High Levels of Interleukin-1 in Patients with Endemic Pemphigus Foliaceus

Endemic pemphigus foliaceus (EPF) is an autoimmune disease characterizedby blister formation with a loss of cohesion and infiltrationof inflammatory cells. We observed that supernatants of peripheralblood mononuclear cells from patients produced significantlymore interleukin-1ß (IL-1ß) than those fromstimulated healthy controls. Furthermore, a Th2 bias was observedin EPF patients when the IL-5/gamma interferon ratio was analyzed.These results indicate that cells from pemphigus patients reactwith a vigorous proinflammatory response.

INTRODUCTION

Top

Introduction

Endemic pemphigus foliaceus (EPF), also known as "fogo selvagem,"is a chronic autoimmune disease characterized by acantholysisand the formation of intraepidermal blisters (23). Lesions appearon the face and can later spread, without reaching mucous tissues(19). In severe cases, it can spread to the scalp, face, dorsum,and arms and legs. The disease can be treated with the administrationof glucocorticosteroids (6, 19) and other immunosuppressivedrugs in more severe cases (3).

The rate of mortality caused by bullous skin diseases, includingEPF, has been estimated to be about 10 to 40% and is mainlydue to complications resulting from secondary infections (15).With the use of glucocorticosteroid therapy, the prognosis forthis disease has been improved significantly.

EPF is associated with the presence of immunoglobulin G4 (IgG4)antibodies against antigens associated with the desmosomes (desmoglein1) that promote an inflammatory reaction in the presence oflymphocytes, neutrophils, and eosinophils (7, 11). The lesionsgenerate a decrease in the adhesion between the keratinocytesof the granular layer of the epidermis, with an increase inthe intercellular space due to the reduction or the total disappearanceof the desmosomes (10, 14, 19, 23, 24). The bullous skin breakupleads to superficial erosions, which result in diffuse erythemaand the formation of crusts (19). Within the blister cavity,the cells observed in association with acantholysis have hyperchromaticnuclei and homogeneous cytoplasms.

The role of antibodies in the pathogenesis of the lesions wasdemonstrated by the injection of serum from patients with pemphigusinto mice. These animals developed a disease that reproducedpemphigus lesions, indicating that the antibodies play an essentialrole in this disease (1).

The participation of the cell-mediated immune response in bullousskin disease has been shown. Peripheral blood mononuclear cells(PBMCs) from EPF patients proliferate after being stimulatedwith desmoglein epitopes. These cells exhibit a Th2 phenotype(17). Moreover, the development of bullous skin lesions afteralpha interferon (IFN- ${alpha}$ ) therapy for hepatitis C or Kaposi'ssarcoma (21, 22) suggests the involvement of cytokines in thedisease.

In the study described here, we addressed other mediating factorsthat may be involved in the development of tissue lesions. Weinvestigated the production of interleukin-1ß (IL-1ß),IL-4, IL-5, IL-10, IFN- ${gamma}$ , and tumor necrosis factor alpha (TNF- ${alpha}$ )in PBMCs from EPF patients. The results show that IL-1ßlevels are significantly higher in EPF patients even duringglucocorticosteroid therapy and that the cytokine balance favorsTh2.

MATERIALS AND METHODS

Top

Materials and Methods

Subjects. Patients diagnosed with pemphigus foliaceus were invited toparticipate in this study. Ten patients with active lesions(9 with blisters and crusts and 1 with crusts only) and 15 controlsubjects were analyzed. The control subjects were matched withthe patients by age and gender. The ages of the patients, whowere receiving therapy with the glucocorticosteroid methylprednisolone,ranged from 21 to 88 years, and five were males.

Blood for PBMC purification was collected by venipuncture witha 20-ml heparinized syringe.

This study was approved by the Ethical Committee of the Faculdadede Medicina do Triângulo Mineiro, Minas Gerais, Brazil.

Cell culture. PBMCs were isolated from heparinized blood by centrifugationat 400 x g for 20 min at room temperature in Ficoll-Paque (Sigma,St. Louis, Mo.), followed by three washes with RPMI (Gibco,Grand Island, N.Y.) medium. The pellets were resuspended inRPMI supplemented with 50 mM 2-mercaptoethanol (Sigma), 2 mML-glutamine (Sigma), 40 µg of gentamicin per ml, and 5%fetal calf serum (Gibco) (complete medium).

