945

ASSOCIATION BETWEEN HLA AND CUTANEOUS NECROTIZING VENULITIS DAVID GLASS, NICHOLAS A. SOTER, DAVID GIBSON, C. B. CARPENTER, and PETER H . SCHUR

A group of patients has been identified with cu- taneous necrotizing venulitis (vasculitis). These patients, some with concomitant connective tissue disorders, have skin lesions that separate them from the arteritis com- monly described as rheumatoid vasculitis. HLA typing has been performed on 31 of these unrelated patients with cutaneous necrotizing venulitis, including 19 with asso- ciated chronic disorders. The antigen pair All,BW35 was found in 5 of these 19 patients and in 11 of 346 controls. This difference in frequency is statistically signif- icant. Because HLA genes appear to be linked to immune response genes, these data suggest that such genes may exist in patients with this form of cutaneous necrotizing venulitis with associated connective tissue disease.

From the Departments of Medicine and Dermatology, Harvard Medical School. Robert B. Brigham Hospital. and Peter Bent Brigham Hospital. Boston. Massachusetts.

Supported by USPHS Grants AM 11414, AMO5577. RR05669. and AM5076. and by the Histocompatibility Research Fund of the Peter Bent Brigham Hospital.

David Glass, M.R.C.P.: Fellow, Robert B. Brigham Hos- pital. supported by a Traveling Grant from the Arthritis and Rheumatism Council, United Kingdom; Nicholas A. Soter, M.D.: Assistant Professor of Dermatology, Harvard Medical School: David Gibson. M.D.: former Fellow, Robert B. Brigham Hospital, presently at the USAF Medical Center, Kessler AFB. Biloxi, Mississippi: C. B. Carpenter. M.D.: Investigator. Howard Hughes Medical Insti- tute. Peter Bent Brigham Hospital: Peter H. Schur, M.D.: Associate Professor of Medicine, Harvard Medical School and Robert B. Brigham Hospital.

Address reprint requests to Peter H. Schur, M.D., Robert B. Brigham Hospital, 125 Parker Hill Avenue. Boston, Massachusetts 02 120.

Submitted for publication December 5 , 1975; accepted May 20. 1976.

A number of forms of vasculitis have been de- scribed in patients with connective tissue diseases. One of these types, which involves venules can be easily recognized in the skin.

Cutaneous necrotizing venulitis may be asso- ciated with connective tissue disorders in which im- munologic mechanisms may be involved. The skin le- sions in some patients contain immunoglobulins and complement proteins; that hypocomplementemia with or without cryoglobulins may be found in the serum suggests that immune complexes may be important in the pathogenesis of tissue injury in these individuals (1-3). That other mechanisms may be operative is sug- gested by the presence of cellular infiltrates containing lymphocytes and hypogranulated mast cells in the le- sions of some patients with cutaneous necrotizing venu- litis (4). Because the immune system is implicated in both vascular and connective tissue disorders, immune response ( IR) genes may be involved. In view of the association between IR genes and the histocompatibility (H2) system in mice ( 5 ) and an apparent association with the HLA system in humans (6,7), the association of vasculitis with particular HLA antigens, or combina- tions of such, might be demonstrable. This study found a significant increase in the frequency of the HLA an- tigen pair, A 11 and BW35, in patients with cutaneous necrotizing venulitis.

MATERIALS AND METHODS Thirty-one unrelated Caucasian patients a t the Robert

B. Brigham Hospital who had cutaneous necrotizing venulitis (vasculitis) were studied. This patient population was selected

Arthritis and Rheumatism, Vol. 19, No. 5 (September-October 1976)

946 GLASS ET AL

from a pool of 134 individuals referred with a possible diag- nosis of cutaneous vasculitis over a period of 3 years. In 62 patients the lesions of cutaneous necrotizing venulitis were documented by characteristic findings on examination of skin biopsy specimens (see below). Of these, 3 1 patients were avail- able for HLA typing. This group included 19 in whom there was an associated disease: 6 with rheumatoid arthritis (RA), 6 with systemic lupus erythematosus (SLE), 4 with Sjogren's syndrome, I with Sjogren's syndrome and lymphoma, 1 with lymphoma, and I with a syndrome of arthritis and glomerulo- nephritis. The 19 patients are subsequently referred to as the complicated uasculitis group. The remaining 12 patients, in whom there was no concomitant disease in that the lesions were restricted to the skin, are referred to as the simple uascu- litis group.

