The Neutrophil and Chronic Allergic Inflammation Immunochemical Localization of Neutrophil Elastase 1- 3
TAKAO FUJISAWA,4 GAIL M. KEPHART, BEULAH H. GRAY, and GERALD J. GLEICH
Introduction
Controversy exists regarding the importance of the neutrophil as an effector cell in chronic allergicinflammation (1). Neutrophil infiltration in cutaneous IgEmediated late-phase reactions (LPR) has been reported (2, 3), and elevations in serum levelsof neutrophil chemotactic factor during the LPR in the lung have also been reported (4, 5). Several animal models have demonstrated neutrophil and eosinophil involvement in the experimental LPR (6-12). On the other hand, eosinophil infiltration is characteristic of chronic allergic inflammation
SUMMARY To test whether neutrophils infiltrate and degranulate in areas of chronic respiratory allergic inflammation, we developed an indirect immunofluorescence technique to localize neutrophil elastase in formalin-fixed, paraffin-embedded tissues. The affinity-purified antielastase stained only neutrephlls on peripheral blood bufly coat smears, and in lung tissue from patients with pneumonia. Weexamined tissue specimens from four patients with fatal asthma, 10patients with chronic sinusitis, and 10 patients with nasal polyposis for the presence of elastase, as well as eosinophil granule major basic protein (MBP). Neutrophil infiltration and extracellular elastase deposition in association with damage to respiratory epithelium were generally sparse in most specimens; the exceptions were one patient with asthma, one patient with chronic sinusitis, and two patients with nasal polyposis. In contrast, eosinophil infiltration and extracellular MBP deposition were generally marked in most specimens; the exceptions were one patient with asthma and one patient with nasal polyps where extracellular MBP deposition did not coincide with damage to respiratory epithelium. The results suggest that the neutrophil does not usually infiltrate tissues showing allergic inflammation; however, on occasion, it may participate in these inflammatory reactions. AM REV RESPIR DIS 1990; 141:689-697
(13-16).
Prior studies of diseases associated with eosinophilia have shown extensive ation between neutrophil degranulation deposition of eosinophil granule major and tissue damage, and whether there is basic protein (MBP) in the absence of a relationship between the involvement a pronounced accumulation of tissue eo- of the eosinophil and the neutrophil in sinophils (17-19). These studies suggest chronic allergic inflammation. that eosinophils commonly lose their morphologic integrity in diseased tissues Methods and indicate that assessment of eosinoTissue Specimens phil involvement in disease cannot be We examined four lung, 10 sinus, and 10 naoased simply on enumeration of intact sal polyp tissue specimens. The lung autopsy eosinophils in tissue. Because neutrophils tissues were obtained from four patients with night play a more prominent role in asthma who died suddenly. Clinical data and chronic allergic inflammation than the length of time between death and autoporesently suspected, possibly because sy for the patients with asthma are shown in .hey also degranulate and are not recog- table 1. Patient 1 was thought to have had iizable by light microscopy,wedeveloped an upper respiratory infection as a complicaof the underlying asthma. The sinus tisIn immunofluorescence technique for tion sue biopsies were obtained from 10 patients .he localization of human neutrophil with chronic sinusitis and asthma (three men, ~lastase. One of the advantages of the imseven women; mean age, 34 yr). Finally, the nunofluorescence technique is the abil- nasal polyp biopsies were obtained from 10 ty to detect extracellular granule proteins patients with nasal polyposis (seven men, hat cannot be detected with the conven- three women; mean age, 48 yr); seven of the ional hematoxylin-eosin (H&E) stain. 10 patients had asthma. All specimens were Jsing an indirect immunofluorescence fixed in 100/0 buffered formalin (pH, 7) and echnique, weexamined sinus, nasal pol- embedded in paraffin for sectioning and hisrp, and lung tissue specimens for the tologic studies. iresence of neutrophil elastase and eoDetection oj Neutrophil Elastase and inophil granule MBP. We determined Eosinophil Granule MBP by he prevalence of neutrophil infiltration, Immunofluorescence vhether neutrophils degranulate and Immunofluorescent localization of elastase hus are not recognized by conventional was accomplished utilizing a method modiI&E staining, whether there is an associ- fied from that previously described for MBP
(20-22); the specificity of anti-MBP and the affinity purification of anti-MBP have been reported elsewhere (21, 22). Four serial sections, 6 J.1m thick, were cut and affixed to microscope slides with LePage's glue (LePage's Ltd., Bramalea, Ontario, Canada). After deparaffinization with xylene and rehydration, the sections were partially digested with 0.1 % trypsin containing 0.1070 CaCI] (pH, 7.8) for 1 hat 37° C. After washing in water, the first and second slides were placed in 10070 normal rabbit serum (Lot 81579; Pel-Freeze Biologicals, Rogers, AR), and the third and fourth slides were placed in 100/0 normal goat
(Received in original form April 3, 1989 and in revised form August 1, 1989) 1 From the Allergic Diseases Research Laboratory and the Department of Immunology, Mayo Clinic and Mayo Foundation, Rochester, and the Department of Microbiology, University of Minnesota, Minneapolis, Minnesota. 2 Supported by Grants AI-15231 and HL-37615 from the National Institutes of Health, and from the Mayo Foundation. 3 Correspondence and requests for reprints should be addressed to Gerald J. Gleich, M.D., Chairman, Department of Immunology, Mayo Clinic, Rochester, MN 55905. 4 Visiting Scientist at the Division of Allergic Diseases and Internal Medicine. Present address: Department of Pediatrics, Mie National Hospital, Mie-ken 51401, Japan; supported by a grant from the Rotary Foundation of Rotary International.
689
690
FWISAWA, KEPHART, GRAY, AND GLEICH
TABLE 1 PATIENTS WITH ASTHMA'
Patient No.
Duration of Asthma
Age
(yry
Sex
(yry
3
59 46 70
4
68
F F F F
10 40 19 18
1 2
Systemic Glucocorticoid Therapy
+ + +
Time Elapsing between Death and Autopsy (h) 19
3 6.5 11
, Three tissue blocks from each patient were studied . All patients died of asthma. Patient 1 died 35 min after hospnal admission; Patients 2, 3, and 4 were dead on arrival at the hospital.
serum (Lot 2978; Pel-Freeze Biologicals). All slides were incubated overnight at 4 0 C, and the next day they were washed in Dulbecco's phosphate-buffered saline (PBS). The first section was overlaid with protein-A-purified normal sheep IgG (NSIgG) (Lot I06F-8806; Sigma Chemical Co., St. Louis, MO) as the negative control for antielastase, the second section was overlaid with affinity-chromatography-purified sheep antibody to human neutrophil elastase (vide infra), the third section was overlaid with affinity-chromatographypurified rabbit antibody to MBP, and the fourth section was overlaid with protein-Apurified normal rabbit IgG (NRIgG) as the negativecontrol for anti-MBP. The slideswere placed in a moist chamber for 30 min at 370 C, washed with PBS, stained with 1% chromotrope 2R for 30 min at room temperature, and washed again. The first two slides were then overlaid with a fluorescein-conjugated IgG fraction of rabbit antisheep IgG (Lot 26481; Cooper Biomedical, West Chester, PA), and the last two slides were overlaid with a fluorescein-conjugated IgG fraction of goat antirabbit IgG (Lot 24682; Cooper Biomedical). After an additional incubation for 30 min at 37 0 C, the slides were washed, mounted with
a 10% PBS:90OJo glycerol solution containing p-phenylenediamine (Aldrich Chemical Co., Milwaukee, WI) (23), coverslipped, and sealed with clear nail polish. Slides wereexamined with a Zeiss standard microscope (Carl Zeiss Inc., Oberkochen, FRG) equipped with Zeiss IV FL vertical illumination for epifluorescence and a fluorescein filter system (48771O:Zeiss).After photographing the immunofluorescence findings, coverslips were removed, the tissues were counterstained with H&E, and the identical fields were rephotographed under transmitted light.
Validation of Neutrophil Elastase Localization by Immunofluorescence Sheep antiserum to human neutrophil elastase (Lot AHP 051) was purchased from Serotec (Kidlington, Oxford, UK) and first tested for its ability to specifically stain neutrophils on cytocentrifuge preparations of buffy coat cells from normal human peripheral blood by immunofluorescence, using the same procedure as described previously for anti-MBP (21). Second, to determine whether the epitopes of elastase survived formalin-fixation and paraffin-embedding, wetested the antielastase
on lung tissue specimens from patients with pneumonia and on sinus tissue specimens from patients with chronic sinusitis. Third, purified antibody to elastase was obtained by affinity chromatography. Purified human neutrophil elastase (vide infra) was coupled to cyanogea-bromide-activated Sepharose 4B (Lot 126FOO58; Sigma) using a procedure described previously (22). The sheep antiserum to human neutrophil elastase was applied to the beads and mixed overnight at 4 0 C. The beads were packed into a column and washed with PBS and with 0.17 M borate containing 0.5 M NaCI (pH, 8.0) to remove nonspecifically bound components. The antielastase was eluted with 0.05 M glycine-HCI(pH, 3.0), and peak fractions were pooled. Equal concentrations of purified antielastase and NSIgG (50 ug/ml), as determined by absorbance at 280 nm, wereused in the immunofluorescence assay.