For supernatant production, 2 x 10⁶ cells per ml were culturedin a 24-well microplate in the presence of medium alone, 5 µgof phytohemagglutinin (PHA; Sigma) per ml, or 5 µg oflipopolysaccharide (LPS; Sigma) per ml. The plates were incubatedat 37°C in a 5% CO₂ atmosphere. At 48 and 120 h, the supernatantswere collected, centrifuged, and stored at -70°C for analysisof cytokine production.

Cytokine titration. For cytokine titration, microplates (Nunc, Roskilde, Denmark)were sensitized overnight with anti-IL-1ß monoclonalantibody (MAb; Genzyme, Cambridge, Mass.), anti-TNF- ${alpha}$ MAb (Genzyme),anti-IL-4 MAb (Mabtech, Nacka, Sweden), anti-IL-5 MAb (Pharmingen,San Diego, Calif.), anti IL-10 MAb (Pharmingen), or anti-IFN- ${gamma}$ MAb (Mabtech). Nonspecific binding was prevented by incubatingthe plates with 3% bovine serum albumin (Sigma) in phosphate-bufferedsaline (PBS). The plates were incubated overnight with 100 µlof a 1:2 dilution of culture supernatant in PBS, 2% bovine serumalbumin, and standard cytokines (Pharmingen and R&D, Minneapolis,Minn.). The plates were then washed four times with 0.05% Tweenin PBS and incubated with rabbit anti-IL-1ß antibody(Genzyme), rabbit anti-TNF- ${alpha}$ antibody (Genzyme), biotinylatedanti-IL-4 MAb (Mabtech), biotinylated anti-IL-5 MAb (Pharmingen),biotinylated anti-IL-10 MAb (Pharmingen), or biotinylated anti-IFN- ${gamma}$ MAb (Mabtec) for 4 h. The plates were then washed and incubatedfor 2 h with alkaline phosphatase-conjugated goat anti-rabbitIgG (Pierce, Rockford, Ill.) or alkaline phosphatase conjugatedto streptavidin. Finally, the plates were washed four times,and enzymatic activity was developed by incubating the plateswith p-nitrophenyl phosphate (Sigma). The absorbance at 405nm was read in a microplate reader (Bio-Rad, Hercules, Calif.).The sensitivity of the tests was 10 to 30 pg/ml.

Statistical analysis. Differences in cytokine levels between patients and controlswere analyzed by the Mann-Whitney test, with significance setat a P value of <0.05. The balance between Th2 and Th1 cellswas expressed by the ratio of IL-5 and IFN- ${gamma}$ levels, and differencesbetween IL-5/IFN- ${gamma}$ ratios were analyzed by an unpaired t test,with significance set at a P value of <0.05.

RESULTS

Top

Results

Production of inflammatory cytokines. All patients included in this study presented with active lesions,and diagnosis was performed by clinical anatomicopathologicalexamination. PBMCs derived from patients and healthy controlswere stimulated with PHA or LPS for 48 or 120 h, and the supernatantswere tested for the presence of regulatory and inflammatorycytokines. The results in Fig. 1 show that after LPS stimulation,the IL-1ß level was significantly higher in the supernatantsfrom pemphigus patients than in those from the healthy controls(P < 0.05). The levels of IL-10 after PHA stimulation didnot differ between the patients and the controls. These resultsindicate a proinflammatory response by pemphigus patients evenwhile they are receiving glucocorticosteroid therapy.

View larger version (24K):
[in this window]
[in a new window]
FIG. 1. Production of cytokines involved in the inflammatory reaction after stimulation with LPS and PHA. PBMCs from patient (hatched bars) and control subjects (gray bars) were cultured in the presence of PHA or LPS for 48 h. The cytokine levels in the supernatants (in picograms per milliliter) were determined by ELISA. Horizontal lines represent median values, boxes represent 25th to 75th percentiles, and vertical lines represent 10th to 90th percentiles. Statistical significance between groups is indicated (*, P < 0.05, Mann-Whitney test).

Th1-Th2 cytokine balance. PBMCs derived from patients and healthy controls were stimulatedwith PHA for 48 or 120 h, and the supernatants were tested forthe presence of regulatory cytokines. After PHA stimulation,the levels of IL-4, IL-5, and IFN- ${gamma}$ did not differ significantlybetween the patients and the healthy controls (Fig. 2a). TheTh1-Th2 cytokine balance was analyzed by determination of theIL-5/IFN- ${gamma}$ ratio. As shown in Fig. 2b, patients had a cytokinebalance that favored Th2, with the levels of Th2 cytokines beingsignificantly higher for the patients than for the controls.