The control population consisted of 346 unrelated Caucasian individuals, 177 normal subjects, and 169 whose kidneys were used in the cadaver kidney donor program of the New England lnterhospital Organ Bank. Individuals known to have vascular, connective tissue, or other diseases that might affect the outcome of transplantation were excluded from the control group.

The diagnosis of cutaneous necrotizing venulitis was based on the presence of an eruption that appeared as either palpable purpura or recurrent urticaria and that was docu- mented as venulitis by examination of skin biopsy specimens. The histopathologic features included fibrinoid necrosis of the venule, a cellular infiltrate rich in neutrophils, fragmentation of nuclei, and extravasation of red blood cells (3,s).

These patients were also examined for clinical evidence of involvement of larger blood vessels, as is seen in rheumatoid arteritis, i.e., peripheral neuropathy, nailfold infarcts, and dig- ital infarcts were sought.

The diagnoses of SLE and RA were based on the criteria of the American Rheumatism Association (9.10). Sjogren's syndrome was confirmed by lip biopsy in 4 of the 5 subjects with this disease.

HLA phenotyping was performed by the microdroplet lymphocyte cytotoxicity test ( I I ) and by using antisera to define 30 specificities. The antisera used for the patients and the 169 kidney donor controls were kindly provided by Dr. Donald Kayhoe, Transplantation Immunology Branch, Na- tional Institute of Allergy and Infectious Diseases, and from other sources. The antisera used to type cells from the other normal subjects in the control group had similar specificity, except that antigens AW25, AW26, and BW 16 were not typed for. All typings of controls were performed by or under the supervision of the Tissue Typing Laboratory of the Peter Bent Brigham Hospital and the lnterhospital Organ Bank. The nomenclature used throughout the text conforms to that re- cently adopted by the Nomenclature Committee of the Sixth International Histocompatibility Workshop (12).

The HLA data were analyzed by comparing the fre- quency of single antigens and of all possible combinations of A and B series in the patient and control populations. To assess significance the x2 test was used; P values are given both with and without a correction for the number of variables tested ( I 3). The P values were corrected by multiplying by the num- ber of variables tested, 30 in the single antigen analysis and 225 in the analysis of antigen pairs. The relative risk ( X ) value was calculated as described previously (14).

Sera from the patient population were stored at -70°C within 2 hours of separation and subsequently assayed for total hemolytic complement (CH50) by the method of Kent and Fife (15). The concentrations of serum complement proteins Clq, CIS, C4, C2, C3, C5, and C6 were assayed by radial immunodiffusion with monospecific antisera (16). An- tinuclear antibodies were determined by immunofluorescence with mouse liver as a substrate. Renal status was evaluated by blood urea nitrogen, serum creatinine, 24-hour urinary pro- tein, and examination of urinary sediment. Blood for the as- sessment of cryoglobulins was drawn, clotted, and separated at 37°C. After 24 to 72 hours of incubation at 4"C, the serum was examined for the presence of a cryoprecipitate (3).

RESULTS

Analysis of Individual HLA Antigens

T h e frequency of the individual HLA ant igens of neither t h e A n o r t h e B series w a s significantly raised in t h e pat ient populat ion as a whole, in the complicated o r in t h e s imple groups,when compared with t h e control populat ion as well as with t h e W H O and UCLA con- trols (Tables 1 a n d 2).

Analysis of Antigen Pairs

T h e combinat ion of t w o A a n d B series antigens, A1 1 and BW35, was found in 5 of the 19 patients with complicated vasculitis a n d in n o n e of t h e 12 with simple vasculitis. These t w o ant igens were inherited as a hap- lotype in 3 of the 5 individuals with A1 l,BW35 on w h o m family studies were possible. These 5 patients represented 16.1% o f t h e ent i re vasculitis g r o u p a n d 26.3% o f t h e 19 complicated vasculitis patients. This antigen pair was found in only 1 1 (3.2%) of the 346 controls. T h e difference in frequency between normals a n d the vasculitis g r o u p as a whole was significant (x' = 11.74, P < 0.0007, X = 6.0), as was the difference between normals a n d t h e complicated vasculitis g roup (x' = 23.0, P < 0.00001, X = 10.92). These P values, when corrected for t h e total number of pairs of A and B series antigens tested for , i.e. 225, were P < 0.16 a n d P < 0.002. O t h e r pairs o f A a n d B series antigens were not present more frequently among either t h e simple o r complicated vasculitis g roup , as compared t o each o ther a n d t o t h e n o r m a l controls .