Purification of Human Neutrophil Elastase Human neutrophil elastase was purified from an extract of cytoplasmic granules from normal human neutrophils by two column chromatography steps as described previously (24, 25). Briefly,proteins of the extract werebound to Matrex-Gel Orange A (Amicon Corp., Danvers, MA) and fractionated by elution with a gradient from 0.1 to 1.6 M NaCI in citrate-phosphate buffer at pH 5.6. The elastase peak eluted between 0.35 and 0.68 M NaCI and was identified by hydrolysis of the chromogenic peptide Sue-ala-ala-alaNPhN0 2 , an elastase-specific substrate (26). Fractions containing elastase were diluted into 0.05 M NaCl, applied to a column of BioRex 70 (Bio-Rad Laboratories, Richmond, CAl, and eluted with a gradient of 0.04 to 1.0M NaCI in citrate-phosphate buffer at pH
Fig. 1. Localization of elastase in a cytocentrifuge preparation of human peripheral blood bufly coat cells. A (left). Cells stained with crude antielastase. B (right). The same cells counterstained with Wright's stain. The results show that the fluorescent polymorphonuclear cells in A are neutrophils; the long black arrows in B indicate the same cells shown by white arrows in A. Lymphocytes (short black arrow) and eosinophils (open black arrows) in B are unstained in A. One monocyte (curved arrows in A and B) stains faintly for elastase. Magnification in A and B: x1 ,428.
691
NEUTROPHIL AND ALLERGIC INFLAMMATION
Fig. 2. Localization of neutrophil elastase in a lung tissue autopsy obtained 4.5 h after death from a patient with pneumon ia. A (/eft). Section stained with affinity·purified antielastase. B (right) . Same section as in A, counterstained with H&E. The black arrows in B indicate the same cells shown by white arrows in A; note that the positively staining cells in A are polymorphonuclear when counterstained with H&E. Note the coarse granulation of the neutrophil's cytoplasm after staining with antielastase in A. The NSlgG·stained serial section was negative (results not shown). Magnification in A and B: x l, 428.
7.0. Elastase (overall yield, 340/0) eluted in a sharp peak at 0.27 M NaC! . By sodium dodecyl sulfate polyacrylamide gel electrophoresis the purified enzyme gave a major band and two minor bands slightly above and below the 29,000 molecular weight carbonic anhydrase marker, and it hydrolyzed 3H-elastin (27) at a rate of 2.6 mg/h/mg of elastase.
graded on a scale of zero to 3. Specimens with no or occasional extracellular staining were scored as zero. If there werescattered foci containing faint staining, the specimen wasscored as 1. Specimens with scattered foci containing marked staining were scored as 2, and 3 was assigned to specimens showing intense deposition throughout the lesion.
Evaluation of Immunofluorescence
Results
The evaluation was made by two of the writers independently (TF and GMK). Cellular infiltration of neutrophils or eosinophils, as revealed by cellular staining, was scored on a scale from zero to 3. A score of zero was assigned when no or occasional neutrophils or eosinophils were present. If a few cells were seen in the lesion, it was scored as 1. A score of 2 was given when many neutrophils or eosinophils werescattered throughout the lesion, and 3 was assigned to specimens demonstrating dense infiltration. The degree of extracellular deposition of MBP and elastase was also
Immunofluorescent Staining for Neutrophil Elastase Analyses of the reactivity of sheep antielastase were initially performed by immunofluorescence on buffy coat cells fixed with 100% methanol. Here, the cells showing bright cytoplasmic staining for elastase (figure lA) were identi fied as neutrophils by counterstaining the same slides with Wright's stain (figure lB). Note that eosinophils are not stained by antielastase. Analyses of 143 mono-
nuclear cells for elastase staining revealed faint staining of seven of 70 monocytes and two of 73 lymphocytes; figure 1 shows faint staining of a monocyte. In contrast, the buffy coat cellsdid not stain with normal sheep serum (results not shown). Second, the reactivity of antielastase was tested on formalin-fixed, paraffin-embedded lung tissue specimens from patients with pneumonia. Here, not only neutrophils but also secretory glands in the airways stained with the antisera (results not shown). Therefore , third, antielastase was purified by affinity chromatography on a column of solid-phase elastase. Testsof the affinity-purified antielastase on buffy coat cells, lung tissue, and sinus tissue specimens showed that only neutrophils stained brightly. A section of lung from a patient with pneumonia is shown in figure 2; cells staining with antielastase are concentrated in the
TABLE 2 IMMUNOFLUORESCENCE FINDINGS IN PATIENTS WITH ASTHMA
Patient No. 1
2 3 4
Extracellular Elastase
Neutrophilia B
M
A
B
M
A
2t 0/1 0 0
3 2 0 0
1
0 0 0
2 1 0 0
2 2 0 0
0 0 0 0
Coexistence of Elastase and Epithelial Damage'
++
Eosinophilia
Extracellular MBP
B
M
A
B
M
A
Coexistence of MBP and Epithelial Damage'
1
1 1 2 0
0 1 1 1
1 2 1 2
1 1 2 1
0 0 0 0
+ ++ +
1 2 2
Deffnltion of abbreviations: B • bronch ial wall; M • mucus in bronchial lumen; A = alveoli . • Coexistence ot epithelial damage and elastase or MBP deposltton was scored on a scale from - to + + . A score of - was assigned when no coexistence was observed . Specimens wnh scattered foci showing coexistence were scored + . A score of + + was given to specimens showing extensive coexistence throughout the lesion. The pathology was semiquanlitatively graded (see METHOOS) by two observers . t Two numbers represe nt differing evaluations by the two observers ; single numbers indicate agreement by the two.
692
FWISAWA, KEPHART, GRAY, AND GLEICH
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Fig. 3. Localization of neutrophil elastase and eosinophil MBP in lung from a patient with asthma (Patient 1). A (top, left). Section stained with anti·MBP. C and E (left, middle and bottom) . Section stained with antielastase. B, 0, and F (right). The same sections as in A, C, and E, respectively, counterstained with H&E. Only scattered eosinophils and minimal extracellular MBP deposition are present in the anti·MBP·stained section (A and B). In contrast, note the intense neutrophil infiltration in a serial section stained with antielastase (C and 0). E and F are high power views of C and 0, respectively, and show the coexistence of diffuse extracellular elastase deposition and damaged epithelium. Elastase deposition is also evident in areas of the mucous plug (arrow in E) and in areas beneath the membrane. The arrow in F identifies a Curschmann's spiral. Magnification in A to 0 : x571; magnification in E and F: x 1,428.
693
NEUTROPHIL AND ALLERGIC INFLAMMATION
TABLE 3 IMMUNOFLUORESCENCE FINDINGS IN PATIENTS WITH CHRONIC SINUSITIS*
Patient No. 5 6 7 8 9 10 11 12 13 14
Neutrophilia
Extracellular Elastase
Coexistence of Elastase and Epithelial Damage t
0
0* 2 0 1 1
++
2 0 0
0
1/0 0
0 1 0 1 0
1 1/0 0
Eosinophilia 2/1 3/2 2 3/2 2 3 3/2 2/1 2 3
Extracellular MBP 2/3 2
2 2 2 2/1 3 2 2 2
Coexistence of MBP and Epithelial Damage t
++ ++ ++ + +/+ + +/+ ++ ++ ++ ++ +
* These cases of chronic sinusitis have been reported elsewhere (30). Patients 5 to 14 correspond to Patients 12, 8, 2,11,7,13,9,5,10, and 6, respectively, in reference 30. t Coexistence of epithelial damage and elastase or major basic protein (MBP) deposition was scored on a scale from - to + +. A score of - was assigned when no coexistence was observed. Specimens with scattered foci of coexistence were scored +. A score of + + was given to specimens showing extensive coexistence throughout the lesion. The pathology was sernlquantitatlvely graded (see METHODS) by two observers. :I: Two numbers represent differing evaluations by the two observers; single numbers indicate agreement by the two.
airway lumen, have polymorphonuclear nuclei, and contain discrete cytoplasmic granules. Finally, NSlgG did not stain tissues (results not shown).
