British Journal of Dermatology (1979) lOO, 389.
Herpes gestationis: immunopathological and ultrastructural studies'^ CHRISTINE I.HARRINGTON AND S.S.BLEEHEN Rupert Hallam Department of Dermatology, Hallamshire Hospital, Sheffield Sro 2JF Accepted for publication 31 July 1978
SUMMARY
Eleven patients with the clinical picture of herpes gestationis were investigated. Biopsies were taken from involved and uninvolved areas of skin and the immunopathological and microscopic changes studied. Direct immunofiuorescence showed a deposition of C3 and/or IgG at the basement membrane zone in the involved skin of nine patients and the uninvolved skin of five. Immuno-electron microscopy using a multistep peroxidase antiperoxidase method revealed the in vivo deposition of IgG at the basal plasma cell membrane that extended into the lamina lucida. Light microscopy of urticarial and vesicular lesions showed a marked oedema of the papillary dermis with an inflammatory cell infiltrate that was mainly perivascular. There was spongiosis of the epidermis with oedema and necrosis of the basal cells and in several specimens sub-epidermal clefts with bulla formation. Electron microscopy confirmed the marked degenerative and necrotic changes of the basal cells in the involved areas of skin.
Herpes gestationis is a vcsico-bullous eruption of pregnancy associated with severe pruritus and with a tendency to recur in subsequent pregnancies. It is a rare condition and occurs in about 113000 to 1:4000 pregnancies (Russell & Thome, 1957). Clinically and histologically, herpes gestationis may resemble bullous pemphigoid, dermatitis herpetiformis and erythema multiforme. However, recent work indicates that herpes gestationis is more akin to bullous pemphigoid, particularly with regard to the pattern of deposition of complement and immunoglobulins at the basement membrane zone (Bushkell, Jordan & Goltz, 1974; Honigsmann et al., 1976; Yaoita, Gullino & Katz, 1976; Carruthers, Black & Ramnarain, 1977). This present study on eleven patients supports these previous reports. PATIENTS AND METHODS
Eleven patients were studied (Table 1), the ages ranging from 16-30 and all except one were primigravidae. The time of onset in pregnancy varied from 28-40 weeks gestation and the duration of the * Presented at the Winter Meeting of the British Association of Dermatologists, January 1978, Sheffield. 0007-0963/79/0400-03S9S02.00 C 1979 British Association of Dermatologists 389
390
C.I.Harrington and S.S.Bleehen TABLE I. Patients with herpes gestationis Patient No. I 2
3 4 5 6 7 8 9 IO :i
Name Age Pregnancy
JC JG JW
27 28 28
IC SD CD FM
27 2O
30 25 i6 23
JD ML VG Lt:
Local steroids Local steroids Local steroids Oral Prednisoione Local steroids Local steroids Local steroids Oral Prednisolone Local steroids Local steroids Local steroids
First First First First First Second First First First First First
26
29
Sex of baby
Treatment
F
M M M F M M M F F M
rash following delivery ranged from 3 days to 2 months (Fig. i). The eruption was intensely itchy and consisted mainly of urticarial, vesicular and polycyclic lesions (Fig. 2) on the trunk and limbs, but in two patients, lesions also occurred on tbe face. In several, tbere were large tense bullae. Two patients that were severely affected required treatment with oral prednisolone, the others were treated with only local applications. One patient had a recurrence of the eruption after delivery shortly after commencing on an oral contraceptive. Even after stopping the 'pill' the patient required to be treated for 8 weeks with prednisolone. There were no fetal complications and none of the babies born were affected.
JC
kWVVN
Delivery \ 11 10
9
9
7
. p,e d e l i v e i y
6
5
4
3
2
1 —
0 1 *"
2
3
4
5
6
7
8
Post p a r t u m
FIGURE I. Duration of herpes gestationis in eleven patients.
Herpes gestationis
391
FIGURE 2. Case 8. Erythematous polycyclic lesions.
Skin biopsies were taken from involved skin and from clinically normal skin distant from a lesion. All were processed for routine light microscopy and for direct immunofluorescence (IF). Portions from the biopsies of eight patients were processed for electron microscopy and from two for immunoelectron microscopy. For direct IF the biopsies were snap-frozen and sections, 4 ^m thick, were cut on a cryostat and stained with fluorescein isothiocyanate (FITC) conjugated rabbit anti-human antisera to IgG, IgA, IgM fibrin C3 and C4 (Behringwerke AG), using routine techniques. The sections were examined with a Leitz Orthoplan fluorescence microscope and photographs taken with an attached Orthomat camera using high speed Ektachrome film. Portions of each biopsy were fixed in 3% glutaraldehyde and later embedded in Araldite. Ultrathin sections were cut on an ultramicrotome and stained with uranyl acetate and lead hydroxide. For the light and electron microscopic localization of m vivo bound immunoglobulin, portions of the skin biopsies were chopped into small fragments and these as well as cryostat sections were exposed to an immunological chain of antibodies as shown in Fig. 3, using the horseradish peroxidase-antihorseradish peroxidase multistep method (Holubar et al., 1975). After incubation in Graham and Karnovsky's cytochemical medium (Graham & Karnovsky, 1966} to reveal the sites of peroxidase activity, the sections were then rinsed thoroughly. Control specimens were treated in the same way except Step 3 in the immunological chain (goat anti-horseradish peroxidase antibody) was omitted. Sections were mounted on slides for light microscopy and fragments of tissue after fixation in 3% glutaraldehyde were processed in the usual way for electron microscopy. Samples of sera were taken from all patients and stored at —20 C. Indirect IF was carried out with dilutions of the sera using cryostat sections of snap frozen normal skin as substrate and after staining with FITC conjugated anti-human antisera.
