Clin. exp. Immunol. (1982) 49, 247-255.

Auto-oxidative damage in Behset's disease-endothelialcell damage following the elevated oxygen radicals

generated by stimulated neutrophils

Y. NIWA,* S. MIYAKEt T. SAKANE,* M. SHINGUI & M. YOKOYAMA§ * NiwaInstitute for Immunology, tDepartment ofInternal Medicine, Kochi Prefectual Hospital;

IDepartment ofInternal Medicine, Institutefor Hot Spring, Kyushu University and §Department ofPathology and Microbiology, Illinois Medical Center, University ofIllinois, USA

(Acceptedfor publication 29 January 1982)

SUMMARY

The functions of phagocytes are enhanced in patients with Behget's disease, therefore, weinvestigated the neutrophil-derived oxygen intermediates (01) and lysosomal enzymesfrom 17 patients receiving glucocorticosteroids (steroids) and colchicine. Culturedendothelial cells were incubated with neutrophils to assess tissue injury. In cases of thecomplete type, in the active stage of the disease, 01 production was markedly increased.The other patients showed significantly higher OI and higher lysosomal enzyme levelsthan patients with other diseases (controls) receiving drug therapy. Cytotoxicity testsshowed that the 5'Cr release was also significantly higher. The destruction ofdesmosomesand cell deformation were demonstrated electron microscopically. The simultaneousaddition of superoxide dismutase and catalase in the cell culture decreased the 51Cr releaseto control levels. These findings suggest that neutrophils from patients with Behqet'sdisease generate high levels of OI, resulting in endothelial tissue damage.

INTRODUCTION

Non-lethally enhanced oxygen intermediates (01; 0 2,H202, OH-, 102) produced by stimulatedneutrophils (Fridovich, 1976; Salin & McCord, 1975; McCord & Fridovich, 1978) induceauto-oxidative damage such as tissue injury (Sacks et al., 1978; Malawista, 1975), haemolyticanaemia (Boxer et al., 1979; Spielberg, Boxer & Corash, 1979; Necheles, Maldonado &Barquet-Chediak, 1969), decreased lysosomal enzyme release, (Skosey et al., 1981), shortenedneutrophil viability (Salin & McCord, 1975), lymphocytic hyporesponse to mitogens (Metzger,Hoffeld & Oppenheim, 1980; Nishida, Tanimoto & Akaoka, 1981) and impaired bactericidalfunction (Murray & Cohn, 1979; Murray et al., 1979).

The association of Behqet's disease with potentiated functions of phagocytes has assumedincreasing importance in view ofthe recent documentations ofenhanced chemotaxis (Matsumura&Mizushima, 1975; Abdulla & Lehner, 1979) amplified phagocytosis (Takabatake, 1975; Takeda,1979) and uptake of immunoglobulins (Shimizu, Katsuta & Oshima, 1965) and other phagocytes(Noguchi et al., 1979) by neutrophils. Furthermore, colchicine, a phagocytosis attenuating agent isclinically effective for treating this disease (Mizushima et al., 1977).

Behget's disease manifests itself symptomatically by tissue injury (Lehner, 1969; O'Duffy,

Correspondence: Dr Y. Niwa MD, PhD, Niwa Institute for Immunology, 4-4 Asahimachi, Tosashimizu,Kochi-ken 787-03, Japan.

0009-9104/82/0700-0247S02.00 C) 1982 Blackwell Scientific Publications

247

Carney & Deodhar, 1971) rather than by lymphocytic abnormalities as are seen in systemic lupuserythematosus. Among the clinical features of Behret's disease, ocular uveitic lesions, primarilyinduced by phlebitis, are the most important (Sanders, 1979).

We studied the capacity of patient and normal neutrophils, stimulated with opsonized zymosan,to produce 01 (0 , H202, OH*) and examined their chemiluminescence and the lysosomal enzyme(LE) levels (f-glucuronidase, lysosome). In addition, the endothelial cell damage induced byincreased OI generation was studied by the cytotoxicity test and electron microscopic investiga-tions, using cultured endothelial cells from human umbilical vein.