HLA AND CUTANEOUS VENULITIS 947

Clinical Associations

In addition to cutaneous necrotizing venulitis, the 5 patients with A1 1,BW35 had SLE in 2 instances, RA in 2 instances, and in 1 Sjogren’s syndrome. Of the 31 patients only 2 had evidence of rheumatoid arteritis. One patient with RA had large vessel involvement by arteriography of the femoral artery, and 1 patient with SLE had digital gangrene. Both of these patients had involvement of venules as well.

There were no clinical features to distinguish these 5 patients from the other 14 with complicated vasculitis. The sex ratio, however, varied in the two groups: 3 of the 19 patients with complicated vasculitis were male, as were 5 of the 12 patients with simple vasculitis and 1 of the 5 with All,BW35. ANA were present in 13 of 19 of the complicated subgroup, in 4 of 12 of the simple group, and in 3 of the 5 All,BW35 group. Hypocomplementemia was noted in 14 of the 19 in the complicated group, in 6 of the 12 in the simple group, and in 3 of the 5 in the A1 1,BW35 group. Strik- ing degrees of hypocomplementemia, particularly of early components, were noted in the complicated group, and very low levels of Clq were found in 1 of the simple group and 4 of the complicated group, including 2 of the All,BW35 patients. Cryoglobulins were found in 3 of

19 patients with complicated vasculitis, 1 of 6 with simple vasculitis, and 1 of 5 with the A1 I,BW35 antigen pair.

DISCUSSION HLA-All and HLA-BW35 were found in 5 of 19

patients with various connective tissue diseases com- plicated by cutaneous necrotizing venulitis, and this was a significantly greater number than found in normal controls. In Caucasian population surveys these two antigens are randomly distributed and do not occur together with any increased frequency (14). When eval- uated individually, the frequency of each H LA antigen in patients was found not to be significantly different from that in the controls. The use of accepted levels of significance, i.e., P values < 0.05-0.01, has been ques- tioned in situations in which multiple comparisons are made, as in studies of HLA antigens. A simple correc- tion factor can be applied by multiplying the P value found by standard tests of significance by the number of comparisons made. Corrected P values have been pre- sented in this report, but the use of such a correction factor is not necessary when pilot studies have been performed (13). The P values for the association be- tween the pair A I I,BW35 and complicated vasculitis is

Table 1. First Series HLA-A

xz Com- WHO Total Complicated Simple Workshop UCLA

Control oarison Relative with Corn- Relative Control Control

H LA Vasculitis xz Com- parison Antigens Vasculitis Vasculitis

~

No. (%) No. (%) No. (7%) Positive with Total Risk plicated Risk Positive Positive New (Old) Positive Positive Positive (%) Group (Xvalue) Group (X value) (70) (% )

A1 ( A l ) A2 (A2) A28 (W28) A3 (A3) A9 (A9) AW23 (W23) AW24 (W24) A10 (AIO) AW25 (W25) AW26 (W26) A l l ( A l l ) AW33 (W19) AW29 (W29) AW30 (W30) AW31 (W31) AW32 (W32)

Total

10 (32) 6 14 (45) I 1 6 (19) 2 3 (10) I 2 (6) 2 1 (3) - 2 (6) -

I (3) 1

6 (19) 5 5 (13) 3 2 (6) 2

6 (19) 3

- -

- - - - - -

31 19

0.98 I .01 1.96 0.32 0.28 3.8 I .7S I .89 0.26

I .46 I .92 1.41

-

- - -

GLASS ET AL

Table 2. Second Series HLA-B

H LA Total Complicated Simple Antigens Vasculitis Vasculitis Vasculitis

No. ( 7 6 ) No. (%) NO. ("/. 1

B5 (A5) 3 (10) 2 (10.5) I (8) BW35 (W5) 6 (19) 6 (31.5) - B l 8 (W18) - - - 8 7 (A7) 3 (10) I ( 5 ) 2 (17) BW22 (W22) 3 (10) I (5 ) 2 (17) 827 (W27) - - - B8 (A8) 10 (32) 6 (31.5) 4 (33) BIZ (A12) 8 (26) 5 (26) 3 (25) B13 (A13) I (.3) 1 (5) - BW40 (WIO) 4 (13) 3 (16) I (8)