392
C.I.Harrington and S.S.Bleehen STEP 4
(HRP) /^~\ \^^/
STEP 3
STEP 2
STEP I
y^
X ^
>Y
Horseradish peromdase [ HRP)
inRPl
Goat anti-horseradish peroxidose antibody
^'''bbit onti-goat IqG
Goat onti-human IgG
//7 f/fo bound IgG
FIGURE 3. Localization of in vivo boutid imraunoglobuHn G iti tissue usitig a peroxidase-antiperoxidase multistep method (Holubar et al., 1975)-
The serum immunoglobulins and complement levels were determined in fresh samples of serum from these patients. Other investigations carried out were white blood cell counts and HLA typing. RESULTS Light microscopy
In involved areas of skin there was marked oedema of the papillary dermis (Fig. 4) that was also seen to a lesser degree in some sections of supposedly uninvolved 'normal' skin. A pronounced dermal infiltrate was present in most sections consisting mainly of lymphocytes and histiocytes with variable numbers of eosinophils and a few neutrophils. The infiltrate was mainly peri-vascular particularly around vessels in the upper dermis. There was in most specimens spongiosis of the epidermis with oedema particularly of the basal cells that appeared vacuolated and necrotic (Fig. 4). In several specimens there was a marked necrosis of the basal layer of the epidermis with cleft formation and in two specimens a well developed sub-epidermal bulla was present that contained eosinophils. Direct immunofiuorescence
The results of direct IF are shown in Table 2. In the involved skin, nine of the eleven patients showed IgG and/or C3 deposition in the basement membrane zone. Patients i, 2 and 8 showed both IgG and C3 deposition; patients 3, 4, 10 and 11, C3 only and patients 6 and 9, IgG only. In the uninvolved skin only two patients, 4 and 8, displayed both IgG and C3, and C3 only was present in patients i, 2 and 3. No immunofiuorescence was present in the uninvolved skin of patients 6, 9, 10 and II. The staining at the basement membrane zone was almost invariably linear and was more intense for C3 than for IgG. (Figs 5 and 6). The pattern of fluorescence appeared to relate to the severity of the disease. Patients 4 and 8 had both IgG and C3 in the involved and uninvolved skin and their rash started earlier in pregnancy and lasted longer after delivery. These were the only two patients who required oral prednisolone to control the eruption. Also, patient 8 had a recurrence
Herpes gestationis
393
FIGURE 4. Skin biopsy of erythematous urticarial lesion showing marked oedema of papillary dermis and damaged basal keratinocytes. (H & E, original magnification x 160.) TABLE 2. Direct immunofluorescence findings Patient No. Name JC JG 3 4
JW
5 6 7 8
SD CD FM
9
ML
10
IC
JD
VG
LC
Involved Skin IgG—basement membrane C3—basement membrane IgG—basement membrane C3—basement membrane C3—basement membrane C3—basement membrane and sub-epidermal region No immunofluorescence IgG—basement membrane No immunofluorescence IgG—basement membrane C3—basement membrane and sub-epidermal region IgG—basement membrane C3—basement membrane and sub-epidermal region C3—basement membrane and sub-epidermal region
Uninvolved Skin C3—basement membrane C3—basement membrane C3—basement membrane IgG—basement membrane C3—basement membrane No immunofluorescence No immunofiuorescence No immunofluorescence IgG—basement membrane C3—basement membrane and sub-epidermal region No immunofluorescence No immunofluorescence No immunofluorescence
394
C.I.Harrington and S.S.Bleehen
FIGURE 5. Direct IF for C3. Uninvolved skin.
of the rash following taking an oral contraceptive and further biopsies taken at that time showed deposition of IgG and C3 in both involved and uninvolved areas of skin. Routine electron microscopy
The most striking changes were found in the basal part of the epidermis. Even in uninvolved areas of skin degenerative changes were found in the basal cells. These showed damage particularly at the dermal side of their plasma membranes. In erythematous areas marked degenerative and necrotic changes were seen in the basal cells. There was intracellular oedema with vacuolar degeneration of the cytoplasm and swelling of the mitochondria as well as dilatation of the rough endoplasmic reticulum (Fig. 7). The plasma membranes of the basal cells showed discontinuities through which the contents of the cell protruded. Though the basal lamina was mainly intact there were in places small breaks through which sometimes pseudopodia of the basal cells protruded (Fig. 7). In some specimens the damaged basal cells formed microvesicles ard in some portions this necrosis was so marked that the basement membrane zone could not be recognized (Fig. 8). Intercellular oedema, though present, was not very marked and oedema of the papillary dermis, though present, was not striking. In some specimens lymphocytes and eosinophils, some of which were fragmented, were present and these were almost entirely around and close to vessels in the papillary dermis. Immunoelectron microscopy
The biopsies were taken of erythematous areas from two patients. Sections examined by light microscopy showed that in one patient (3) there was a linear brown staining of the basement membrane zone. This brown reaction product indicated in vivo deposits of IgG. The direct IF for IgG was
Herpes gestationis
395
FIGURE 6. Direct IF for IgG. Uninvolved skin.