MATERIALS AND METHODS

Seventeen Japanese Behqet's disease patients who had received colchicine (1-2 mg/day) andprednisolone (10-20 mg/day) for 2-10 consecutive months were classified into complete (involve-ment of four major sites) and incomplete (three major sites, or ocular lesions and one major site)types (Lehner & Barnes, 1979). Each group was further divided into the active and inactive stage(Table 1). Clinical activity was assessed at the time of blood sampling; patients in the active stagemanifested a worsening of the clinical symptoms, a CRP titre above 4 mm, leucocyte counts above104/ml, an erythrocyte sedimentation rate exceeding 50 mm/l hr, and increased complementcomponents and CH50. The controls were 26 patients with dermatosis or asthma; 13 had beentreated with prednisolone (10-20 mg/day) and colchicine (1-2 mg/day) for 2 months to 3 years,consecutively; the other 13 had received no drug therapy. Twelve normal adults served as thehealthy controls (Table 1).

Table 1. Patients and controls

Classification No. cases Sex Ages Drugs(years)

complete type and in active stage 5 4 27-39 +I 31

complete type and in inactive stage {2 33,37 +1 Y 50

Behcet's disease

incomplete type and in active stage 42 +34,40

2 Y 36,52

incomplete type and in inactive stage{ 2 C 38,52

+3 Y 39-59

psoriasis vulgaris 4 J 42-56 +dermatosis

pustulosis palmaris 4 1 28-45 + druget plantaris 1 3 j 2 patients

asthma 5{ 44-58 +

Controls

dermatosis 9 26-56 -

2no-drug

asthma 4 C 42-58 - patients6 S 17-45

healthy individuals 12 16 Y 22-49

248 Y. Niwa et al.

Tissue damage in Behfet's diseaseNeutrophil preparation. Neutrophils (PMNs), isolated from the peripheral blood (Skosey et al.,

1973) were suspended in Krebs-Ringer phosphate buffer (KRP) (Cohen, 1957) containing glucose(5 mM) and gelatin (1 mg/ml) forOI and lysosomal enzyme generation assay; they were suspended inHank's solution (Nissui Pharmaceutical Co., Tokyo, Japan) for chemiluminescence assay, and inRPMI medium (GIBCO, Grand Island, New York, USA) for the cytotoxicity test.

Olgeneration assays. 0 formation was determined according to Johnston & Lehmeyer (1976).Ferricytochrome C (type III, Sigma, St Louis, Missouri, USA) reduction by 02 produced from4 x 106 PMNs stimulated with I mg/ml opsonized zymosan (Sigma) (Cohen, 1957) was assayed bymeasuring the absorbance at 550 nm (Johnston & Lehmeyer, 1976; Massey, 1959).

H202 generation was determined by using 2 5 x 106 PMNs stimulated with 1 mg/ml opsonizedzymosan, 0 1 ml of 50 mm scopoletin (Sigma) in KRP and 0 1 ml of 1 mg/ml horseradish peroxidase(type II, Sigma) (Johnston & Lehmeyer, 1976; Root & Metcalf, 1979): the rate of decrease influorescent intensity of the scopoletin within 30 min was quantitated in a fluorescencespectrophotometer (Hitachi Co. Ltd., Tokyo, Japan).OH was quantitated by the amount of ethylene gas formed from a-keto-methiol butylic acid

(KMB) (Sigma) and by the PMN-generated OH- (Klebanoff & Rosen, 1978); 2 x 106 PMNsstimulated with 1 mg/ml opsonized zymosan and 1 mm KMB were used and the total amount ofOH- gas formed at 10, 20 and 30 min was determined on a gas-chromatograph (Hitachi).

Chemiluminescence was measured in a scintillation spectrometer (Packard, Illinois, USA)according to a modification of the method of Allen & Loose (1976). Five million PMNs in 3 mlcolourless Hank's solution containing 1 mg/ml opsonized zymosan, but no luminol, were incubatedunder shaded conditions. Chemiluminescence was monitored on the spectrometer, which wasoperated in the out-of-coincidence summation mode.

Lysosomal enzyme assay. The activity of f-glucuronidase was determined as has been described;phenolphthalein glucuronide was the substrate (Baehner, Karnovsky & Karnovsky, 1969).Lysozyme activity was measured as described by Wright & Malawista (1972), using theturbidimetric method and Micrococcus lysodeikticus. LDH activity, measured to estimatethe leakage of cytoplasmic enzymes resulting from non-specific cell damage, was alsodetermined (Bergmeyer, Bernt & Hess, 1963). The enzyme activity released by PMNs wasexpressed as a percentage of total enzyme activity released from an equal number of PMNs bysonification.

