These studies were carried out to examine the presence of the inflammatory cytokines IL-6 and TNFalpha in kidneys of patients with lupus nephritis as an indicator of their possible role in its pathogenesis. A total of 19 kidney biopsies from patients with type III or IV lupus nephritis were processed by direct immunofluorescence using monoclonal anti-IL-6 and TNFalpha antibodies. Local expression of these genes was demonstrated both by in situ hybridization and by reverse transcriptase-PCR amplification of total RNA isolated from kidney tissue. Fifty-two percent of the biopsies exhibited IL-6 and TNFalpha deposited along the glomeruli and tubules; in situ expression of these cytokines was demonstrated in 6 biopsies with type IV, and 1 with type III nephritis. Inflammatory cytokines are actively synthesized in the kidneys of patients with lupus nephritis and therefore, may play a role in its pathogenesis.

RIASSUNTO La formazione di depositi di materiale immune lungo la giunzione dermo-epidermica nei pazienti affetti da lupus eritematoso sistemico (LES), è il risultato dall'interazione in situ degli anticorpi antinucleari con i loro rispettivi antigeni. L'esposizione al sole determina la liberazione di autoantigeni cutanei e simultaneamente la produzione di heat shock proteins (Hsp). In questo studio abbiamo valutato mediante immunofluorescenza la presenza di Hsp70 lungo la giunzione dermo-epidermica in 20 biopsie cutanee di pazienti affetti da LES ed in 20 soggetti di controllo. I depositi immuni cutanei erano pricipalmente composti da IgG, IgM e C3. Tali depositi sono stati evidenziati lungo la giunzione dermo-epidermica in tutte le biopsie dei pazienti affetti da LES. Erano inoltre evidenti depositi di materiale immune nei vasi cutanei ed in misura minore nei keratinociti epidermici. La presenza di Hsp70 è stata evidenziata nel 60% delle biopsie dei pazienti affetti da LES. Questa proteina era pricipalmente distribuita lungo la giun-view that Hsp are induced in the skin after sun exposure, we theorized that Hsp would shuttle autoantigens to the dermo-epidermal junction. To assess our hypothesis the presence of Hsp70 concurrent with lupus band immunoreagents was studied in skin biopsies from lupus patients. MATERIAL AND METHODSPatients. Twenty patients (18 females and 2 males; mean age 23 years, range 16-35 years) with systemic lupus erythematosus diagnosed according to ACR classification criteria were studied (6). Skin biopsies were performed after informed consent had been obtained. Twenty biopsies taken from the skin of the back of healthy individuals during orthopaedic surgery were used as controls.Skin Biopsies. Samples were obtained from non-lesional and unexposed skin using a 5 mm punch, under local anaesthesia with 1% xilocaine (Astra). The biopsies were rinsed in 0.15 M NaCl pH 7.5 buffer solution (PBS), and immediately included in Tissue-Tek (Ames), and frozen at -20°C. The specimens were then cryosectioned at 4 µm, and used for immunofluorescent assays. Direct immunofluorescence (IF). Skin biopsies were examined by IF as follows: 4 µm skin sections were fixed in cold acetone, washed in PBS and incubated 30 minutes with monospecific goat anti human-IgG, IgA, IgM, C3, C4, C1q and fibrinogen antibodies (Sigma. St Louis MO). After washings with PBS, the slides were evaluated using an Olympus B-Max microscope equipped with epifluorescence. Controls were processed equally. Double labelling Assay. Skin sections were incubated with FITC (green tagged) monospecific anti-IgG, IgA and IgM antibodies; after washing the slides were incubated with a monoclonal antiHsp70 antibody (Sigma); next, specimens were washed and reincubated with a second antibody tagged in red (rabbit anti-mouse rodamin conjugated IgG) to disclose the Hsp70. Fluorescein and rodamine filter combinations were used, with excitations of 488 and 546 nm and emissions of 520 and 568 nm respectively. RESULTS Patients.None of SLE patients were under ...

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