negative though C3 was present. The other biopsy showed that there was no reaction product present and no deposits of IgG or C3 were found in this patient (5) on direct IF. All the controls carried out were negative, there being no peroxidase staining. On electron microscopy electron dense reaction product appeared to be deposited in a linear band along the basal lamina particularly at the plasma membrane of the basal keratinocytes and at their hemidesmosomes (Fig. 9a). The granular product extended into the lamina lucida. In a few areas there were granular deposits found in the papillary dermis but no reaction product was observed in the intercellular spaces between the basal cells of the epidermis. Similarly, no reaction product was found in the controls (Fig. 9b). Indirect Immunofluorescence
No IgG anti-basement membrane zone antibodies were demonstrated in the sera of these patients using as substrate normal human skin from controls. Other Investigations
The serum immunoglobulins were mainly normal but three patients (8, 9 and 10) had low IgA levels and one patient (i) had a raised IgM. Patient i also had 2 g/l serum of IgG cryoglobulin. A heat labile IgG subclass was found in patients i and 4 at concentrations of o-i and 07 g/l serum. Serum complement levels were all normal apart from an elevated C3 level in patient 2. A positive anti-reticulin antibody was present in patient 5, otherwise other indirect immunofluorescent tests were all negative.
396
C.I.Harrington and S.S.Bleehen
FIGURE 7. Erythematous skin. Basal keratmocytes showing degenerative and necrotic changes. The basal lamina (BL) is mainly intact though a few discontinuities are present ( X 6400) (inset x 12,800).
There was no consistent pattern of HLA types or blood groups. Two patients had a marked eosinophilia in the peripheral blood. DISCUSSION
Immunopathological and light and electron microscopic studies on patients with herpes gestationis show that there is a close relationship between this condition and bullous pemphigoid. This study on eleven patients supports previous reports (Provost & Tomasi, 1973; Bushkell et al., 1974; Schaumburg-Lever et al., 1973; Hertz et al., 1976; Yaoita et al., 1976; Honigsmann et al., 1976; Carruthers £/a/., 1977). Light microscopy indicates that the histopathology of herpes gestationis has certain distinctive features that are clearly different from other bullous disorders such as dermatitis herpetiformis and erythema multiforme. The marked necrosis of basal cells is characteristic (Schaumburg-Lever etal., 1973; Hertz era/., 1976) and though sometimes seen in bullous pemphigoid, it is not as apparent. However, there is sometimes difficulty in distinguishing between the two conditions (Hertz et al., 1976)-
Herpes gestationis
397
FIGURE 8. Involved non-buUous skin. Disruption of basal keratinocytes with formation of a microvesicle(X2ioo).
FIGURE 9. Perilesional skin. Case 3. Tissue damage is due to no cryoprotective agent being used. (a) The electron dense reaction product is mainly at the dermal side of the plasma membrane of the basal cells, particularly at the hemidesmosomes indicating the in vivo deposition of IgG ( X 12,800). (b) Control. Tissue was incubated with serum instead of goat, antihorseradish peroxidase antibody. No reaction product is seen at basement membrane zone (BL) ( X 12,800). The findings on direct IF in herpes gestationis are very similar to those in bullous pemphigoid but ditfer sharply from those seen in other bullous conditions. In most patients studied there is in involved areas of skin a linear deposition of C3 with or without IgG at the basement membrane zone.