In each 01 assay system, PMNs not stimulated by opsonized zymosan were also tested.Preparation ofendothelial cells. As described by Jaffe, Nackman & Becker (1973), the umbilical

vein obtained from the placenta was cannulated with a syringe adapter, infused with collagenaseand trypsin (GIBCO), clamped shut, and incubated for 40 min at 37°C under 5% CO2. The cells wereflushed from the cord, washed, sedimented, resuspended in culture medium containing RPMImedium, fetal calf serum, L-glutamine and antibiotics, and incubated at 37°C under 5% CO2.Cultures were checked for integrity after approximately 1 week using an electron microscope, theperoxidase reaction and the reaction with fluorescein-conjugated rabbit antisera to human FactorVIII (Johnston & Erdos, 1977; Hoyer, Santos & Hoyer, 1976; Jaffe, 1976).

Determination of endothelial cell damage. Cultured cells (3 x 105), labelled with 5 pCi ofradiolabelled sodium chromate (= 50 m Ci/mg, E. R. Squibb & Sons, Inc., Princeton, New Jersey,USA) and 3 x 106 PMNs/ml RPMI medium were incubated for 90 min at 37°C with or without 1mg/ml opsonized zymosan. Four patients manifesting markedly high 01 production and threehealthy controls were the PMN donors. The control for spontaneous 5"Cr release consisted of alabelled cell culture that had been treated as described above before the addition of the PMNmedium. 51Cr levels were assayed in a gamma scintillation spectrometer (Packard). In someexperiments, 100 units/ml superoxide dismutase (SOD) and 400 units/ml catalase were added to themedium to ascertain the specific effect of 01 on the elevated 51Cr release.

After incubation in the presence of PMNs, endothelial cell preparations were made; these wereexamined under an electron microscope (HU-IID and HS-9; Hitachi) according to the method ofKomura et al. (1980).

Triplicate assays were performed simultaneously for each experiment and the results wereexpressed as mean + s.e. The statistical significance was ascertained by the Student's t-test.

249

RESULTS

In our study, there were four groups: group 1 consisted of 17 Behqet's disease patients (Behqet'spatients), group 2 of 13 patients with dermatosis or asthma who were on drug therapy (drugpatients), group 3 of 13 dermatosis or asthma patients who received no drug (no-drug patients),group 4 was made up of 12 healthy individuals. Groups 2-4 served as the controls.

OI and enzyme generation by zymosan-stimulated PMNsSimilar results were obtained for no-drug patients and healthy controls; none of the no-drugpatients manifested 01 and enzyme levels lower than healthy controls (data not shown). On theother hand, OI (P < 0 01) and enzyme (0l0I <P< 0-05) values were significantly lower when PMNsfrom drug patients were stimulated with zymosan (Fig. 1).

________0'2 0 4 0 6 0-8 1-0 nmot/min/4 x l0& PMN

_0 | |2 0-020

(Ferricytochrome C F= 0-023

reduction)10-0202 4 6 8 10 pmotxlO /min/2.5xO6 PMN

_~~~~~~~~~~~1~-0-912

H202 _I

0-07

5 10 5 20 25 pmoLx12/2x106 PMN

OH 0-7

(Ethylene formation), 0.1

__________I 20 30 40 5Qc.p.m.x104/5x 106 PMNl*2

Chemiluminescence i-41-4

i_.2

Fig. 1. Oxygen intermediates generated by PMNs with stimulation of zymosan. U =complete type in activestage; 0=complete type in inactive stage; incomplete type in active stage; incomplete type in inactive stage;0 = diseased control groups receiving drugs; H = healthy controls.

Increases in O2, H202 and chemiluminescence produced by zymosan stimulated PMNs were

statistically significant (P<0-01) and OH- generation was markedly (P<0001) elevated in

Behqet's patients of the complete type in an active stage compared to healthy controls. This increasewas even more significant compared to the drug patients (O , H202, chemiluminescence: P < 0-001,OH *: P < 0 00001) (Fig. 1). Lysosomal enzyme release by zymosan stimulated PMNs from the aboveBehset's patients was also increased compared to the healthy controls (001 <P< 0 05) and to thedrug patients (P<0 01) (Fig. 2).

