Role of tumor necrosis factor-a inSneddon..Wilkinson subcorneal pustular dermatosisA model ofneutrophil priming in vivo

J. J. Grob, MD,a J. L. Mege, MD,b C. Capo, MD,b E. Jancovicci, MD,aJ. R. Fournerie, MD,a P. Bongrand, MD,b and J. J. Bonerandi, MDa Marseille, France

A patient with IgG-kappa-associated subcorneal pustular dermatosis (Sneddon-Wilkinsondisease) refractory to dapsone, etretinate, and plasma exchange was successfully treated withcorticosteroids. A study of neutrophils from both blood and pustules was carried out beforeand during treatment. Levels of tumor necrosis factor-a were measured in serum, pustules,content, and supernatant of monocytes. The results suggest that a hyperactivation of neu­trophils in the skin is due at least partly to excessive production of tumor necrosis factor-a.(J AM ACAD DERMATOL 1991;25:944-7.)

Subcorneal pustular dermatosis (SPD) was firstdescribed in 1956.1Its pathogenesis, particularly themigration of neutrophils toward the epidermis, ispoorly understood. There is growing evidence that anetwork of cytokines2 mediates an inflammatoryprocess in the skin. Tumor necrosis factor-a (TNF­a), synthetized and released by monocytes and alsoprobably by keratinocytes, is known to activate neu­trophils.3 We therefore studied TNF-a and neutro­phil function in a typical case ofSPD associated witha monoclonal IgO-kappa (IgO-x).

CASE REPORT

An 83-year-old man had a chronic relapsing eruptionofnumerous flaccid pustules. The lesions formed circinateand serpiginous patterns on the trunk and shoulders. Thepatient had no personal or family history of skin diseaseand was taking no medications. Several skin biopsy spec­imens showed subcomeal pustules. Plasma electrophore­sis revealed monoclonal IgG-K gammopathy. IgG level inserum was high (28.1 gm/L), but other classes of immu­noglobulins were decreased. Direct immunofluorescencewith anti-IgG, -lgA, -IgM, and -C3 showed linear depos­its of IgG and C3 at the level of the basement membrane.Indirect immunofluorescence findings were negative,however. Results ofculture of the pustules were also neg-

From Service de Dermato}ogie,· and Laboratoired'Immuno}ogie,b Ho­pital Sainte Marguerite.

Reprint requests: J. J. Grob, MD, Service de Dermatologie, Hopital SteMarguerite. 270 Bd de Ste Marguerite,BP29, 13277 Marseille,CEDEX 9 France,

16/4j'1J?J217

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ative. The results of laboratory studies before treatmentshowed 4400 white blood cells, 59% neutrophils, 1%eosinophils, 31% lymphocytes, and 6% monocytes. Thefollowing tests showed negative or normal results: plasmaglucose, blood urea nitrogen, serum creatinine, alkalinephosphatase, aspartate and alanine transaminase, iodideand bromideserumlevels, glucagon level, and antinuclearantibodies. Bone scintigraphy, thorax and abdominalcomputed tomography scan, bone marrow biopsy. biopsyofan axillary node, and colonoscopy also showed negativefindings.

The patient's disease was successively treated withdapsone (100 rog/day), etretinate (60 mg/day), andthree plasma exchanges. Because these treatments failed,roethylprednisone (64 mg/day) was administered, withexcellent results. After 3 weeks no new pustules had ap­peared. The patient still requires methylprednisolone (12mg/day) because pustules reappear when he is given alower dose.

Methods

Immunoglobulin levels were measured in both plasmaand pustule content. Neutrophils and monocytes wereisolated from the blood according to the usual procedure.4

Pustulecontentwas collectedby aspiration, and cells wereseparated by centrifugation and were extensively washed.More than 90% of the cells from pustules were neutro­phils, and trypan blue exclusion showed more than 85%to be viable.

Neutrophil mobility in response to 10-8moljL formyl­methionyl-leucyl-phenylalanine (fMet-Leu-Phe) and zy­mosan-activated plasma was studied by the modifiedBoyden chamber technique as described elsewhere.4 Re­sults were expressed as the number of cells migrating at

Volume 25Number 5, Part 2November 1991 Role ofTNF-a in Sneddon-Wilkinson SPD 945

Nanomoles 02per 106 cells

100

90

8070

60

50

40

30

20

10

O+----:.........----=-""------'----..........---......;..l'-------'

Before

treatment

Dapsone Etretinale Plasma Corticosteroids Normal

exchanges controls

Fig. 1. Influence of treatment on f-Met-Leu-Phe-induced superoxide anion production byneutrophils.•--------, Circulating neutrophils; 0 , neutrophils from pustules.