398
C.I.Harrington and S.S.Bleehen
Apart from two patients, who had negative IF but clinically had the typical eruption of herpes gestationis, this pattern is found in our series. In many reports (Provost & Tomasi, 1973; Jordon et al., 1976; Katz, Hertz & Yaoita, 1976; Carruthers et al., 1977) a circulating complement fixing antibody to the basement membrane zone is also present. This anti-basement zone antibody is seldom detected by the usual IgG method, as is observed in our study, but is seen in the majority of patients using the method of indirect complement IF (Jordon et al., 1976; Katz et al., 1976; Hodge et ai, 1978). Why it is not demonstrable by the conventional indirect IF is unknown while in bullous pemphigoid it is frequently found. In herpes gestationis and less commonly bullous pemphigoid (Ahmed, Maize & Provost, 1977) when in remission, there is a disappearance of IgG and C3 deposition at the basement membrane zone and of circulating basement membrane zone antibodies. The ultrastructural changes seen in this study correspond to those reported (Schaumburg-Lever et al, 1973; Yaoita et al., 1976). The main finding is a necrosis of basal cells. There is in the involved areas of skin the formation of microvesicles that often have an intact basal lamina at their base. Though these findings are also found in bullous pemphigoid they are unusual (Bleehen, unpublished observations). They certainly do not resemble the changes seen in dermatitis herpetiformis. Immuno-electron microscopy demonstrated the in vivo deposition of IgG at the epidermal basal lamina in one of our patients. This patient showed the direct IF of the involved skin for IgG to be negative and the finding of immunoglobulin using the multistcp peroxidase anti-peroxidase staining methods confirms that it is much more sensitive than immunofiuorescence. A similar finding to our case is reported (Holubar, Konrad & StingI, 1977). The localization of deposits of C3 (Yaoita et al., 1976) and IgG (Honigsmann et al., 1976) on immunoelectron microscopy, using a multistep horseradish peroxidase antibody technique, is similar in both herpes gestationis and bullous pemphigoid. In our patient the reaction product is associated with the cytomembranes of the basal cells particularly at the hemidesmosomes. This is the location as observed also in bullous pemphigoid (Holubar et al., 1975). However, Yaoita and his colleagues observed that the reaction product indicating the deposition of C3 is uniformly distributed throughout the lamina lucida in herpes gestationis whereas in bullous pemphigoid it is preferentially associated with the hemidesmosomes and more heavily deposited on the epidermal side of the lamina lucida. Further studies on more cases are necessary using modified techniques that allow for better preservation of the tissues. The exact pathogenesis of herpes gestationis remains unknown. Hormonal and possibly also immunological infiuences (Reunala et al, i^if) m some way stimulate the formation of herpes gestationis factor, an IgG subclass, which activates complement (Jordon et al, 1976; Katz et al, 1976) and crosses the placenta. Complement activation at the basement membrane zone produces destruction of the basal cells with the subsequent protrusion of epidermal and junctional substances into the dermis. This may result in inflammatory cell infiltration and blister formation. ACKNOWLEDGMENTS
We wish to thank all our colleagues who referred patients to us and Miss Jennifer Senior and Miss Lisa Hartley for their technical assistance. This study was supported by a L.O.H.S. research grant from the Trent Regional Health Authority.
REFERENCES AHMED, A.R., MAIZE, J.C. & PROVOST, T.T. (1977) Bullous pemphigoid. Clinical and immunological follow-up after successful therapy. Archives of Dermatology, 113,1043.
Herpes gestationis
399
BUSHKELL, L.L., JORDON, R.E. & GOLTZ, R.W. (1974} Herpes gestationis: new immunoiogical findings. Archives of Dermatology, IIO, 65. CARRUTHERS, J.A., BLACK, M.M. & RAMNARAIN, N . (1977) Immunopathological studies in herpes gestationis. British Journal of Dermatology, 96, 35. GRAHAM, R.C. & KARNOVSKY, M.S. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. Journal of Histochemistry and Cytochemistry, 14, 291. HERTZ, K.C., KATZ, S.L, MAIZE, J. & ACKERMAN, A.B. (1976) Herpes gestationis; a clinicopathologic study.
Archives of Dermatology, 112,1543. HODGE, L , , BLACK, M.M., RAMNARAIN, N . & BHOGAL, B. (1978) Indirect complement immunofluorescence in
the immunopathological assessment of bullous pemphigoid, cicatricial pemphigoid and herpes gestationis. Clinical and Experimental Dermatology, 3,61. HOLUBAR, K . , WOLFF, K., KONRAD, K . & BEUTNER, E.H. (1975} Ultrastructural localization of immunoglobulins
in bullous pemphigoid skin. Journal of Investigative Dermatology, 64, 220. HOLUBAR, K., KONRAD, K. & STINGL, G . (1977) Detection by immunoelectron microscopy of immunoglobuiin G deposits in skin in immunofluorescence negative herpes gestationis. Bn'tis/jJoMrfja/t)/Dermuro%_V, 96, 569, HONIGSMANN, J., STINGL, G., HOLUBAR, K. & WOLFF, K . (1976) Herpes gestationis: fine structural pattern of immunoglobulin deposits in the skin in vivo. Journal of Investigative Dermatology, 66,389. JORDON, R.E., HEINE, K.G., TAPPEINER, G . , BUSHKELL, L . L . & PROVOST, T.T. (1976) The immunopathology of
herpes gestationis: immunofluorescence studies and characterization of the HG factor. Journal of Clinical Investigation, 57,1426. KATZ, S.I., HERTZ, K . C . & YAOITA, H . (1976) Herpes gestationis. Immunopathology and characterization of the HG faczor. Journal of Clinical Investigation, 57, 1434. PROVOST, T.T. & TOMASI, T.B. (1973) Evidence for complement activation via the alternate pathway in skin disease, i. Herpes gestationis, systemic lupus erythematosus and bullous pemphigoid. Journal of Clinical Investigation, 52, 1779. REUNALA, T . , KARVONEN, J., TIILIKAINEN, A. & SALO, O.P. (1977) Herpes Gestationis. A high titre of antiHLA-B8 antibody in the mother and pemphigoid-like immunohistological findings in the mother and the child. Britishjournal of Dermatology, 96, 563. RUSSELL, B. & THORNE, N . A . (1957) Herpes gestationis. Britishjournal of Dermatology, 69, 339. SCHAUMBURG-LEVER, G., SAFFOLD, O.E., ORFANOS, C E . & LEVER, W . E . (1973) Herpes gestationis. Histology and
ultrastructure. Archives of Dermatology, 107, 888. YAOITA, H . , GULLINO, M . & KATZ, S.I. (1976) Herpes gestationis. Ultrastructure and ultrastructural localization of i'w vivo bound complement. Jourjial of Investigative Dermatology, 66,383.