Lung Tissue from Patients with Fatal Asthma The H&E stained lung tissue specimens from all the patients with fatal asthma showed the characteristic findings of asthma (28), namely, thickened basement membranes, mucous plugs in the airway with or without Creola bodies, various degrees of bronchial epithelial damage (including epithelial shedding and squamous cell metaplasia), and smooth muscle hyperplasia. Mononuclear cell infiltration was also seen in varying degrees. None of the specimens demonstrated other pathologic lesions such as pneumonia or emphysema. Sections stained with antielastase by immunofluorescence showed evidence of extracellular deposition in two of the four
patients (table 2). In Patient 1, there was striking neutrophil infiltration and extracellular elastase deposition in the bronchial wall, peribronchial tissue, and, particularly, in the bronchial mucus. It can be seen in figure 3 that the region with prominent extracellular elastase deposition also displayed striking damage characterized by loss of epithelium and pyknosis of remaining epithelial cells. In contrast, deposition of MBP was not as prominent as elastase deposition. In Patient 2, extracellular elastase was deposited in the bronchial mucus (2 +) and in the bronchial wall (1+), but it was not localized in any regions of epithelial damage. Extracellular MBP, however, was found on areas of damaged epithelium. In Patients 3 and 4, there were only occasional foci of intracellular and extracellular elastase deposition, but extracellular MBP and eosinophil infiltration were observed in the bronchial wall, where extensive epithelial damage was evident.
Neither extracellular elastase nor MBP was observed in the alveoli of any tissue.
Specimens from Patients with Sinusitis and Nasal Polyps Marked eosinophil infiltration and extracellular deposition of MBP were observed in all 10 patients with chronic sinusitis and in all 10 patients with nasal polyposis by immunofluorescence (tables 3 and 4). Representative photomicrographs from Patient 16 with nasal polyposis are shown in figure 4. Marked extracellular deposition of MBP (figure 4A) coincided with the presence of epithelial damage (figure 4B). Note the denuded epithelial layer and only a few remaining basal cells in figure 4B. In this case, only a few elastase-positive cells were identified (figure 4C). Marked elastase deposition (2+) was seen in only one of 10 patients with sinusitis and in one of 10 patients with nasal polyposis. The coexistence of striking, focal elastase deposi-
TABLE 4 IMMUNOFLUORESCENCE FINDINGS IN PATIENTS WITH NASAL POLYPS
Patient No. 15 16 17 18 19 20 21 22 23 24
Neutrophilia
Extracellular Elastase
1t 1 3 1 1 0 0 0 2 0
1 1 2/1 1 0 0 0 0 1 0
Coexistence of Elastase and Epithelial Damage *
+/+ +/+
++
Eosinophilia
Extracellular MBP
2 3 2/1 3 3 3 3 3/2 2/3 3/2
2 3/2 1 3 2 3 2 2/1 3 1
Coexistence of MBP and Epithelial Damage *
++ ++ + ++ ++ ++ ++ + +/+ ++
* Coexistence of epithelial damage and elastase or major basic protein (MBP) deposition was scored on a scale from - to + + . A score of - was assigned when no coexistence was observed. Specimens with scattered foci of coexistence were scored +. A score of + + was given to specimens showing extensive coexistence throughout the lesion. The pathology was semlquantitatively graded (see METHODS) by two observers. t Two numbers represent differing evaluations by the two observers; single numbers indicate agreement by the two.
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Fig . 4. Localization of MBP and elastase in a nasal polyp biopsy from Patient 16. A (top, leff). Section stained with anti·MBP. B (top, right). Same section as in A counter, stained with H&E. C (bottom . left). Serial section to A stained with antielastase. Note the striking eosinophil infiltration and extracellular MBP deposition beneath the basement membrane in association with epithelial damage (A and B). In con, trast, only minimal elastase staining is present (C). Magnification in A to C: x 571.
tion and epithelial damage in Patient 6 with chronic sinusitis and asthma is shown in figure 5. Note that in the area of epithelial damage, intact neutrophils could not be identified by H&E, suggesting that neutrophil degranulation had occurred. The presence of extracellular elastase deposition is shown in figure 6A, and striking neutrophil infiltration is shown in figure 6B beneath largely intact respiratory epithelium of Patient 17 with nasal polyps. Interestingly, the extracellular elastase deposition is concentrated just beneath the respiratory epithelium, whereas intact neutrophils are present in the deeper stroma. In other areas of this specimen, extracellular elastase deposition coincided with epithelial damage (table 4). As can be seen from tables 3 and 4, there was no obvious relationship between the degree of infiltration by eosinophils and neutrophils.
Discussion
These results show that neutrophil granule elastase can be localized in formalinfixed paraffin-embedded tissue by immunofluorescence using essentially the same method employed for localization of MBP (20-22), Initial studies with buffy coat cells showed localization of elastase to neutrophils and its absence in eosinophils and mononuclear cells (figure 1).Staining of rnonocytes byantielastase was seen in only 10070 of cellseventhough the peripheral blood monocyte has been shown to contain a molecule immunochemically similar to neutrophil elastase (29). Preliminary analyses of formalinfixed tissue from patients with pneumonia showedstaining of neutrophils as well as bronchial glands. Therefore, the anti elastase serum was affinity-purified on a column of solid-phase elastase to remove the unwanted antibodies. The pu-
rified anti elastase stained peripheral blood neutrophils in a manner comparable to that shown in figure 1 and also stained neutrophils in formalin-fixed, paraffin-embedded tissue from a patient with pneumonia, as shown in figure 2; the ability of the crude anti elastase to stain bronchial glands was removed by affinity purification. In contrast to the results obtained with affinity-purified antielastase, NSIgG did not stain tissue from patients with pneumonia. Thus, these results indicate that this method permits staining of neutrophil elastase and suggest that this method is useful for detection of neutrophil infiltration and degranulation in inflammatory reactions, especially in situations where eosinophil infiltration is prominent and neutrophils might be overlooked or in situations where neutrophils might have degranulated. As stressed above, eosinophils lose
695
NEUTROPHIL AND ALLERGIC INFLAMMATION
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Fig. 5. Localizationofelastasein a sinus tissue biopsyfrom Patient6. A (left). Sectionstainedwith antielastase. B (right). Samesection as in A counterstainedwith H&E. Notethe coexistence of intense elastase deposition and epithelial denudation. Magnification in A and B: x 1,428.
their morphologic integrity in certain tissues (17-19, 22), and their involvement in these intlammatory reactions cannot be recognized by light microscopy after staining with H&E. Using the immunofluorescence assay for elastase, we have examined tissues from patients with fatal asthma, chronic sinusitis with asthma, and nasal polyps to determine whether neutrophil infiltration and degranulation commonly occur in these conditions. Because of the pathologic similarity of chronic sinusitis and nasal polyposis to asthma (28, 30, 31), we believe that these tissues can be included in the category of allergic inflammation. In previous studies, we have
shown that eosinophil granule proteins are toxic to respiratory epithelium (3234), and eosinophil degranulation (as judged by MBP deposition) occurs in areas of desquamation in bronchial asthma and chronic sinusitis (21, 30). These observations support the hypothesis that the eosinophil is an effector cell in allergic inflammation. Here, in general, we were impressed by the degree of eosinophil infiltration, by the frequency and intensity of extracellular MBP deposition, and by the coexistence of extracellular MBP deposition and epithelial damage (figure 4). Concerning localization of the neutrophil and neutrophil elastase, our results show that neutrophils are far less
prominent in the specimens studied (tables 2 to 4); marked neutrophil infiltration was seen in two of four patients with asthma, in one of 10 patients with sinusitis, and in two of 10 patients with nasal polyps . However, in Patients 1,6, 17, and 23, extracellular elastase deposition coexisted with respiratory epithelial damage (as illustrated in figures 3 and 5). Elastase, a neutral serine protease, is localized to the azurophilic or primary granules in neutrophils and plays an important role in host defense (35). Besides its beneficial function, it may also have various harmful effects and it has been implicated as an effector of tissue damage in emphysema (36-38) and other dis-
Fig. 6. Localizationof elastasein a nasal polyp biopsy from Patient 17. A (left). Section stained with antielastase. B (right). Same section as in A counterstainedwith H&E. The tissueis denselyinfiltratedwith intact neutrophils;the whitearrowin A and the black arrowin B identifyan areaof apparentdegranulation. Magnificationin A and B: x 1,428.