the lower side of the ffiter. Superoxide anion (02-) gen­eration was estimated by superoxide dismutase-in­hibitable reduction of ferricytochrome C, in response to50 ngjml phorbol 12-myristate 13-acetate or 10-7

rnoljL fMet-Leu-Phe.S Immunoradiometric assay wasused to evaluate TNF-a and interleukin 1 (IL-l) levels inserum and pustule content as well as in the supematantof monocytes stimulated by lipopolysaccharide (LPS,Escherichia coli extract, 20 /oLg/ml) for 18 hours. TNF-areceptors of neutrophils were studied on 106 cells bybinding of iodine 125- TNF-a with and without a 1000­fold excess ofhuman recombinantTNF-a.6These studieswere conducted before and during treatment. All resultswere the mean of three experiments and were comparedwith those of eight normal controls.

RESULTS

Levels of IgG, 19A, and albumin were approxi­matively half as much in pustules (IgG, 14.20gmjL;IgA, 0.71 gmjL; albumin, 14 gmjL) as in serum(IgG, 28.10 gmjL; IgA, 1.02 gmjL; albumin, 36gmjL). Levels ofIgM (0.13 gmjL) in pustules wereabout three times lower than in serum (IgM, 0.42gmjL).

Compared with circulating neutrophils from con­trol subjects, superoxide production by neutrophilsfrom the patient's pustules as well as from hisperipheral blood was significantly elevated in re­sponse to fMet-Leu-Phe (p < 0.02) (Fig. 1) orphorboll2-myristate 13-acetate (59 ± 3 nmol O2­

per 106 circulating neutrophils in the patient vs43 ± 2 nmol O2- in control subjects, p < 0.05;

62 ± 3 nmol 02- per 106 pustule neutrophils).Chemotaxis in response to activated plasma (datanot shown) or fMet-Leu-Phe (Fig. 2) was markedlyenhanced (p < 0.001).

The patient's serum TNF-a levels (Table I) werehigher than those of control subjects. TNF-a levelsinpustules wereconsiderably higher (p < 0.01) thanin serum (see Table I). Production ofTNF-a by un­stimulated monocytes was greater (p <0.0l) in thepatient than in the control subjects (see Table I).LPS stimulation of monocytes induced a larger in­crease of TNF-a production in the patient than incontrol subjects (p < 0.001) (see Table I). Com­pared with circulating neutrophils from control sub­jects, TNF-a receptor expression in the patient wasonly slightly decreased (p < 0.05) in circulatingneutrophils, whereas it was markedly decreased inpustule neutrophils (Table II).

IL-l in the serum was below the detection thresh­old of the method. In the patient spontaneous andLPS-stimulated production of IL-1 by monocyteswas similar to that of control subjects (l00 ± 70vs 300 ± 50 pgjml and 10.000 ± 1045 vs8376 ± 1113 pgjml, respectively).

During dapsone therapy, superoxide productionby circulating neutrophils and their chemotaxis weresignificantly decreased while superoxide productionby pustule neutrophils remained high (Figs. 1 and2). During etretinate therapy, superoxide produc­tion and chemotaxis were even higher than beforetreatment (see Figs. I and 2). After plasma ex-

946 Grob et ai.

Journal of theAmerican Academy of

Dermatology

mean numberof cells

800

700

600

500,

,1: ~111I1IIII

400

-r-+87-~- ,

,11111o 4----.1-1..-----'-' ---1.....1.-- ..:- -'- --'

100

200

300

Beforetreatment

Dapsone Etretinate Plasma Corticosteroids Normalexchanges controls

Fig. 2. Influence of treatment on neutrophil chemotaxis in response to f-Met-Leu-Phe.a---------, Circulating neutrophils; 0 , neutrophils from pustules.

Table I. TNF in serum, pustules, and supernatant of monocytes and influence of treatment*

Control subjectsPatients

Before treatmentReceiving corticosteroids

10 ± 3

45 ± 510 ± 2

TNF-ain pustules

3193 ± 260260 ± 70

Spontaneous productionof TNF-a by monocytes

180 ± 20

425 ± 50102 ± 30

Production of TNF-aby LPS-stimuIated monocytes

1950 ± 400

9600 ± 7505000 ± 510

*Results are expressed in picograms per milliliter.

Table II. TNF-a receptor expression onneutrophils and influence of treatment

change, findings were the same as during dapsonetherapy (see Figs. 1 and 2). These treatments did notsignificantly affect TNF-a levels in the serum and inpustule content or TNF-a production by monocytes(data not shown).

Eight days after the first intake of corticosteroids,superoxide production by circulating as well as pus­tule neutrophils was markedly decreased (see Fig.