Herpes gestationis: immunopathological and ultrastructural studies'^ CHRISTINE I.HARRINGTON AND S.S.BLEEHEN Rupert Hallam Department of Dermatology, Hallamshire Hospital, Sheffield Sro 2JF Accepted for publication 31 July 1978
SUMMARY
Eleven patients with the clinical picture of herpes gestationis were investigated. Biopsies were taken from involved and uninvolved areas of skin and the immunopathological and microscopic changes studied. Direct immunofiuorescence showed a deposition of C3 and/or IgG at the basement membrane zone in the involved skin of nine patients and the uninvolved skin of five. Immuno-electron microscopy using a multistep peroxidase antiperoxidase method revealed the in vivo deposition of IgG at the basal plasma cell membrane that extended into the lamina lucida. Light microscopy of urticarial and vesicular lesions showed a marked oedema of the papillary dermis with an inflammatory cell infiltrate that was mainly perivascular. There was spongiosis of the epidermis with oedema and necrosis of the basal cells and in several specimens sub-epidermal clefts with bulla formation. Electron microscopy confirmed the marked degenerative and necrotic changes of the basal cells in the involved areas of skin.
Herpes gestationis is a vcsico-bullous eruption of pregnancy associated with severe pruritus and with a tendency to recur in subsequent pregnancies. It is a rare condition and occurs in about 113000 to 1:4000 pregnancies (Russell & Thome, 1957). Clinically and histologically, herpes gestationis may resemble bullous pemphigoid, dermatitis herpetiformis and erythema multiforme. However, recent work indicates that herpes gestationis is more akin to bullous pemphigoid, particularly with regard to the pattern of deposition of complement and immunoglobulins at the basement membrane zone (Bushkell, Jordan & Goltz, 1974; Honigsmann et al., 1976; Yaoita, Gullino & Katz, 1976; Carruthers, Black & Ramnarain, 1977). This present study on eleven patients supports these previous reports. PATIENTS AND METHODS
Eleven patients were studied (Table 1), the ages ranging from 16-30 and all except one were primigravidae. The time of onset in pregnancy varied from 28-40 weeks gestation and the duration of the * Presented at the Winter Meeting of the British Association of Dermatologists, January 1978, Sheffield. 0007-0963/79/0400-03S9S02.00 C 1979 British Association of Dermatologists 389
390
C.I.Harrington and S.S.Bleehen TABLE I. Patients with herpes gestationis Patient No. I 2
3 4 5 6 7 8 9 IO :i
Name Age Pregnancy
JC JG JW
27 28 28
IC SD CD FM
27 2O
30 25 i6 23
JD ML VG Lt:
Local steroids Local steroids Local steroids Oral Prednisoione Local steroids Local steroids Local steroids Oral Prednisolone Local steroids Local steroids Local steroids
First First First First First Second First First First First First
26
29
Sex of baby
Treatment
F
M M M F M M M F F M
rash following delivery ranged from 3 days to 2 months (Fig. i). The eruption was intensely itchy and consisted mainly of urticarial, vesicular and polycyclic lesions (Fig. 2) on the trunk and limbs, but in two patients, lesions also occurred on tbe face. In several, tbere were large tense bullae. Two patients that were severely affected required treatment with oral prednisolone, the others were treated with only local applications. One patient had a recurrence of the eruption after delivery shortly after commencing on an oral contraceptive. Even after stopping the 'pill' the patient required to be treated for 8 weeks with prednisolone. There were no fetal complications and none of the babies born were affected.
JC
kWVVN
Delivery \ 11 10
9
9
7
. p,e d e l i v e i y
6
5
4
3
2
1 —
0 1 *"
2
3
4
5
6
7
8
Post p a r t u m
FIGURE I. Duration of herpes gestationis in eleven patients.
Herpes gestationis
391
FIGURE 2. Case 8. Erythematous polycyclic lesions.
Skin biopsies were taken from involved skin and from clinically normal skin distant from a lesion. All were processed for routine light microscopy and for direct immunofluorescence (IF). Portions from the biopsies of eight patients were processed for electron microscopy and from two for immunoelectron microscopy. For direct IF the biopsies were snap-frozen and sections, 4 ^m thick, were cut on a cryostat and stained with fluorescein isothiocyanate (FITC) conjugated rabbit anti-human antisera to IgG, IgA, IgM fibrin C3 and C4 (Behringwerke AG), using routine techniques. The sections were examined with a Leitz Orthoplan fluorescence microscope and photographs taken with an attached Orthomat camera using high speed Ektachrome film. Portions of each biopsy were fixed in 3% glutaraldehyde and later embedded in Araldite. Ultrathin sections were cut on an ultramicrotome and stained with uranyl acetate and lead hydroxide. For the light and electron microscopic localization of m vivo bound immunoglobulin, portions of the skin biopsies were chopped into small fragments and these as well as cryostat sections were exposed to an immunological chain of antibodies as shown in Fig. 3, using the horseradish peroxidase-antihorseradish peroxidase multistep method (Holubar et al., 1975). After incubation in Graham and Karnovsky's cytochemical medium (Graham & Karnovsky, 1966} to reveal the sites of peroxidase activity, the sections were then rinsed thoroughly. Control specimens were treated in the same way except Step 3 in the immunological chain (goat anti-horseradish peroxidase antibody) was omitted. Sections were mounted on slides for light microscopy and fragments of tissue after fixation in 3% glutaraldehyde were processed in the usual way for electron microscopy. Samples of sera were taken from all patients and stored at —20 C. Indirect IF was carried out with dilutions of the sera using cryostat sections of snap frozen normal skin as substrate and after staining with FITC conjugated anti-human antisera.