696
eases (39-42). In airways, elastase could alter tracheal mucociliary activity (43) and cause bronchial secretory cell metaplasia (37, 44). The association between the deposition of elastase and the occurrence of epithelial damage suggests that neutrophils injure respiratory tissue in allergic inflammation. The importance of neutrophils in the pathogenesis of allergic inflammation in humans is under active study in several laboratories. Histologic examinations of the human cutaneous LPR provoked by intradermal injection of allergen or antiIgE have shown that both neutrophils and eosinophils contribute to the inflammatory response (2, 3). We have examined tissue from the cutaneous human LPR for neutrophil degranulation using the same assay described here and have observed neutrophil infiltration and striking extracellular elastase deposition at 1 hand 8 h after challenge (Leiferman KM, Fujisawa T, Gray BH, Gleich, GJ, unpublished observations); eosinophil infiltration and MBP deposition occurred in these same tissues. These results suggest that neutrophils, as well as eo sinophils, are active participants in the allergic inflammation associated with the cutaneous LPR. In human respiratory tracts of patients with asthma, analyses of cells by bronchoalveolar lavage have shown an increase of eosinophils and, in some instances, of neutrophils in the lavage fluid (15, 16, 45). Overall, neutrophils were relatively scant in the tissues showing chronic allergic inflammation examined here. How can one reconcile this finding with the occasional association of elastase deposition and asthma? First, the pathogenesis of allergic inflammation may be heterogeneous. In a recent study of localization of MBP in patients with fatal bronchial asthma, some specimens showing striking epithelial damage in the absence of eosinophil degranulation have been found (46). Thus, the pathologic features of allergic inflammation may not be uniform in terms of cellular infiltration. Second, neutrophil infiltration in chronic allergic disease may be evanescent. Histologic studies of the LPR in experimental animals have shown that neutrophils predominate at 6 to 8 h after challenge, and after this are replaced by eosinophils (10, 11). If the neutrophil infiltrates and degranulates in the acute phase of allergic inflammation, then our specimens from patients with chronic allergic inflammation might not contain neutrophils and their granules. Last, the
FWISAWA, KEPHART, GRAY, AND GLEICH
possibility that neutrophils might have appeared because of infection, as suspected in Patient 1, must be considered. In a prior study, we enumerated neutrophils in nasal polyps and found a highly significant positive correlation (r = +0.9; P < 0.001) between tissue neutrophilia and the number of bacteria cultured from the tissue (47). Acknowledgment The writers thank Dr. Thomas Colby for his valuable suggestions, Mrs. Cheryl Adolphson for editorial assistance, and Mrs. Linda H. Arneson for secretarial assistance. References 1. Henson PM. Neutrophils in acute allergic reactions. In: Lessof MH, Lee TH, Kemeny DM, eds. Allergy: an international textbook. New York: John Wiley & Sons, 1987; 157-68. 2. Dolovich J, Hargreave FE, Chalmers R, Shier KJ, Gauldie J, Bienenstock J. Late cutaneous allergic responses in isolated Igfi-dependent reactions. J Allergy Clin Immunol 1973; 52:38-46. 3. Solley GO, Gleich OJ, Jordon RE, Schroeter AL. The late phase of the immediate wheal and flare skin reaction. Its dependence upon IgE antibodies. J Clin Invest 1976; 58:408-20. 4. Nagy L, LeeTH, Kay AB. Neutrophil chemotactic activity in antigen-induced late asthmatic reactions. N Engl J Med 1982; 306:497-501. 5. Lee TH, Nagakura T, Papageorgiou N, likura Y, Kay AB. Exercise-induced late asthmatic reactions with neutrophil chemotactic activity. N Engl J Med 1983; 308:1502-05. 6. Oertel HL, Kaliner M. The biologic activity of mast cell granules. III. Purification of inflammatory factors of anaphylaxis (IF-A) responsible for causing late phase reactions. J Immunol 1981; 127:1398-1402. 7. Lemanske RF Jr, Guthman DA, Oertel H, Barr L, Kaliner M. The biologic activity of mast cell granules. VI. The effect of vinblastine-induced neutropenia on rat cutaneous late phase reactions. J Immunol 1983; 130:2837-42. 8. Lemanske RF Jr, Guthman DA, Kaliner M. The biologic activity of mast cell granules. VII. The effect of anti-neutrophil antibody-induced neutropenia on rat cutaneous late phase reactions. J Immunol 1983; 131:929-33. 9. Murphy KR, Wilson MC, Irvin CG, et ale The requirement for polymorphonuclear leukocytes in the late asthmatic response and heightened airway reactivity in an animal model. Am Rev Respir Dis 1986; 134:62-8. 10. Behrens BL, Clark RAF, Presley DM, Graves JP, Feldsien DC, Larsen GL. Comparison of the evolving histopathology of early and late cutaneous and asthmatic responses in rabbits after a single antigen challenge. Lab Invest 1987; 56:101-13. 11. Iijima H, Ishii M, Yamauchi K, et ale Bronchoalveolar lavage and histologic characterization of late asthmatic response in guinea pigs. Am Rev Respir Dis 1987; 136:922-9. 12. Dunn CJ, Elliott GA, Oostveen JA, Richards 1M. Development of a prolonged eosinophil-rich inflammatory leukocyte infiltration in the guineapig asthmatic response to ovalbumin inhalation. Am Rev Respir Dis 1988; 137:541-7. 13. Frigas E, Gleich GJ. The eosinophil and the pathophysiology of asthma. J Allergy Clin Immunol 1986; 77:527-37.
14. de Monchy JOR, Kauffman HK, Venge P, et 01. Bronchoalveolar eosinophilia during allergeninduced late asthmatic reactions. Am Rev Respir Dis 1985; 131:373-6. 15. Kirby JG, Hargreave FE, Gleich GJ, O'Byrne PM. Bronchoalveolar cell profiles of asthmatic and nonasthmatic subjects. Am Rev Respir Dis 1987; 136:379-83. 16. Wardlaw AJ, Dunnette S, Gleich GJ, Collins, JV, Kay AB. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma: relationship to bronchial hyperreactivity. Am Rev Respir Dis 1988; 137:62-9. 17. Leiferman KM, Ackerman SJ, Sampson HA, Haugen HS, Venencie PY, Gleich GJ. Dermal deposition of eosinophil-granule major basic protein in atopic dermatitis. N Engl J Med 1985; 313:282-5. 18. LeifermanKM, PetersMS, GleichGJ. The eosinophil and cutaneous edema. J Am Acad ' Dermatol 1986; 15:513-7. 19. Gleich GJ, Adolphson CR. The eosinophilic leukocyte: structure and function. Adv Immunol 1986; 39:177-253. 20. Filley WV, Ackerman SJ, Gleich GJ. An immunofluorescent method for specific staining of eosinophil granule major basic protein. J Immunol Methods 1981; 47:227-38. 21. Filley WV, Holley KE, Kephart GM, Gleich GJ. Identification by immunofluorescence of eosinophil granule major basic protein in lung tis-' sues of patients with bronchial asthma. Lancet 1982; 2:11-6. 22. Peters MS, Schroeter AL, Kephart GM, Gleich GJ. Localization of eosinophil granule major basic protein in chronic urticaria. J Invest Dermatol 1983; 81:39-43. 23. Krenik KD, Kephart OM, Offord KP, Dunnette SL, Gleich GJ. Comparison of antifading agents used in immunofluorescence. J Immunol Methods 1989; 117:91-7. 24. Hovde CJ, Gray BH. Characterization of a protein from normal human polymorphonuclear leukocytes with bactericidal activity against Pseudomonas aeruginosa. Infect Immun 1986; 54:142-8. 25. Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal JR. Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988; 82:1963-73. 26. Wenzel HR, Engelbrecht S, Reich H, Mondry W, Tschesche H. Synthesis and analytical use of 3-carboxypropionyl-alanyl-alanyl-valine-4-nitroanilide: a specific substrate for human leukocyte elastase. Hoppe-Seylers Z Physiol Chern 1980; 361:1413-6. 27. Stone PJ, Franzblau C, Kagan HM. Proteolysis of insoluble elastin. Methods Enzymol 1982; 82:588-605. 28. Dunnill MS. The pathology of asthma. In: Middleton E ~r, Reed CE, Ellis EF, eds. Allergy: principles and practice. 1st ed, vol 2. St. Louis: ey. Mosby Co., 1978; 678-86. 29. Senior RM, Campbell EJ, Landis JA, Cox FR, Kuhn C, Korert HS. Elastase of U-937 monocytelike cells. Comparisons with elastases derived from human monoeytes and neutrophils and murine macrophagelike cells. J Clin Invest 1982; 69:384-93. 30. Harlin SL, Ansel DO, Lane SR, Myers J, Kephart GM, Gleich GJ. A clinical and pathologic study of chronic sinusitis: The role of the eosinophil. J Allergy Clin Immunol 1988; 81:867-75. 31. Slavin RG. Relationship of nasal disease and sinusitis to bronchial asthma. Ann Allergy 1982; 49:76-80. 32. Gleich GJ, Frigas E, Loegering DA, Wassom DL, Steinmuller D. Cytotoxic properties of the eosinophil major basic protein. J Immunol 1979;