In the other Behqet's disease patients (complete type in inactive stage; incomplete type in bothactive and inactive stages), although three showed OI and enzyme levels as high as those of patientswith the complete type in the active stage (data not shown), mean 01 levels (except for OH *) werenot significantly different from the healthy controls (O , H202, chemiluminescence: P> 0 05, OH*:0-0 <P <0-05). They were, however, elevated over those in the drug patients (01, H202,chemiluminescence: 0 01 <cP<0 05, OH*: P<0-01) (Fig. 1).

The lysozyme and f3-glucuronidase levels released by the stimulated PMNs from the latter groupof Behqet's disease patients was not significantly higher than in the healthy controls (P > 0 05), but

did differ from the levels noted in the drug patients (0 01 < P < 0 05) (Fig. 2). In general, the degreeof increase in lysosomal enzymes was considerably lower than in OI (Figs 1 & 2). The lack of

significant LDH increase (Fig. 2) indicates that our cell treatment induced neither non-specificpermeability nor cell damage.

Y. Niwa et al.250

Tissue damage in Behfet's diseaseEnzymes 10 20 30%*

LysozymeF--- 1

8

MIOR 1. 30%*._~~~~~

.

B-glucuronidase0.45

0-553Q% *

L

LDH =10.1009 -

251

Fig. 2. Lysosomal enzymes and LDH released from stimulated PMNs with opsonized zymosan. For Key see Fig.1. (*expressed as percentage of total enzyme activity).

OI generation in the abscence ofPMN stimulationWhen OI generation by unstimulated PMNs was assayed, similar findings to those made withstimulated PMNs were obtained. There was a statistically significant difference between OIproduction by unstimulated PMNs from drug patients and healthy controls (0-01 <P <0-05).Patients with Behqet's disease of the complete type and in the active stage showed significantlyhigher OI production than the healthy controls (0-01 < P < 0-05); 01 generation in the former groupwas also significantly higher than in the drug patients (P < 0-01) (Fig. 3).

Cytotoxicity test5'Cr release from endothelial cells incubated with zymosan stimulated PMNs from four Behqet'spatients of the complete type with active disease (showing highly increased 01 production) was

markedly elevated (P <0-01) compared to controls (5tCr released from endothelial cells incubatedby themselves). This increase was greater (0-01 < P < 0-05) than when labelled endothelial cells wereincubated with zymosan-stimulated PMNs from healthy controls, although, in the presence ofzymosan-stimulated PMNs from healthy controls, 5'Cr release from endothelial cells was higher(0-01 < P < 0-05) than in their absence (Table 2).

0-05 0-1 0-15 0-2 nmoL/min/4xl06 PMN'0.006

0_ __ 0-005(Ferricytochrome C 0-002

reduction 0-0030-5 1.0 1i5 2 0 2-5 30 pmoLxi02/min/2-5x 106 PMN

_ G0045D Al 0B~~~~O025H202 010013

__0-022 pmoL x 102/2 x106 PMN. _ .. 0-09

OH _ i 010(Ethylene formation) 0-06

_ ^ 0 0 8~~~.0____________ 2 3 4 5 c.pm. x 104/5 x 6PMN

Chemilurrinescence WHO-12

Fig. 3. Oxygen intermediates generated by PMNs without stimulation of zymosan. For Key, see Fig. 1.

252 Y. Niwa et al.Table 2. Stimulated PMNs induced 5tCr release from endothelial cells and protection of cells by SOD andcatalase

Medium of EndothelialCell Cultures N 5ICr Release* Pt Added SOD and catalase:

PMNs from Pt+Zys+51Cr-cells 4 21 5+ 1o% <001 9.2+0.88%PMNs from normals+Zys+51Cr-cells 3 125 +088% 0 01 -005 8 5+0.72%PMNs from Pt+51Cr-cells 4 103±+0 68% >0-05 n.d.PMNs from normals+5lCr-cells 3 7-7+0 45% >0-05 n.d.5'Cr-cells alone 3 6 7+022% n.d.

* 51Cr release is expressed as mean percentage release of 5ICr (mean+ s.e.m.).t Probability compared to per cent release of 5tCr in the medium of 5 Cr-cells (51Cr-labelled

endothelial cells) alone.$ 200 units/ml SOD and 400 units/ml catalase were added to the medium.n= Number of preparations; PMNs= polymorph neutrophils; Cells = endothelial cells; SOD=

superoxide dismutase; Zys=opsonized zymosan (Cohen, 1957); n.d. =not done.

