Immunohistochemical study of the early histopathologic changes occurring in trauma-injured skin of psoriatic patients

ARTICLE

Psoriasis is a proliferative disorder of unknown aetiology, although many observations indicate that it might be a T cell-mediated disease triggered by streptococcal superantigen [1-2]. A number of important factors have been identified as relevant in the pathogenesis of psoriatic skin lesions: keratinocyte proliferation, vascular alterations [3-5] and activation of T lymphocytes, dermal macrophages and dendritic cells [6-10]. The close association in psoriatic lesions of the inflammatory infiltrates with cytokine and chemokine production, and the basal keratinocyte hyperplasia has suggested a role of immunological processes in the pathogenesis of the disease [11-13].

It is still debated which are the earliest events leading to psoriatic skin lesions. Previous immunohistochemical studies of very early pre-psoriatic lesions have suggested that the alterations of T lymphocytes, endothelial cells, dendritic cells and macrophages seem to precede epidermal proliferation [14].

To investigate this issue, previous reports have used the Koebner responses which were classically induced by sellotape stripping repeated until clinical erythema is produced [15-17]. In the present study, we have induced a Koebner reaction by needle scarification of uninvolved skin of 23 psoriatic patients. This type of trauma for inducing a Koebner reaction allowed us to investigate small-well, defined areas of injured skin and to compare them with the nearby normal skin, as early as 2-3 days after the injury.

Materials and methods

Patients and biopsies

Twenty-three patients with chronic plaque type psoriasis (14 males, 9 females, age 21-85 years) and 7 control subjects (4 males, 3 females, age 18-73 years) affected by chronic dermatitis other than psoriasis, underwent scarification of uninvolved skin with a 21 gauge sterile needle to reach the dermis as demonstrated by bleeding, after having given oral informed consent. Fifteen psoriatic patients received a 4-mm punch skin biopsy of the scratched area under local anaesthesia with lidocaine 2-3 days after injury. Eight psoriatic and 7 control patients underwent skin biopsy 7 days after scarification. Immediately after removal, skin biopsies were embedded in OCT compound (Miles Diagnostic Division, Elkhart, USA), snap frozen in liquid nitrogen and stored at ­ 80° C until sectioning. All the psoriatic and control samples were studied by immunohistochemistry.

Immunohistochemical studies

Serial frozen sections of 5 µm were acetone-fixed and then immunostained with the monoclonal antibodies listed in Table I. The sections were then incubated with a biotin-conjugated anti-mouse IgG and then with avidin-biotin peroxidase-complex (LSAB, Dako, Denmark). The reaction product was developed using 0.06% 3, 3'diaminobenzidine (Sigma Chemicals, St.Louis, MO) and 0.03% hydrogen peroxide. In control slides the primary antibody was omitted.

Results

A Koebner reaction was induced by scarification in uninvolved skin of psoriatic and control patients. Clinically, after 7 days, in 2 out of 8 cases the lesions were obviously psoriatic with the typical scales of a fully developed Koebner reaction. In Koebner negative psoriatic patients and in controls, the scratched areas clinically appeared as a healing wound.

Thirteen out of 23 psoriatic patients (56.5%) showed peculiar histological features characterised by a keratinocyte hyperplasia, leading to a "papillary" intradermal projection just beneath the scarification (Table II) (Fig.1). The detection of Ki67⁺ cells in the epidermal suprabasal layer of the papillary projection was interpreted as indicative of keratinocyte proliferation. The keratinocytes of the deeper layers in the scratched areas were positive for the intercellular adhesion molecule-1 (ICAM-1) in 9 out 13 cases (70%) (Fig. 2), whereas consistently negative in uninvolved skin. In about 30% of positive cases, just beneath the scarification, a marked reduction or absence in collagen type IV and laminin content in the epidermal basal membrane was observed (Fig. 3). The alterations of the epidermal basal membrane were also highlighted by tenascin immunostaining. Tenascin was accumulated in the extracellular matrix of the superficial dermis in 12 out of the 13 cases in which a hyperplastic reaction of the skin was present, while in the nearby normal-looking skin, it was observed in smaller quantities and located at the dermo-epidermal junction (Fig. 4).