392
C.I.Harrington and S.S.Bleehen STEP 4
(HRP) /^~\ \^^/
STEP 3
STEP 2
STEP I
y^
X ^
>Y
Horseradish peromdase [ HRP)
inRPl
Goat anti-horseradish peroxidose antibody
^'''bbit onti-goat IqG
Goat onti-human IgG
//7 f/fo bound IgG
FIGURE 3. Localization of in vivo boutid imraunoglobuHn G iti tissue usitig a peroxidase-antiperoxidase multistep method (Holubar et al., 1975)-
The serum immunoglobulins and complement levels were determined in fresh samples of serum from these patients. Other investigations carried out were white blood cell counts and HLA typing. RESULTS Light microscopy
In involved areas of skin there was marked oedema of the papillary dermis (Fig. 4) that was also seen to a lesser degree in some sections of supposedly uninvolved 'normal' skin. A pronounced dermal infiltrate was present in most sections consisting mainly of lymphocytes and histiocytes with variable numbers of eosinophils and a few neutrophils. The infiltrate was mainly peri-vascular particularly around vessels in the upper dermis. There was in most specimens spongiosis of the epidermis with oedema particularly of the basal cells that appeared vacuolated and necrotic (Fig. 4). In several specimens there was a marked necrosis of the basal layer of the epidermis with cleft formation and in two specimens a well developed sub-epidermal bulla was present that contained eosinophils. Direct immunofiuorescence
The results of direct IF are shown in Table 2. In the involved skin, nine of the eleven patients showed IgG and/or C3 deposition in the basement membrane zone. Patients i, 2 and 8 showed both IgG and C3 deposition; patients 3, 4, 10 and 11, C3 only and patients 6 and 9, IgG only. In the uninvolved skin only two patients, 4 and 8, displayed both IgG and C3, and C3 only was present in patients i, 2 and 3. No immunofiuorescence was present in the uninvolved skin of patients 6, 9, 10 and II. The staining at the basement membrane zone was almost invariably linear and was more intense for C3 than for IgG. (Figs 5 and 6). The pattern of fluorescence appeared to relate to the severity of the disease. Patients 4 and 8 had both IgG and C3 in the involved and uninvolved skin and their rash started earlier in pregnancy and lasted longer after delivery. These were the only two patients who required oral prednisolone to control the eruption. Also, patient 8 had a recurrence
Herpes gestationis
393
FIGURE 4. Skin biopsy of erythematous urticarial lesion showing marked oedema of papillary dermis and damaged basal keratinocytes. (H & E, original magnification x 160.) TABLE 2. Direct immunofluorescence findings Patient No. Name JC JG 3 4
JW
5 6 7 8
SD CD FM
9
ML
10
IC
JD
VG
LC
Involved Skin IgG—basement membrane C3—basement membrane IgG—basement membrane C3—basement membrane C3—basement membrane C3—basement membrane and sub-epidermal region No immunofluorescence IgG—basement membrane No immunofluorescence IgG—basement membrane C3—basement membrane and sub-epidermal region IgG—basement membrane C3—basement membrane and sub-epidermal region C3—basement membrane and sub-epidermal region
Uninvolved Skin C3—basement membrane C3—basement membrane C3—basement membrane IgG—basement membrane C3—basement membrane No immunofluorescence No immunofiuorescence No immunofluorescence IgG—basement membrane C3—basement membrane and sub-epidermal region No immunofluorescence No immunofluorescence No immunofluorescence
394
C.I.Harrington and S.S.Bleehen
FIGURE 5. Direct IF for C3. Uninvolved skin.
of the rash following taking an oral contraceptive and further biopsies taken at that time showed deposition of IgG and C3 in both involved and uninvolved areas of skin. Routine electron microscopy
The most striking changes were found in the basal part of the epidermis. Even in uninvolved areas of skin degenerative changes were found in the basal cells. These showed damage particularly at the dermal side of their plasma membranes. In erythematous areas marked degenerative and necrotic changes were seen in the basal cells. There was intracellular oedema with vacuolar degeneration of the cytoplasm and swelling of the mitochondria as well as dilatation of the rough endoplasmic reticulum (Fig. 7). The plasma membranes of the basal cells showed discontinuities through which the contents of the cell protruded. Though the basal lamina was mainly intact there were in places small breaks through which sometimes pseudopodia of the basal cells protruded (Fig. 7). In some specimens the damaged basal cells formed microvesicles ard in some portions this necrosis was so marked that the basement membrane zone could not be recognized (Fig. 8). Intercellular oedema, though present, was not very marked and oedema of the papillary dermis, though present, was not striking. In some specimens lymphocytes and eosinophils, some of which were fragmented, were present and these were almost entirely around and close to vessels in the papillary dermis. Immunoelectron microscopy
The biopsies were taken of erythematous areas from two patients. Sections examined by light microscopy showed that in one patient (3) there was a linear brown staining of the basement membrane zone. This brown reaction product indicated in vivo deposits of IgG. The direct IF for IgG was
Herpes gestationis
395
FIGURE 6. Direct IF for IgG. Uninvolved skin.