Control subjectsPatients

Before treatmentReceiving corticosteroids

cpm, Counts per minute.

Specific binding Specific binding(in cpm), (in cpm)

on circulating on pustuleneutrophils neutrophils

3661 ± 154

2635 ± 150 1017 ± 853725 ± 120 2995 ± 95

1). During this treatment chemotaxis of blood neu­trophils was lower than before treatment but simi­lar to that measured during dapsone therapy andafter plasma exchange (seeFig. 2). Conversely, corti­costeroids were the only treatment able to decreaseTNF-a levels simultaneously in the serum and pus­tules, lower TNF-a production by monocytes (seeTable I), and increase TNF-a receptor expressionon neutrophils in the pustules (see Table II).

DISCUSSION

Our patient's findings were typical of the diseasedescribed by Sneddon and Wilkinson, but twounusual features should be noted. The first is the as­sociation with a monoclonal IgG. This has been re­ported only three times, once with IgG cryoglobulin1

and twice with IgG myeloma,?,8 About 20 othercases of SPD with monoclonal gammopathy havebeen reported to date, most involving IgA.9

The second is the presence of a bullous pemphi­goid-like deposit of IgG and C3 at the dermoepi-

Volume 25Number 5, Part 2November 1991

dermal junction. This finding has not been reportedbefore. In three previously described cases1D-12 C3was present at the dermoepidermal junction, but intwo of them10, 11 there were also pemphigus-like in~

tercellular deposits of IgA in the skin. This suggeststhat the SPD may include several different subsetsof neutrophilic dermatosis.

The pathogenesis of SPD is completely unknown.Up to now the only data available were reported byTagami et al.,13 who extracted a 1200 molecularweight "chemotactic factor" thought to be related toC5.

Because neutrophils were highly activated in ourpatient's pustules and, to a lesser degree, in his blood,we studied mediators that could potentiate neutro­phil responses. TNF-a,14 IL-l, and granulocyte­macrophage colony-stimulating factors may be re­sponsible for neutrophil potentiation (also calledpriming). Increased TNF-a production by mono­cytes and serum TNF-a levels in the patient com­pared with control subjects, as well as elevatedTNF-a level in the patient's pustules compared withhis serum, suggested that this cytokine might playarole in this disorder. Conversely, from our results in­volvement of IL-l was unlikely.

Two results support the hypothesis that TNF-awas at least one of the mediators responsible forneutrophil priming in SPD. First, the only treatmentto bring a clear clinical response, that is corticoster­oids, was also the only one that affected TNF-aproduction. Second, TNF-a probably interactedwith its receptors on neutrophils. It has been docu­mented that, once TNF-a has bound to its specificreceptors, the resulting complex is rapidly internal­ized and degraded. 1S In our patient, when TNF-alevel in pustules and serum decreased during corti­costeroid therapy, TNF-a receptor expression onpustule and circulating neutrophils increased. Themost likely hypothesis to account for these findingsis that excess TNF-a bound its receptors, which thusdisappeared.

Neutrophil priming by TNF-a is well establishedin vitro.14, 15 To our knowledge this is the first studyin man that demonstrated involvement of this phe­nomenon in vivo.

Role ofTNF-a in Sneddon-Wilkinson SPD 947

Although SPD is often associated with mono­clonal gammopathy, we were unable to show that, inour patient, the monoclonal protein played a directrole in the pathogenesis of this disease. Plasma ex­change led to a decrease in immunoglobulin levelsbut did not bring clinical improvement or correctionof TNF-a production.

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10. Wallach D, Foldes C, Cottenot F. Pustulose sous-cornee,acantholyse superficielle et IgA monoclonale. Ann Derma­tol VenereoI1982;109:959-63.

11. Burrows D, Bingham EA. Subcorneal pustular dermatosisand IgA gammopathy. Br 1 Dermatol 1984;111(supp126):91-3.

12. Marsden JR, Millard LG. Pyoderma gangrenosum, sub­corneal pustular dermatosis and 19A paraproteinaemia. Br1 Dermatol 1986;114:125-9.

13. Tagami H, Iwatsuki K, Iwase Y, et al. Subcorneal pustu­lar dermatosis with vesiculo-bullous eruption. Demonstra­tion of subcorneal IgA deposits and a leukocyte chemotac­tic factor. Br J DermatoI1973;109:581-7.

14. Klebanoff JJ, Vadas MA. Harlan JM, et aI. Stimulation ofneutrophils by tumor necrosis factor. 1 Immunol 1986;136:4220-5.

15. Beutler B, Cerami A. The biology of cachectin/TNF: aprimary mediator of the host response. Ann Revlmmunol1989;7:625-55.