697
NEUTROPHIL AND ALLERGIC INFLAMMATION
123:2925-7. 33. Frigas E, Loegering DA, Gleich OJ. Cytotoxic effects of the guinea pig eosinophil major basic protein on tracheal epithelium. Lab Invest 1980; 42:35-43. 34. Motojima S, Frigas E, Loegering DA, Oleich OJ. Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. Am Rev Respir Dis 1989; 139:801-5. 35. Falloon J, Oallin JI. Neutrophil granules in health and disease. J Allergy Clin Immunol1986; 77:653-62. 36. Janoff A. Elastases and emphysema: current assessment of the protease-antiprotease hypothesis. Am Rev Respir Dis 1985; 132:417-33. 37. Snider OL, Lucey EC, Christensen TO, et al. Emphysema and bronchial secretory cell metaplasia induced in hamsters by human neutrophil products. Am Rev Respir Dis 1984; 129:155-60. 38. Damiano VV, Tsang A, Kucich U, et al. Im-
munolocalization of elastasein human emphysematous lungs. J Clin Invest 1986; 78:482-93. 39. OarciaJON, JamesHL, ZinkgrafS, Perlman MB, Keogh BA. Lower respiratory tract abnormalities in rheumatoid interstitial lung disease: potential role of neutrophils in lung injury. Am RevRespir Dis 1987; 136:811-7. 40. Niederman MS, Merrill WW, Polomski LM, Reynolds HY, Oee JBL. Influence of sputum IgA and elastase on tracheal cell bacterial adherence. Am Rev Respir Dis 1986; 133:255-60. 41. Suter S, Schaad DB, TegnerH, Ohlsson K, Desgrandchamps D, WaldvogelFA. Levelsof free granulocyte elastase in bronchial secretions from patients with cystic fibrosis: effect of antimicrobial treatment against Pseudomonas aeruginosa. J Infect Dis 1986; 153:902-9. 42. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989; 320:365-76. 43. Tegner H, Ohlsson K, Toremalm NO, von
Mecklenburg C. Effect of human leukocyteenzymes on tracheal mucosa and its mucociliary activity. Rhinology 1979; 17:199-206. 44. Breuer R, Lucey EC, Stone PJ, Christensen TO, Snider OL. Proteolytic activity of human neutrophil elastase and porcine pancreatic trypsin causes bronchial secretory cell metaplasia in hamsters. Exp Lung Res 1985; 9:167-75. 45. Metzger WJ, Moseley P, Nugent K, Richerson HB, Hunninghake OW. Local antigen challenge and bronchoalveolar lavage of allergic asthmatic lungs. Chest 1985; 87(Suppl:155-6). 46. Oleich OJ, Motojima S,Frigas E, Kephart OM, Fujisawa T, Kravis LP. The eosinophilic leukocyte and the pathology of fatal bronchial asthma: evidence for pathologic heterogeneity. J Allergy Clin Immunol 1987; 80:412-5. 47. Dunnette SL, Hall MM, Washington JA II, et al. Microbiologic analyses of nasal polyp tissue. J Allergy Clin Immunol 1986; 78:102-8.
TAKAO FUJISAWA,4 GAIL M. KEPHART, BEULAH H. GRAY, and GERALD J. GLEICH
Introduction
Controversy exists regarding the importance of the neutrophil as an effector cell in chronic allergicinflammation (1). Neutrophil infiltration in cutaneous IgEmediated late-phase reactions (LPR) has been reported (2, 3), and elevations in serum levelsof neutrophil chemotactic factor during the LPR in the lung have also been reported (4, 5). Several animal models have demonstrated neutrophil and eosinophil involvement in the experimental LPR (6-12). On the other hand, eosinophil infiltration is characteristic of chronic allergic inflammation
SUMMARY To test whether neutrophils infiltrate and degranulate in areas of chronic respiratory allergic inflammation, we developed an indirect immunofluorescence technique to localize neutrophil elastase in formalin-fixed, paraffin-embedded tissues. The affinity-purified antielastase stained only neutrephlls on peripheral blood bufly coat smears, and in lung tissue from patients with pneumonia. Weexamined tissue specimens from four patients with fatal asthma, 10patients with chronic sinusitis, and 10 patients with nasal polyposis for the presence of elastase, as well as eosinophil granule major basic protein (MBP). Neutrophil infiltration and extracellular elastase deposition in association with damage to respiratory epithelium were generally sparse in most specimens; the exceptions were one patient with asthma, one patient with chronic sinusitis, and two patients with nasal polyposis. In contrast, eosinophil infiltration and extracellular MBP deposition were generally marked in most specimens; the exceptions were one patient with asthma and one patient with nasal polyps where extracellular MBP deposition did not coincide with damage to respiratory epithelium. The results suggest that the neutrophil does not usually infiltrate tissues showing allergic inflammation; however, on occasion, it may participate in these inflammatory reactions. AM REV RESPIR DIS 1990; 141:689-697
(13-16).
Prior studies of diseases associated with eosinophilia have shown extensive ation between neutrophil degranulation deposition of eosinophil granule major and tissue damage, and whether there is basic protein (MBP) in the absence of a relationship between the involvement a pronounced accumulation of tissue eo- of the eosinophil and the neutrophil in sinophils (17-19). These studies suggest chronic allergic inflammation. that eosinophils commonly lose their morphologic integrity in diseased tissues Methods and indicate that assessment of eosinoTissue Specimens phil involvement in disease cannot be We examined four lung, 10 sinus, and 10 naoased simply on enumeration of intact sal polyp tissue specimens. The lung autopsy eosinophils in tissue. Because neutrophils tissues were obtained from four patients with night play a more prominent role in asthma who died suddenly. Clinical data and chronic allergic inflammation than the length of time between death and autoporesently suspected, possibly because sy for the patients with asthma are shown in .hey also degranulate and are not recog- table 1. Patient 1 was thought to have had iizable by light microscopy,wedeveloped an upper respiratory infection as a complicaof the underlying asthma. The sinus tisIn immunofluorescence technique for tion sue biopsies were obtained from 10 patients .he localization of human neutrophil with chronic sinusitis and asthma (three men, ~lastase. One of the advantages of the imseven women; mean age, 34 yr). Finally, the nunofluorescence technique is the abil- nasal polyp biopsies were obtained from 10 ty to detect extracellular granule proteins patients with nasal polyposis (seven men, hat cannot be detected with the conven- three women; mean age, 48 yr); seven of the ional hematoxylin-eosin (H&E) stain. 10 patients had asthma. All specimens were Jsing an indirect immunofluorescence fixed in 100/0 buffered formalin (pH, 7) and echnique, weexamined sinus, nasal pol- embedded in paraffin for sectioning and hisrp, and lung tissue specimens for the tologic studies. iresence of neutrophil elastase and eoDetection oj Neutrophil Elastase and inophil granule MBP. We determined Eosinophil Granule MBP by he prevalence of neutrophil infiltration, Immunofluorescence vhether neutrophils degranulate and Immunofluorescent localization of elastase hus are not recognized by conventional was accomplished utilizing a method modiI&E staining, whether there is an associ- fied from that previously described for MBP
(20-22); the specificity of anti-MBP and the affinity purification of anti-MBP have been reported elsewhere (21, 22). Four serial sections, 6 J.1m thick, were cut and affixed to microscope slides with LePage's glue (LePage's Ltd., Bramalea, Ontario, Canada). After deparaffinization with xylene and rehydration, the sections were partially digested with 0.1 % trypsin containing 0.1070 CaCI] (pH, 7.8) for 1 hat 37° C. After washing in water, the first and second slides were placed in 10070 normal rabbit serum (Lot 81579; Pel-Freeze Biologicals, Rogers, AR), and the third and fourth slides were placed in 100/0 normal goat
(Received in original form April 3, 1989 and in revised form August 1, 1989) 1 From the Allergic Diseases Research Laboratory and the Department of Immunology, Mayo Clinic and Mayo Foundation, Rochester, and the Department of Microbiology, University of Minnesota, Minneapolis, Minnesota. 2 Supported by Grants AI-15231 and HL-37615 from the National Institutes of Health, and from the Mayo Foundation. 3 Correspondence and requests for reprints should be addressed to Gerald J. Gleich, M.D., Chairman, Department of Immunology, Mayo Clinic, Rochester, MN 55905. 4 Visiting Scientist at the Division of Allergic Diseases and Internal Medicine. Present address: Department of Pediatrics, Mie National Hospital, Mie-ken 51401, Japan; supported by a grant from the Rotary Foundation of Rotary International.