negative though C3 was present. The other biopsy showed that there was no reaction product present and no deposits of IgG or C3 were found in this patient (5) on direct IF. All the controls carried out were negative, there being no peroxidase staining. On electron microscopy electron dense reaction product appeared to be deposited in a linear band along the basal lamina particularly at the plasma membrane of the basal keratinocytes and at their hemidesmosomes (Fig. 9a). The granular product extended into the lamina lucida. In a few areas there were granular deposits found in the papillary dermis but no reaction product was observed in the intercellular spaces between the basal cells of the epidermis. Similarly, no reaction product was found in the controls (Fig. 9b). Indirect Immunofluorescence
No IgG anti-basement membrane zone antibodies were demonstrated in the sera of these patients using as substrate normal human skin from controls. Other Investigations
The serum immunoglobulins were mainly normal but three patients (8, 9 and 10) had low IgA levels and one patient (i) had a raised IgM. Patient i also had 2 g/l serum of IgG cryoglobulin. A heat labile IgG subclass was found in patients i and 4 at concentrations of o-i and 07 g/l serum. Serum complement levels were all normal apart from an elevated C3 level in patient 2. A positive anti-reticulin antibody was present in patient 5, otherwise other indirect immunofluorescent tests were all negative.
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FIGURE 7. Erythematous skin. Basal keratmocytes showing degenerative and necrotic changes. The basal lamina (BL) is mainly intact though a few discontinuities are present ( X 6400) (inset x 12,800).
There was no consistent pattern of HLA types or blood groups. Two patients had a marked eosinophilia in the peripheral blood. DISCUSSION
Immunopathological and light and electron microscopic studies on patients with herpes gestationis show that there is a close relationship between this condition and bullous pemphigoid. This study on eleven patients supports previous reports (Provost & Tomasi, 1973; Bushkell et al., 1974; Schaumburg-Lever et al., 1973; Hertz et al., 1976; Yaoita et al., 1976; Honigsmann et al., 1976; Carruthers £/a/., 1977). Light microscopy indicates that the histopathology of herpes gestationis has certain distinctive features that are clearly different from other bullous disorders such as dermatitis herpetiformis and erythema multiforme. The marked necrosis of basal cells is characteristic (Schaumburg-Lever etal., 1973; Hertz era/., 1976) and though sometimes seen in bullous pemphigoid, it is not as apparent. However, there is sometimes difficulty in distinguishing between the two conditions (Hertz et al., 1976)-
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FIGURE 8. Involved non-buUous skin. Disruption of basal keratinocytes with formation of a microvesicle(X2ioo).
FIGURE 9. Perilesional skin. Case 3. Tissue damage is due to no cryoprotective agent being used. (a) The electron dense reaction product is mainly at the dermal side of the plasma membrane of the basal cells, particularly at the hemidesmosomes indicating the in vivo deposition of IgG ( X 12,800). (b) Control. Tissue was incubated with serum instead of goat, antihorseradish peroxidase antibody. No reaction product is seen at basement membrane zone (BL) ( X 12,800). The findings on direct IF in herpes gestationis are very similar to those in bullous pemphigoid but ditfer sharply from those seen in other bullous conditions. In most patients studied there is in involved areas of skin a linear deposition of C3 with or without IgG at the basement membrane zone.