Simultaneous addition of SOD and catalase reduced the 5'Cr release to near control levels,thereby indicating the specificity of the effect of 01 on endothelial cell damage.

Electron microscopic observationsObvious histological changes in both the cell membrane and the cytoplasm were noted. Some of thedesmosomes around adjacent cells were destroyed (Fig. 4a). Cellular changes, i.e. membrane defect,membrane deformity, cytoplasmic herniation and cytoplasmic vacuole formation were evident(Figs 4a,b,c). Some neutrophil adherence to the membrane ofendothelial cells was present (Fig. 4c).These findings were frequent upon the incubation of endothelial cells with opsonized zymosan-treated PMNs from Behcet's disease patients. Similar observations were occasionally made whenendothelial cells were incubated with zymosan-untreated PMNs from these patients. On the otherhand, the above changes were not noted when they were incubated with non-stimulated PMNs fromhealthy controls (Fig. 5).

DISCUSSION

Our results suggest that tissue injury in Behqet's disease may be related to the excessive generation ofOI by stimulated PMNs. In patients with the complete type of the disease and in the active stage,markedly increased 01 production was present. The cytotoxicity test revealed significantly elevated51Cr release; desmosome destruction and cell deformation were demonstrated electron microscopi-cally. Our findings suggest that PMNs from Behqet's disease patients generate high 01 levels,resulting, by oxidative damage, in endothelial cell injury.

PMN-mediated tissue injury has been thought to be a consequence of lysosomal degranulation(Cochrane, 1968; Weissman, 1972; Janoff, 1975), however, other factors from PMNs may play a

role in the inflammatory tissue damage (Cochrane, Unanue & Dixon, 1965). In Behqet's disease, 01seems to contribute to tissue damage more than lysosomal enzymes. We base this suggestion on our

observation that in Behcet's disease patients, 01 production was increased more than lysosomalenzyme production. Furthermore, the simultaneous presence of SOD and catalase duringincubation, reduced the 51Cr release to the control levels. It is interesting that umbilical veinendothelial cells acted as target cells for PMNs from patients in the cytotoxicity tests since phlebitisis usually found in the uveitis of the complete type of Behqet's disease as an associated ocular lesion(Sanders, 1979).

We also found that colchicine and steroids suppressed the generation of the 01 and lysosomalenzymes; this may explain the comparably mild elevation of 01 production noted in patients with

Tissue damage in Behfet's disease 253

L

Fig. 4. Histological findings under electron microscope following incubation of endothelial cells with zymosanstimulated PMNs from the patients. (a) Destruction of desmosome, vacuole formation and cytoplasmicherniation (arrow). Original x 5000. (b) Membrane deformity and defect. Original x 5000. (c) Adherence ofPMNs (N) to the endothelial cell (E), and membrane deformity and cell damage of the cell. Original x 5000.

Fig. 5. Histological findings in the endothelial cells following incubation with non-stimulated PMNs fromhealthy control. Original x 5000.

254 Y. Niwa et al.incomplete disease and patients with inactive disease. This observation is consistent with findings inpatients receiving steroids; they manifested decreased score in the NBT test (Miller & Kaplan, 1970;Chretien & Garagusi, 1972).

The elevated 01 production in Behget's disease, may be attributable to circulating immunecomplexes (ICs). Johnston & Lehmeyer (1976) demonstrated a marked increase in OI generationupon contacting PMNs with microfilter-bound ICs. The presence of ICs has been confirmed in40-70% of Behget's disease patients (Williams & Lehner, 1977; Levinsky & Lehner, 1978; Lehner,Alemeida & Levinsky, 1978). However, we also noted increased OI levels in patients withoutcirculating ICs (data not shown), and 01 levels generated by PMNs from healthy individuals uponstimulation with patient serum exceeded only in some cases, the levels produced upon stimulationwith normal serum (not shown); these rule out the possibility that enhanced 01 production wasascribable to ICs. Based on our findings, we suggest that tissue injury in Behcet's disease isattributable to the enhanced function ofthe patients' PMNs although further studies are required toexplain why PMNs in Behqet's disease have a potential for 01 generation.

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