View larger version (29K):
[in this window]
[in a new window]
FIG. 2. Production of regulatory cytokines after stimulation with PHA. PBMCs from patients (hatched bars) and control subjects (gray bars) were cultured in the presence of PHA for 120 h. (a) The cytokine levels in the supernatants (in picograms per milliliter) were determined by ELISA. Horizontal lines represent median values, boxes represent 25th to 75th percentiles, and vertical lines represent 10th to 90th percentiles. (b) IL-5/IFN- ${gamma}$ ratios were obtained by dividing the IL-5 levels by the IFN- ${gamma}$ levels in cultured supernatants stimulated with PHA. Bars represent averages, and vertical lines represent standard deviations. Statistical significance between groups is indicated (*, P < 0.05, the Student t test).

DISCUSSION

Top

Discussion

The prevalence of antibodies against desmoglein 1 is higheramong healthy individuals who live in areas where pemphigusfoliaceus is endemic. The beginning of the disease is precededby the continuous production of these types of antibodies (24).This suggests that mediating factors other than antibodies maybe involved in disease development. Due to its endemic nature,it is suspected that some environmental factor has a role inthe pathogenesis of the disease. Since some subjects live inareas of endemicity and do not develop skin lesions, the involvementof genetic factors may also contribute to the susceptibilityto this disease. There is evidence that disease susceptibilityis under the control of multiple genes (5) and is associatedwith HLA-DR1, which is a known susceptibility factor (20).

In this study, significantly higher levels of IL-1ßwere observed in EPF patients after stimulation of their PBMCswith LPS. IL-1ß is a cytokine involved in inflammatoryresponses, and its presence may influence the lesion formationmechanism because of its ability to stimulate the expressionof intercellular adhesion molecules and the production of chemotacticpeptides, such as monocyte chemoattractant protein 1, by fibroblastsand epithelial cells (13, 16). Inflammatory mediators such asIL-1, prostaglandins, thromboxanes, and leukotrienes were previouslyobserved in patients with bullous skin diseases. Moreover, therole of IL-1 has been demonstrated in vivo and in vitro (2).Furthermore, the development of lesions by injection of patientserum into mice was less intense in IL-1-knockout mice thanin wild-type mice (8). These mediators are present at variousconcentrations, depending on the duration of blister development(10). Keratinocytes have been recognized as a source and targetof a large variety of cytokines. Keratinocytes express typeI and type II IL-1 receptors, and the presence of caspase 1in keratinocytes has been observed under inflammatory conditions,suggesting its ability to activate IL-1 (12).

Although the levels of regulatory cytokines were not significantlydifferent between the two groups evaluated in this study, IFN- ${gamma}$ levels were lower in EPF patients, while IL-5 levels were higherin these patients. A significant difference in the Th1-Th2 cytokinebalance was observed when cytokine levels were analyzed on thebasis of IL-5/IFN- ${gamma}$ ratios in supernatants after stimulationwith PHA for 120 h. EPF patients produced cytokines with boththe Th1 and the Th2 patterns, although a balance in favor ofTh2-like cytokines was observed. Conversely, healthy controlshad a balance in favor of Th1-like cytokines. This may reflectthe importance of Th2 cytokines in the production of antibodies,especially IgG4, a Th2-related immunoglobulin isotype (9). Regulatorycytokines were previously observed to play a role in the productionof cytokines with both the Th1 and the Th2 patterns in patientswith pemphigus vulgaris, with the Th2 cytokine response beingdominant (4, 18).

In conclusion, the results presented here support the hypothesisthat patients with EPF present a Th2-like cytokine responseand that other mediators, such as IL-1ß, a proinflammatorycytokine, may participate in the tissue damage observed in patientswith this disease. If the role of IL-1ß is confirmedby further investigations, it may allow the introduction ofnew therapeutic approaches for the management of EPF patients.

ACKNOWLEDGMENTS

This work was supported by FAPEMIG. G. Paschoini was supportedby CNPq/PIBIC.

FOOTNOTES

* Corresponding author. Mailing address: Faculdade de Medicina do Triângulo Mineiro, Laboratory of Immunology, 30, Frei Paulino, Uberaba, Minas Gerais 38025-180, Brazil. Phone: 55 34 3318 52 89. Fax: 55 34 3318 56 51. E-mail: vrodrigues{at}mednet.com.br.

REFERENCES

Top