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Apart from two patients, who had negative IF but clinically had the typical eruption of herpes gestationis, this pattern is found in our series. In many reports (Provost & Tomasi, 1973; Jordon et al., 1976; Katz, Hertz & Yaoita, 1976; Carruthers et al., 1977) a circulating complement fixing antibody to the basement membrane zone is also present. This anti-basement zone antibody is seldom detected by the usual IgG method, as is observed in our study, but is seen in the majority of patients using the method of indirect complement IF (Jordon et al., 1976; Katz et al., 1976; Hodge et ai, 1978). Why it is not demonstrable by the conventional indirect IF is unknown while in bullous pemphigoid it is frequently found. In herpes gestationis and less commonly bullous pemphigoid (Ahmed, Maize & Provost, 1977) when in remission, there is a disappearance of IgG and C3 deposition at the basement membrane zone and of circulating basement membrane zone antibodies. The ultrastructural changes seen in this study correspond to those reported (Schaumburg-Lever et al, 1973; Yaoita et al., 1976). The main finding is a necrosis of basal cells. There is in the involved areas of skin the formation of microvesicles that often have an intact basal lamina at their base. Though these findings are also found in bullous pemphigoid they are unusual (Bleehen, unpublished observations). They certainly do not resemble the changes seen in dermatitis herpetiformis. Immuno-electron microscopy demonstrated the in vivo deposition of IgG at the epidermal basal lamina in one of our patients. This patient showed the direct IF of the involved skin for IgG to be negative and the finding of immunoglobulin using the multistcp peroxidase anti-peroxidase staining methods confirms that it is much more sensitive than immunofiuorescence. A similar finding to our case is reported (Holubar, Konrad & StingI, 1977). The localization of deposits of C3 (Yaoita et al., 1976) and IgG (Honigsmann et al., 1976) on immunoelectron microscopy, using a multistep horseradish peroxidase antibody technique, is similar in both herpes gestationis and bullous pemphigoid. In our patient the reaction product is associated with the cytomembranes of the basal cells particularly at the hemidesmosomes. This is the location as observed also in bullous pemphigoid (Holubar et al., 1975). However, Yaoita and his colleagues observed that the reaction product indicating the deposition of C3 is uniformly distributed throughout the lamina lucida in herpes gestationis whereas in bullous pemphigoid it is preferentially associated with the hemidesmosomes and more heavily deposited on the epidermal side of the lamina lucida. Further studies on more cases are necessary using modified techniques that allow for better preservation of the tissues. The exact pathogenesis of herpes gestationis remains unknown. Hormonal and possibly also immunological infiuences (Reunala et al, i^if) m some way stimulate the formation of herpes gestationis factor, an IgG subclass, which activates complement (Jordon et al, 1976; Katz et al, 1976) and crosses the placenta. Complement activation at the basement membrane zone produces destruction of the basal cells with the subsequent protrusion of epidermal and junctional substances into the dermis. This may result in inflammatory cell infiltration and blister formation. ACKNOWLEDGMENTS
We wish to thank all our colleagues who referred patients to us and Miss Jennifer Senior and Miss Lisa Hartley for their technical assistance. This study was supported by a L.O.H.S. research grant from the Trent Regional Health Authority.
REFERENCES AHMED, A.R., MAIZE, J.C. & PROVOST, T.T. (1977) Bullous pemphigoid. Clinical and immunological follow-up after successful therapy. Archives of Dermatology, 113,1043.
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BUSHKELL, L.L., JORDON, R.E. & GOLTZ, R.W. (1974} Herpes gestationis: new immunoiogical findings. Archives of Dermatology, IIO, 65. CARRUTHERS, J.A., BLACK, M.M. & RAMNARAIN, N . (1977) Immunopathological studies in herpes gestationis. British Journal of Dermatology, 96, 35. GRAHAM, R.C. & KARNOVSKY, M.S. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. Journal of Histochemistry and Cytochemistry, 14, 291. HERTZ, K.C., KATZ, S.L, MAIZE, J. & ACKERMAN, A.B. (1976) Herpes gestationis; a clinicopathologic study.
Archives of Dermatology, 112,1543. HODGE, L , , BLACK, M.M., RAMNARAIN, N . & BHOGAL, B. (1978) Indirect complement immunofluorescence in
the immunopathological assessment of bullous pemphigoid, cicatricial pemphigoid and herpes gestationis. Clinical and Experimental Dermatology, 3,61. HOLUBAR, K . , WOLFF, K., KONRAD, K . & BEUTNER, E.H. (1975} Ultrastructural localization of immunoglobulins
in bullous pemphigoid skin. Journal of Investigative Dermatology, 64, 220. HOLUBAR, K., KONRAD, K. & STINGL, G . (1977) Detection by immunoelectron microscopy of immunoglobuiin G deposits in skin in immunofluorescence negative herpes gestationis. Bn'tis/jJoMrfja/t)/Dermuro%_V, 96, 569, HONIGSMANN, J., STINGL, G., HOLUBAR, K. & WOLFF, K . (1976) Herpes gestationis: fine structural pattern of immunoglobulin deposits in the skin in vivo. Journal of Investigative Dermatology, 66,389. JORDON, R.E., HEINE, K.G., TAPPEINER, G . , BUSHKELL, L . L . & PROVOST, T.T. (1976) The immunopathology of
herpes gestationis: immunofluorescence studies and characterization of the HG factor. Journal of Clinical Investigation, 57,1426. KATZ, S.I., HERTZ, K . C . & YAOITA, H . (1976) Herpes gestationis. Immunopathology and characterization of the HG faczor. Journal of Clinical Investigation, 57, 1434. PROVOST, T.T. & TOMASI, T.B. (1973) Evidence for complement activation via the alternate pathway in skin disease, i. Herpes gestationis, systemic lupus erythematosus and bullous pemphigoid. Journal of Clinical Investigation, 52, 1779. REUNALA, T . , KARVONEN, J., TIILIKAINEN, A. & SALO, O.P. (1977) Herpes Gestationis. A high titre of antiHLA-B8 antibody in the mother and pemphigoid-like immunohistological findings in the mother and the child. Britishjournal of Dermatology, 96, 563. RUSSELL, B. & THORNE, N . A . (1957) Herpes gestationis. Britishjournal of Dermatology, 69, 339. SCHAUMBURG-LEVER, G., SAFFOLD, O.E., ORFANOS, C E . & LEVER, W . E . (1973) Herpes gestationis. Histology and
ultrastructure. Archives of Dermatology, 107, 888. YAOITA, H . , GULLINO, M . & KATZ, S.I. (1976) Herpes gestationis. Ultrastructure and ultrastructural localization of i'w vivo bound complement. Jourjial of Investigative Dermatology, 66,383.