689
690
FWISAWA, KEPHART, GRAY, AND GLEICH
TABLE 1 PATIENTS WITH ASTHMA'
Patient No.
Duration of Asthma
Age
(yry
Sex
(yry
3
59 46 70
4
68
F F F F
10 40 19 18
1 2
Systemic Glucocorticoid Therapy
+ + +
Time Elapsing between Death and Autopsy (h) 19
3 6.5 11
, Three tissue blocks from each patient were studied . All patients died of asthma. Patient 1 died 35 min after hospnal admission; Patients 2, 3, and 4 were dead on arrival at the hospital.
serum (Lot 2978; Pel-Freeze Biologicals). All slides were incubated overnight at 4 0 C, and the next day they were washed in Dulbecco's phosphate-buffered saline (PBS). The first section was overlaid with protein-A-purified normal sheep IgG (NSIgG) (Lot I06F-8806; Sigma Chemical Co., St. Louis, MO) as the negative control for antielastase, the second section was overlaid with affinity-chromatography-purified sheep antibody to human neutrophil elastase (vide infra), the third section was overlaid with affinity-chromatographypurified rabbit antibody to MBP, and the fourth section was overlaid with protein-Apurified normal rabbit IgG (NRIgG) as the negativecontrol for anti-MBP. The slideswere placed in a moist chamber for 30 min at 370 C, washed with PBS, stained with 1% chromotrope 2R for 30 min at room temperature, and washed again. The first two slides were then overlaid with a fluorescein-conjugated IgG fraction of rabbit antisheep IgG (Lot 26481; Cooper Biomedical, West Chester, PA), and the last two slides were overlaid with a fluorescein-conjugated IgG fraction of goat antirabbit IgG (Lot 24682; Cooper Biomedical). After an additional incubation for 30 min at 37 0 C, the slides were washed, mounted with
a 10% PBS:90OJo glycerol solution containing p-phenylenediamine (Aldrich Chemical Co., Milwaukee, WI) (23), coverslipped, and sealed with clear nail polish. Slides wereexamined with a Zeiss standard microscope (Carl Zeiss Inc., Oberkochen, FRG) equipped with Zeiss IV FL vertical illumination for epifluorescence and a fluorescein filter system (48771O:Zeiss).After photographing the immunofluorescence findings, coverslips were removed, the tissues were counterstained with H&E, and the identical fields were rephotographed under transmitted light.
Validation of Neutrophil Elastase Localization by Immunofluorescence Sheep antiserum to human neutrophil elastase (Lot AHP 051) was purchased from Serotec (Kidlington, Oxford, UK) and first tested for its ability to specifically stain neutrophils on cytocentrifuge preparations of buffy coat cells from normal human peripheral blood by immunofluorescence, using the same procedure as described previously for anti-MBP (21). Second, to determine whether the epitopes of elastase survived formalin-fixation and paraffin-embedding, wetested the antielastase
on lung tissue specimens from patients with pneumonia and on sinus tissue specimens from patients with chronic sinusitis. Third, purified antibody to elastase was obtained by affinity chromatography. Purified human neutrophil elastase (vide infra) was coupled to cyanogea-bromide-activated Sepharose 4B (Lot 126FOO58; Sigma) using a procedure described previously (22). The sheep antiserum to human neutrophil elastase was applied to the beads and mixed overnight at 4 0 C. The beads were packed into a column and washed with PBS and with 0.17 M borate containing 0.5 M NaCI (pH, 8.0) to remove nonspecifically bound components. The antielastase was eluted with 0.05 M glycine-HCI(pH, 3.0), and peak fractions were pooled. Equal concentrations of purified antielastase and NSIgG (50 ug/ml), as determined by absorbance at 280 nm, wereused in the immunofluorescence assay.
Purification of Human Neutrophil Elastase Human neutrophil elastase was purified from an extract of cytoplasmic granules from normal human neutrophils by two column chromatography steps as described previously (24, 25). Briefly,proteins of the extract werebound to Matrex-Gel Orange A (Amicon Corp., Danvers, MA) and fractionated by elution with a gradient from 0.1 to 1.6 M NaCI in citrate-phosphate buffer at pH 5.6. The elastase peak eluted between 0.35 and 0.68 M NaCI and was identified by hydrolysis of the chromogenic peptide Sue-ala-ala-alaNPhN0 2 , an elastase-specific substrate (26). Fractions containing elastase were diluted into 0.05 M NaCl, applied to a column of BioRex 70 (Bio-Rad Laboratories, Richmond, CAl, and eluted with a gradient of 0.04 to 1.0M NaCI in citrate-phosphate buffer at pH
Fig. 1. Localization of elastase in a cytocentrifuge preparation of human peripheral blood bufly coat cells. A (left). Cells stained with crude antielastase. B (right). The same cells counterstained with Wright's stain. The results show that the fluorescent polymorphonuclear cells in A are neutrophils; the long black arrows in B indicate the same cells shown by white arrows in A. Lymphocytes (short black arrow) and eosinophils (open black arrows) in B are unstained in A. One monocyte (curved arrows in A and B) stains faintly for elastase. Magnification in A and B: x1 ,428.
691
NEUTROPHIL AND ALLERGIC INFLAMMATION
Fig. 2. Localization of neutrophil elastase in a lung tissue autopsy obtained 4.5 h after death from a patient with pneumon ia. A (/eft). Section stained with affinity·purified antielastase. B (right) . Same section as in A, counterstained with H&E. The black arrows in B indicate the same cells shown by white arrows in A; note that the positively staining cells in A are polymorphonuclear when counterstained with H&E. Note the coarse granulation of the neutrophil's cytoplasm after staining with antielastase in A. The NSlgG·stained serial section was negative (results not shown). Magnification in A and B: x l, 428.
7.0. Elastase (overall yield, 340/0) eluted in a sharp peak at 0.27 M NaC! . By sodium dodecyl sulfate polyacrylamide gel electrophoresis the purified enzyme gave a major band and two minor bands slightly above and below the 29,000 molecular weight carbonic anhydrase marker, and it hydrolyzed 3H-elastin (27) at a rate of 2.6 mg/h/mg of elastase.
graded on a scale of zero to 3. Specimens with no or occasional extracellular staining were scored as zero. If there werescattered foci containing faint staining, the specimen wasscored as 1. Specimens with scattered foci containing marked staining were scored as 2, and 3 was assigned to specimens showing intense deposition throughout the lesion.
Evaluation of Immunofluorescence
Results
The evaluation was made by two of the writers independently (TF and GMK). Cellular infiltration of neutrophils or eosinophils, as revealed by cellular staining, was scored on a scale from zero to 3. A score of zero was assigned when no or occasional neutrophils or eosinophils were present. If a few cells were seen in the lesion, it was scored as 1. A score of 2 was given when many neutrophils or eosinophils werescattered throughout the lesion, and 3 was assigned to specimens demonstrating dense infiltration. The degree of extracellular deposition of MBP and elastase was also
Immunofluorescent Staining for Neutrophil Elastase Analyses of the reactivity of sheep antielastase were initially performed by immunofluorescence on buffy coat cells fixed with 100% methanol. Here, the cells showing bright cytoplasmic staining for elastase (figure lA) were identi fied as neutrophils by counterstaining the same slides with Wright's stain (figure lB). Note that eosinophils are not stained by antielastase. Analyses of 143 mono-
nuclear cells for elastase staining revealed faint staining of seven of 70 monocytes and two of 73 lymphocytes; figure 1 shows faint staining of a monocyte. In contrast, the buffy coat cellsdid not stain with normal sheep serum (results not shown). Second, the reactivity of antielastase was tested on formalin-fixed, paraffin-embedded lung tissue specimens from patients with pneumonia. Here, not only neutrophils but also secretory glands in the airways stained with the antisera (results not shown). Therefore , third, antielastase was purified by affinity chromatography on a column of solid-phase elastase. Testsof the affinity-purified antielastase on buffy coat cells, lung tissue, and sinus tissue specimens showed that only neutrophils stained brightly. A section of lung from a patient with pneumonia is shown in figure 2; cells staining with antielastase are concentrated in the
TABLE 2 IMMUNOFLUORESCENCE FINDINGS IN PATIENTS WITH ASTHMA
Patient No. 1
2 3 4
Extracellular Elastase
Neutrophilia B
M
A
B
M
A
2t 0/1 0 0
3 2 0 0
1
0 0 0
2 1 0 0
2 2 0 0
0 0 0 0
Coexistence of Elastase and Epithelial Damage'
++
Eosinophilia
Extracellular MBP
B
M
A
B
M
A
Coexistence of MBP and Epithelial Damage'
1
1 1 2 0
0 1 1 1
1 2 1 2
1 1 2 1
0 0 0 0
+ ++ +
1 2 2
Deffnltion of abbreviations: B • bronch ial wall; M • mucus in bronchial lumen; A = alveoli . • Coexistence ot epithelial damage and elastase or MBP deposltton was scored on a scale from - to + + . A score of - was assigned when no coexistence was observed . Specimens wnh scattered foci showing coexistence were scored + . A score of + + was given to specimens showing extensive coexistence throughout the lesion. The pathology was semiquanlitatively graded (see METHOOS) by two observers . t Two numbers represe nt differing evaluations by the two observers ; single numbers indicate agreement by the two.
