. Accordingly, patients with sepsis have increased nitrite and nitrate plasma levels that cannot be explained by renal dysfunction alone (38). In experimental models of the disease, iNOS-deficient mice were found to be superior compared with wild-type mice in terms of survival rate and maintenance of systemic blood pressure (39). With regard to these findings, our results appear to be paradoxical: maxillary sinus iNOS expression and NO release are considerably high in control subjects without infection whereas they are almost completely absent in patients with RMS and sepsis. This is, to our knowledge, the first in vivo demonstration of locally reduced iNOS expression in patients with RMS and sepsis during the course of the disease. The absence of sinus iNOS indicates the significance of other forms of inflammatory response. Accordingly, we found lymphocytes and neutrophils in maxillary sinus biopsies of patients with RMS and sepsis. Investigations suggest that high levels of NO inhibit neutrophilic chemotaxis (40) and proliferative responses in human lymphocytes (41). Therefore, reduction of the intense expression may favor the inflammatory response. There is evidence from in vitro and experimental in vivo studies that certain cytokines such as interleukin-4, interleukin-6, interleukin-10, interleukin-11, interleukin-13, and, in particular, transforming growth factor-ß1 are able to reduce iNOS expression (42). Administration of exogenous transforming growth factor-ß1 markedly reduced iNOS messenger ribonucleic acid in several organs in an experimental model of septic shock (43).

It might be argued that reduced maxillary iNOS expression in patients with RMS and sepsis might be a consequence of the immunomodulatory effects of corticosteroid therapy. First, however, only 6 of the 11 patients with RMS received hydrocortisone and there was no significant difference in maxillary NO net excretion when compared with RMS patients without this treatment. Second, the dose we administered to patients with RMS and septic shock was low and control patients also received glucocorticosteroids before the measurements. Third, all patients with RMS and sepsis increased their maxillary sinus NO release in the time period after fenestration despite ongoing corticosteroid therapy. In particular, the one patient who reached a maxillary sinus NO concentration in the range of the control subjects was still receiving hydrocortisone at a dosage of 250 mg/day at the time of measurement. On the basis of these arguments a decisive effect of corticosteroids on our results appears to be unlikely.

Our data are in line with results of a study of children with acute sinusitis, whose nasal NO levels were found to be significantly lower than in healthy control subjects (44). In addition, Lindberg and co-workers reported reduced NO levels in the nasal cavity of patients with chronic sinusitis (45). Up to now, it has remained uncertain whether these findings are a consequence of reduced maxillary NO production or are caused mainly by an obstruction of the sinus ostia. The latter interpretation has been preferred (44) and was supported by further investigations of patients with nasal polyposis, in whom the nasally measured NO concentration was inversely correlated with the number of occluded paranasal sinuses (46). Our data, however, indicate that reduced iNOS expression might also contribute to reduced NO release as an important factor. This view is supported by the detection of messenger ribonucleic acids for the iNOS-suppressing cytokines interleukin-4, interleukin-6, and transforming growth factor-ß1 in maxillary sinus mucosa of patients with chronic sinusitis (47).

It should, however, be considered that the etiology of RMS in septic patients may be different from that of infectious or allergic sinusitis in nonventilated patients. A characteristic feature of sepsis is that it can induce local inflammatory reactions in all tissues by circulating mediators. In this context, it is conceivable that mucous membranes in particular, including paranasal sinus mucosa, may become affected. The limited number of patients with histologic and microbiological evidence of infection, and the absence of any indication of allergic genesis in our patients, suggest in fact a major role for a sepsis-related systemic trigger rather than an endolumenal invasion by bacteria or other pathogens. Therefore, a possible extension of our results to sinusitis in other clinical situations should be the subject of future investigations.

Brett and Evans described decreased exhaled NO concentrations in patients with acute respiratory distress syndrome (48). At first glance, this seems to be an analogous situation in which the regional synthesis (i.e., production at the alveolar level) of NO is impaired. On the basis of our findings, it could be argued that the seemingly reduced alveolar NO synthesis might be a consequence of reduced iNOS expression within the lung. Careful analysis, however, reveals that on the basis of the published data, these situations cannot be compared. There is no evidence from experimental or human studies of decreased pulmonary iNOS expression in the acute respiratory distress syndrome. In contrast, various experimental models of acute lung injury revealed increased pulmonary NO synthesis (42, 49). This supports the interpretation of Brett and Evans (48) that the low pulmonary NO concentrations in patients with acute respiratory distress syndrome resulted primarily from scavenging reactions with other reactive species.

In conclusion, we found that high maxillary NO levels in human subjects without airway inflammation result from iNOS expression, which is ultrastructurally located in cilia and microvilli of the lumenal sinus epithelium. This most likely serves to improve ciliary function and nonspecific immune defense. In patients with RMS and sepsis, maxillary NO production is almost completely suppressed by downregulation of iNOS messenger ribonucleic acid. This highly impaired production needs several weeks to recover. In patients with RMS and sepsis, a sustained impairment in local host defense and an increased risk for secondary infections can be anticipated.


   Acknowledgments
 
The authors are indebted to Mrs. G. Holland and Mrs. P. Schrade for excellent assistance in electron microscopy, to Priv.-Doz. Dr. U. Keske for analysis of the computerized tomography scans, and to Dr. S. Laudi for assistance in the submission procedure.


   FOOTNOTES
 
Supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Fa 139/4-3).

M.D. and T.B. contributed equally to this study.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form July 2, 2002; accepted in final form April 14, 2003


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 DISCUSSION
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