BW15 (W15) 6 (19) 4 (21) 2 (17) BW16 (W16) 8 (26) 4 (21) 4 (33) 817 (W17) 2 (6) I ( 5 ) I (8) BW2I (W21) - - -

New (Old) Positive Positive Positive

814 (W14) 3 (10) 3 (16) -

Total 31 19 12

Control Positive

(76 )

x2 Com- WHO x 2 Com- parison Workshop UCLA parison Relative with Com- Relative Control Control

with Total Risk plicated Risk Positive Positive Group (Xvalue) Group (Xvalue) (%) (% 1

906

significant after correction; that for the vasculitis group as a whole is not.

Cutaneous necrotizing venulitis in our experience appears to be an uncommon association of rheumatic disease, the 19 patients within the complicated group being drawn from a population of several thousand who have attended the Robert B. Brigham Hospital over a 3- year period.

This type of vasculitis is clinically and patholog- ically distinct from the arteritis in patients with RA that is generally labeled as rheumatoid vasculitis. In these patients with rheumatoid arteritis the nailfold lesions, digital infarcts, and peripheral neuropathy are clinical features distinct from those associated with cutaneous necrotizing venulitis. The pathologic alterations can likewise be separated; there is an obliterative vasculitis of small arteries in the patients with seropositive RA that contrasts with the inflammatory lesions seen in cutaneous necrotizing venulitis (17,18). However, as was evident in 2 of 31 patients, the two forms of vasculitis may coexist.

It is possible that the combination of HLA A1 1,BW35 is associated with some connective tissue diseases rather than with venulitis. The A1 1,BW35 hap- lotype has been reported to be increased in incidence in patients with chronic glomerulonephritis (18). This an- tigen combination was not present to a greater degree than expected in patients with RA, JRA, or SLE (18).

No data of possible HLA vasculitis associations have been presented by others (6,19). BW35 alone has, how- ever, been reported to occur more frequently than ex- pected among black females with SLE (20), but not in Caucasian SLE patients or those with RA, JRA, or Sjogren's syndrome (20-22). In an earlier report (23), the present authors noted an apparent association be- tween All ,W5 (now All,BW35) and markedly de- pressed Clq levels. Further expansion of this series to the present study failed to confirm this initial impression, but suggested that the association was with venulitis. The Al I,BW35 antigen combination was found in this series in 2 patients with SLE, 2 with RA, and 1 with Sjogren's syndrome, a distribution reflecting that of the complicated group as a whole.

I t would seem likely, therefore, that the associa- tion of A1 1,BW35 is with the combination of venulitis and the connective tissue disease rather than with either connective tissue of vascular disease alone. This view is reinforced by the present finding of only I patient with All,BW35 among 39 Caucasian patients with SLE without associated necrotizing venulitis. The results in this report suggest that patients with cutaneous necrotiz- ing venulitis are divisible because of the presence or absence of associated disease, and some of this division appears to be made on the basis of these particular genetic markers. There is possible value in these findings because the classification of vasculitis presents consid-

HLA AND CUTANEOUS VENULITIS 949

erable problems, b lood vessel inf lammat ion being a common occurrence in a very heterogeneous group of diseases (24).

Cutaneous necrotizing venulitis has also been called allergic angiitis, hypersensitivity angiitis, and mi- croscopic periarteritis nodosa. It occurs in pat ients with anaphylactoid purpura (Henoch-Schonlein syndrome) and benign hypergammaglobul inemic purpura. The demonstrat ion of particular tissue ant igens in pat ients with different forms of vasculitis may be a means of clarifying their relationships and classification. T h i s present s tudy also suggests t h a t IR genes may exist in cu taneous necrotizing venulitis.

ACKNOWLEDGMENT The authors would like to thank Ms. Marilyn Kardon

for her excellent technical assistance.

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