692
FWISAWA, KEPHART, GRAY, AND GLEICH
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Fig. 3. Localization of neutrophil elastase and eosinophil MBP in lung from a patient with asthma (Patient 1). A (top, left). Section stained with anti·MBP. C and E (left, middle and bottom) . Section stained with antielastase. B, 0, and F (right). The same sections as in A, C, and E, respectively, counterstained with H&E. Only scattered eosinophils and minimal extracellular MBP deposition are present in the anti·MBP·stained section (A and B). In contrast, note the intense neutrophil infiltration in a serial section stained with antielastase (C and 0). E and F are high power views of C and 0, respectively, and show the coexistence of diffuse extracellular elastase deposition and damaged epithelium. Elastase deposition is also evident in areas of the mucous plug (arrow in E) and in areas beneath the membrane. The arrow in F identifies a Curschmann's spiral. Magnification in A to 0 : x571; magnification in E and F: x 1,428.
693
NEUTROPHIL AND ALLERGIC INFLAMMATION
TABLE 3 IMMUNOFLUORESCENCE FINDINGS IN PATIENTS WITH CHRONIC SINUSITIS*
Patient No. 5 6 7 8 9 10 11 12 13 14
Neutrophilia
Extracellular Elastase
Coexistence of Elastase and Epithelial Damage t
0
0* 2 0 1 1
++
2 0 0
0
1/0 0
0 1 0 1 0
1 1/0 0
Eosinophilia 2/1 3/2 2 3/2 2 3 3/2 2/1 2 3
Extracellular MBP 2/3 2
2 2 2 2/1 3 2 2 2
Coexistence of MBP and Epithelial Damage t
++ ++ ++ + +/+ + +/+ ++ ++ ++ ++ +
* These cases of chronic sinusitis have been reported elsewhere (30). Patients 5 to 14 correspond to Patients 12, 8, 2,11,7,13,9,5,10, and 6, respectively, in reference 30. t Coexistence of epithelial damage and elastase or major basic protein (MBP) deposition was scored on a scale from - to + +. A score of - was assigned when no coexistence was observed. Specimens with scattered foci of coexistence were scored +. A score of + + was given to specimens showing extensive coexistence throughout the lesion. The pathology was sernlquantitatlvely graded (see METHODS) by two observers. :I: Two numbers represent differing evaluations by the two observers; single numbers indicate agreement by the two.
airway lumen, have polymorphonuclear nuclei, and contain discrete cytoplasmic granules. Finally, NSlgG did not stain tissues (results not shown).
Lung Tissue from Patients with Fatal Asthma The H&E stained lung tissue specimens from all the patients with fatal asthma showed the characteristic findings of asthma (28), namely, thickened basement membranes, mucous plugs in the airway with or without Creola bodies, various degrees of bronchial epithelial damage (including epithelial shedding and squamous cell metaplasia), and smooth muscle hyperplasia. Mononuclear cell infiltration was also seen in varying degrees. None of the specimens demonstrated other pathologic lesions such as pneumonia or emphysema. Sections stained with antielastase by immunofluorescence showed evidence of extracellular deposition in two of the four
patients (table 2). In Patient 1, there was striking neutrophil infiltration and extracellular elastase deposition in the bronchial wall, peribronchial tissue, and, particularly, in the bronchial mucus. It can be seen in figure 3 that the region with prominent extracellular elastase deposition also displayed striking damage characterized by loss of epithelium and pyknosis of remaining epithelial cells. In contrast, deposition of MBP was not as prominent as elastase deposition. In Patient 2, extracellular elastase was deposited in the bronchial mucus (2 +) and in the bronchial wall (1+), but it was not localized in any regions of epithelial damage. Extracellular MBP, however, was found on areas of damaged epithelium. In Patients 3 and 4, there were only occasional foci of intracellular and extracellular elastase deposition, but extracellular MBP and eosinophil infiltration were observed in the bronchial wall, where extensive epithelial damage was evident.
Neither extracellular elastase nor MBP was observed in the alveoli of any tissue.
Specimens from Patients with Sinusitis and Nasal Polyps Marked eosinophil infiltration and extracellular deposition of MBP were observed in all 10 patients with chronic sinusitis and in all 10 patients with nasal polyposis by immunofluorescence (tables 3 and 4). Representative photomicrographs from Patient 16 with nasal polyposis are shown in figure 4. Marked extracellular deposition of MBP (figure 4A) coincided with the presence of epithelial damage (figure 4B). Note the denuded epithelial layer and only a few remaining basal cells in figure 4B. In this case, only a few elastase-positive cells were identified (figure 4C). Marked elastase deposition (2+) was seen in only one of 10 patients with sinusitis and in one of 10 patients with nasal polyposis. The coexistence of striking, focal elastase deposi-
TABLE 4 IMMUNOFLUORESCENCE FINDINGS IN PATIENTS WITH NASAL POLYPS
Patient No. 15 16 17 18 19 20 21 22 23 24
Neutrophilia
Extracellular Elastase
1t 1 3 1 1 0 0 0 2 0
1 1 2/1 1 0 0 0 0 1 0
Coexistence of Elastase and Epithelial Damage *
+/+ +/+
++
Eosinophilia
Extracellular MBP
2 3 2/1 3 3 3 3 3/2 2/3 3/2
2 3/2 1 3 2 3 2 2/1 3 1
Coexistence of MBP and Epithelial Damage *
++ ++ + ++ ++ ++ ++ + +/+ ++
* Coexistence of epithelial damage and elastase or major basic protein (MBP) deposition was scored on a scale from - to + + . A score of - was assigned when no coexistence was observed. Specimens with scattered foci of coexistence were scored +. A score of + + was given to specimens showing extensive coexistence throughout the lesion. The pathology was semlquantitatively graded (see METHODS) by two observers. t Two numbers represent differing evaluations by the two observers; single numbers indicate agreement by the two.
694
FWISAWA. KEPHART, GRAY, AND GLEICH
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Fig . 4. Localization of MBP and elastase in a nasal polyp biopsy from Patient 16. A (top, leff). Section stained with anti·MBP. B (top, right). Same section as in A counter, stained with H&E. C (bottom . left). Serial section to A stained with antielastase. Note the striking eosinophil infiltration and extracellular MBP deposition beneath the basement membrane in association with epithelial damage (A and B). In con, trast, only minimal elastase staining is present (C). Magnification in A to C: x 571.
tion and epithelial damage in Patient 6 with chronic sinusitis and asthma is shown in figure 5. Note that in the area of epithelial damage, intact neutrophils could not be identified by H&E, suggesting that neutrophil degranulation had occurred. The presence of extracellular elastase deposition is shown in figure 6A, and striking neutrophil infiltration is shown in figure 6B beneath largely intact respiratory epithelium of Patient 17 with nasal polyps. Interestingly, the extracellular elastase deposition is concentrated just beneath the respiratory epithelium, whereas intact neutrophils are present in the deeper stroma. In other areas of this specimen, extracellular elastase deposition coincided with epithelial damage (table 4). As can be seen from tables 3 and 4, there was no obvious relationship between the degree of infiltration by eosinophils and neutrophils.