In association with the papillary projections, aggregates of CD68⁺ cells were detected (Fig. 5). About 30% of CD68⁺ cells expressed FXIIIa. CD3⁺/CD4⁺ T lymphocytes were consistently present and were grouped in perivascular aggregates; CD8⁺ cells were few and localised in the same areas. CD1a⁺ Langerhans cells were scattered in the basal and suprabasal layers of the epidermis, without any significant difference in number and distribution as compared with normal skin. Munro's microabscesses were observed in only 1 out of 23 patients; in all cases CD15⁺ granulocytes were rare.

As shown in Table II, these histopathological changes were observed in 6/15 psoriatic patients on whom skin biopsy was performed 2-3 days after scarification, in 7/8 psoriatic patients who underwent skin biopsy 7 days after injury and in only 1 out of 7 control patients. In this positive control patient the hyperplastic skin showed the same immunohistochemical features observed in psoriatic skin. The other control patients did not show the hyperplastic skin reaction or any of the immunohistochemical features observed in psoriatic patients. Interestingly, the positive control patient was affected by urticaria and had a family history of psoriasis.

In the scratched areas of 10 out of 23 psoriatic patients, the above described histopathological changes were not detected. In the skin biopsies of negative cases, only mild inflammatory infiltrates were observed in the upper dermis, not associated with epidermal alterations. Similar features were observed in the skin biopsies of the control non psoriatic patients.

Discussion

The term "Koebner phenomenon" is used to indicate the development of a psoriatic lesion following an injury to previously normal-appearing skin. This phenomenon, generally induced by sellotape stripping, was used for studying the pathogenesis of early psoriatic lesions [15-17]. In the present study we used a different technique to induce the trauma, namely needle scarification, which allowed us to study the histopathological changes occurring in small well-defined areas and to compare them with the nearby normal skin. We performed skin biopsies 7 days after scarification, which is the time generally thought to be necessary for the development of a manifest psoriatic lesion [15, 16], or after 2-3 days, for observing the very early phases of the process. Clinically, 7 days after scarification, the complete Koebner phenomenon with the typical psoriatic scales was detected in 2 out of 8 psoriatic patients. The relatively low frequency of Koebner reaction has been already reported for sellotape stripping and it has been related to the generally accepted concept of psoriasis as a multifactorial disease [17, 18].

The keratinocyte proliferation is one of the histopathological features characteristic of chronic psoriatic lesions. In the present study we have shown that this feature is already detectable at 2-3 days after scarification, as shown by the hyperplastic reaction of the rete ridge just beneath the site of scarification. We have also demonstrated that the keratinocytes of the scratched areas are activated, as indicated by the ICAM-1 expression. ICAM-1 is an adhesion molecule not constitutively expressed by keratinocytes, whose expression is induced by inflammatory cytokines [10, 11]. Moreover, keratinocyte activation is associated with a marked tenascin accumulation in the upper dermis. Tenascin is an extracellular matrix glycoprotein, also termed hexabrachion protein, which is characterised by a unique six-armed structure [19]. Tenascin is extensively expressed during embryogenesis, whereas it is absent in many adult tissues. In normal human skin, tenascin is present as a thin, sometimes discontinuous, band in the papillary dermis immediately beneath the derma-epidermal junction [20]. Tenascin production has been demonstrated in hyperproliferative skin diseases and its expression has been associated with morphogenesis and keratinocyte migration [21, 22]. Our results suggest that keratinocyte activation and re-organisation of the extracellular matrix proteins represent early events in the pathogenesis of the psoriatic lesions accounting for the elongation of rete ridge, acantosis and papillomatosis, which are hallmarks of chronic psoriatic lesions.

The above described histopathological features were detected in more than 50% of the psoriatic patients and in a single positive control with a family history of psoriasis. These results indicate that trauma-induced hyperplastic reactions are more frequently detected in psoriatic patients and confirm previous reports showing that psoriatic skin is more prone to be stimulated by the Koebner phenomenon.

Numerous investigations have pointed out the prominent role of dendritic cells in the pathophysiology of psoriasis [23]. We did not find a significant difference in the number and distribution of CD1a⁺ Langerhans cells in the scratched areas as compared to normal skin. However, it must be pointed out that our results concern only the presence of CD1a⁺ Langerhans cells in the skin biopsies taken 2-3 or 7 days after scarification, without any further evaluation of their activation status.

CONCLUSION

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