Discussion
These results show that neutrophil granule elastase can be localized in formalinfixed paraffin-embedded tissue by immunofluorescence using essentially the same method employed for localization of MBP (20-22), Initial studies with buffy coat cells showed localization of elastase to neutrophils and its absence in eosinophils and mononuclear cells (figure 1).Staining of rnonocytes byantielastase was seen in only 10070 of cellseventhough the peripheral blood monocyte has been shown to contain a molecule immunochemically similar to neutrophil elastase (29). Preliminary analyses of formalinfixed tissue from patients with pneumonia showedstaining of neutrophils as well as bronchial glands. Therefore, the anti elastase serum was affinity-purified on a column of solid-phase elastase to remove the unwanted antibodies. The pu-
rified anti elastase stained peripheral blood neutrophils in a manner comparable to that shown in figure 1 and also stained neutrophils in formalin-fixed, paraffin-embedded tissue from a patient with pneumonia, as shown in figure 2; the ability of the crude anti elastase to stain bronchial glands was removed by affinity purification. In contrast to the results obtained with affinity-purified antielastase, NSIgG did not stain tissue from patients with pneumonia. Thus, these results indicate that this method permits staining of neutrophil elastase and suggest that this method is useful for detection of neutrophil infiltration and degranulation in inflammatory reactions, especially in situations where eosinophil infiltration is prominent and neutrophils might be overlooked or in situations where neutrophils might have degranulated. As stressed above, eosinophils lose
695
NEUTROPHIL AND ALLERGIC INFLAMMATION
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Fig. 5. Localizationofelastasein a sinus tissue biopsyfrom Patient6. A (left). Sectionstainedwith antielastase. B (right). Samesection as in A counterstainedwith H&E. Notethe coexistence of intense elastase deposition and epithelial denudation. Magnification in A and B: x 1,428.
their morphologic integrity in certain tissues (17-19, 22), and their involvement in these intlammatory reactions cannot be recognized by light microscopy after staining with H&E. Using the immunofluorescence assay for elastase, we have examined tissues from patients with fatal asthma, chronic sinusitis with asthma, and nasal polyps to determine whether neutrophil infiltration and degranulation commonly occur in these conditions. Because of the pathologic similarity of chronic sinusitis and nasal polyposis to asthma (28, 30, 31), we believe that these tissues can be included in the category of allergic inflammation. In previous studies, we have
shown that eosinophil granule proteins are toxic to respiratory epithelium (3234), and eosinophil degranulation (as judged by MBP deposition) occurs in areas of desquamation in bronchial asthma and chronic sinusitis (21, 30). These observations support the hypothesis that the eosinophil is an effector cell in allergic inflammation. Here, in general, we were impressed by the degree of eosinophil infiltration, by the frequency and intensity of extracellular MBP deposition, and by the coexistence of extracellular MBP deposition and epithelial damage (figure 4). Concerning localization of the neutrophil and neutrophil elastase, our results show that neutrophils are far less
prominent in the specimens studied (tables 2 to 4); marked neutrophil infiltration was seen in two of four patients with asthma, in one of 10 patients with sinusitis, and in two of 10 patients with nasal polyps . However, in Patients 1,6, 17, and 23, extracellular elastase deposition coexisted with respiratory epithelial damage (as illustrated in figures 3 and 5). Elastase, a neutral serine protease, is localized to the azurophilic or primary granules in neutrophils and plays an important role in host defense (35). Besides its beneficial function, it may also have various harmful effects and it has been implicated as an effector of tissue damage in emphysema (36-38) and other dis-
Fig. 6. Localizationof elastasein a nasal polyp biopsy from Patient 17. A (left). Section stained with antielastase. B (right). Same section as in A counterstainedwith H&E. The tissueis denselyinfiltratedwith intact neutrophils;the whitearrowin A and the black arrowin B identifyan areaof apparentdegranulation. Magnificationin A and B: x 1,428.
696
eases (39-42). In airways, elastase could alter tracheal mucociliary activity (43) and cause bronchial secretory cell metaplasia (37, 44). The association between the deposition of elastase and the occurrence of epithelial damage suggests that neutrophils injure respiratory tissue in allergic inflammation. The importance of neutrophils in the pathogenesis of allergic inflammation in humans is under active study in several laboratories. Histologic examinations of the human cutaneous LPR provoked by intradermal injection of allergen or antiIgE have shown that both neutrophils and eosinophils contribute to the inflammatory response (2, 3). We have examined tissue from the cutaneous human LPR for neutrophil degranulation using the same assay described here and have observed neutrophil infiltration and striking extracellular elastase deposition at 1 hand 8 h after challenge (Leiferman KM, Fujisawa T, Gray BH, Gleich, GJ, unpublished observations); eosinophil infiltration and MBP deposition occurred in these same tissues. These results suggest that neutrophils, as well as eo sinophils, are active participants in the allergic inflammation associated with the cutaneous LPR. In human respiratory tracts of patients with asthma, analyses of cells by bronchoalveolar lavage have shown an increase of eosinophils and, in some instances, of neutrophils in the lavage fluid (15, 16, 45). Overall, neutrophils were relatively scant in the tissues showing chronic allergic inflammation examined here. How can one reconcile this finding with the occasional association of elastase deposition and asthma? First, the pathogenesis of allergic inflammation may be heterogeneous. In a recent study of localization of MBP in patients with fatal bronchial asthma, some specimens showing striking epithelial damage in the absence of eosinophil degranulation have been found (46). Thus, the pathologic features of allergic inflammation may not be uniform in terms of cellular infiltration. Second, neutrophil infiltration in chronic allergic disease may be evanescent. Histologic studies of the LPR in experimental animals have shown that neutrophils predominate at 6 to 8 h after challenge, and after this are replaced by eosinophils (10, 11). If the neutrophil infiltrates and degranulates in the acute phase of allergic inflammation, then our specimens from patients with chronic allergic inflammation might not contain neutrophils and their granules. Last, the
FWISAWA, KEPHART, GRAY, AND GLEICH
possibility that neutrophils might have appeared because of infection, as suspected in Patient 1, must be considered. In a prior study, we enumerated neutrophils in nasal polyps and found a highly significant positive correlation (r = +0.9; P < 0.001) between tissue neutrophilia and the number of bacteria cultured from the tissue (47). Acknowledgment The writers thank Dr. Thomas Colby for his valuable suggestions, Mrs. Cheryl Adolphson for editorial assistance, and Mrs. Linda H. Arneson for secretarial assistance. References 1. Henson PM. Neutrophils in acute allergic reactions. In: Lessof MH, Lee TH, Kemeny DM, eds. Allergy: an international textbook. New York: John Wiley & Sons, 1987; 157-68. 2. Dolovich J, Hargreave FE, Chalmers R, Shier KJ, Gauldie J, Bienenstock J. Late cutaneous allergic responses in isolated Igfi-dependent reactions. J Allergy Clin Immunol 1973; 52:38-46. 3. Solley GO, Gleich OJ, Jordon RE, Schroeter AL. The late phase of the immediate wheal and flare skin reaction. Its dependence upon IgE antibodies. J Clin Invest 1976; 58:408-20. 4. Nagy L, LeeTH, Kay AB. Neutrophil chemotactic activity in antigen-induced late asthmatic reactions. N Engl J Med 1982; 306:497-501. 5. Lee TH, Nagakura T, Papageorgiou N, likura Y, Kay AB. Exercise-induced late asthmatic reactions with neutrophil chemotactic activity. N Engl J Med 1983; 308:1502-05. 6. Oertel HL, Kaliner M. The biologic activity of mast cell granules. III. Purification of inflammatory factors of anaphylaxis (IF-A) responsible for causing late phase reactions. J Immunol 1981; 127:1398-1402. 7. Lemanske RF Jr, Guthman DA, Oertel H, Barr L, Kaliner M. The biologic activity of mast cell granules. VI. The effect of vinblastine-induced neutropenia on rat cutaneous late phase reactions. J Immunol 1983; 130:2837-42. 8. Lemanske RF Jr, Guthman DA, Kaliner M. The biologic activity of mast cell granules. VII. The effect of anti-neutrophil antibody-induced neutropenia on rat cutaneous late phase reactions. J Immunol 1983; 131:929-33. 9. Murphy KR, Wilson MC, Irvin CG, et ale The requirement for polymorphonuclear leukocytes in the late asthmatic response and heightened airway reactivity in an animal model. Am Rev Respir Dis 1986; 134:62-8. 10. Behrens BL, Clark RAF, Presley DM, Graves JP, Feldsien DC, Larsen GL. Comparison of the evolving histopathology of early and late cutaneous and asthmatic responses in rabbits after a single antigen challenge. Lab Invest 1987; 56:101-13. 11. Iijima H, Ishii M, Yamauchi K, et ale Bronchoalveolar lavage and histologic characterization of late asthmatic response in guinea pigs. Am Rev Respir Dis 1987; 136:922-9. 12. Dunn CJ, Elliott GA, Oostveen JA, Richards 1M. Development of a prolonged eosinophil-rich inflammatory leukocyte infiltration in the guineapig asthmatic response to ovalbumin inhalation. Am Rev Respir Dis 1988; 137:541-7. 13. Frigas E, Gleich GJ. The eosinophil and the pathophysiology of asthma. J Allergy Clin Immunol 1986; 77:527-37.
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