Differential Expression of Cytokine mRNA in Skin Specimens from Patients with Erythema Migrans or Acrodermatitis Chronica Atrophicans
2000; Elsevier BV; Volume: 115; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2000.00198.x
ISSN1523-1747
AutoresRobert R. Müllegger, Gail McHugh, Robin Ruthazer, Barbara Binder, Helmut Kerl, Allen C. Steere,
Tópico(s)Dermatology and Skin Diseases
ResumoErythema migrans, the characteristic skin manifestation of acute Lyme borreliosis, is a self-limited lesion. In contrast, acrodermatitis chronica atrophicans, the typical cutaneous manifestation of late Lyme borreliosis, is a chronic skin condition. In an effort to understand pathogenic factors that lead to different outcomes in dermatoborrelioses, skin biopsy samples from 42 patients with erythema migrans and 27 patients with acrodermatitis chronica atrophicans were analyzed for mRNA expression of five pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β, interleukin-6, interferon-γ, and interleukin-2) and two anti-inflammatory cytokines (interleukin-4 and interleukin-10) by in situ hybridization with cytokine-specific riboprobes. Among the 27 patients who had erythema migrans alone with no associated signs or symptoms, the major cytokines expressed in perivascular infiltrates of T cells and macrophages were the pro-inflammatory cytokine interferon-γ and the anti-inflammatory cytokine interleukin-10. In the 15 erythema migrans patients who had associated signs and symptoms, including headache, elevated temperature, arthralgias, myalgias, or fatigue, a larger number of macrophages and greater expression of macrophage-derived pro-inflammatory cytokines, tumor necrosis factor α, interleukin-1β, and interleukin-6, were also found. In comparison, infiltrates of T cells and macrophages in the skin lesions of acrodermatitis chronica atrophicans patients had very little or no interferon-γ expression. Instead, they usually expressed only the pro-inflammatory cytokine tumor necrosis factor α and the anti-inflammatory cytokine interleukin-4. Thus, the activation of pro-inflammatory cytokines in erythema migrans lesions, particularly interferon-γ, seems to be important in the control of the spirochetal infection. In contrast, the restricted pattern of cytokine expression in acrodermatitis chronica atrophicans, including the lack of interferon-γ, may be less effective in spirochetal killing, resulting in the chronicity of this skin lesion. Erythema migrans, the characteristic skin manifestation of acute Lyme borreliosis, is a self-limited lesion. In contrast, acrodermatitis chronica atrophicans, the typical cutaneous manifestation of late Lyme borreliosis, is a chronic skin condition. In an effort to understand pathogenic factors that lead to different outcomes in dermatoborrelioses, skin biopsy samples from 42 patients with erythema migrans and 27 patients with acrodermatitis chronica atrophicans were analyzed for mRNA expression of five pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β, interleukin-6, interferon-γ, and interleukin-2) and two anti-inflammatory cytokines (interleukin-4 and interleukin-10) by in situ hybridization with cytokine-specific riboprobes. Among the 27 patients who had erythema migrans alone with no associated signs or symptoms, the major cytokines expressed in perivascular infiltrates of T cells and macrophages were the pro-inflammatory cytokine interferon-γ and the anti-inflammatory cytokine interleukin-10. In the 15 erythema migrans patients who had associated signs and symptoms, including headache, elevated temperature, arthralgias, myalgias, or fatigue, a larger number of macrophages and greater expression of macrophage-derived pro-inflammatory cytokines, tumor necrosis factor α, interleukin-1β, and interleukin-6, were also found. In comparison, infiltrates of T cells and macrophages in the skin lesions of acrodermatitis chronica atrophicans patients had very little or no interferon-γ expression. Instead, they usually expressed only the pro-inflammatory cytokine tumor necrosis factor α and the anti-inflammatory cytokine interleukin-4. Thus, the activation of pro-inflammatory cytokines in erythema migrans lesions, particularly interferon-γ, seems to be important in the control of the spirochetal infection. In contrast, the restricted pattern of cytokine expression in acrodermatitis chronica atrophicans, including the lack of interferon-γ, may be less effective in spirochetal killing, resulting in the chronicity of this skin lesion. acrodermatitis chronica atrophicans erythema migrans immunohistochemistry in situ hybridization Lyme borreliosis Lyme borreliosis (LB) is a complex multisystem disease caused by infection with the tick-borne spirochete Borrelia burgdorferi sensu lato (Steere et al., 1983aSteere A.C. Grodzicki R.L. Kornblatt A.N. et al.The spirochetal etiology of Lyme disease.N Engl J Med. 1983; 308: 733-740Crossref PubMed Scopus (1109) Google Scholar). Three pathogenic borrelial genospecies, B. burgdorferi sensu stricto, B. afzelii, and B. garinii, are known to cause human LB (Baranton et al., 1992Baranton G. Postic D. Saint Girons I. Boerlin P. Piffaretti J.C. Assous M. Grimont P.A.D. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov. & group VS 461 associated with Lyme borreliosis.Int J Syst Bacteriol. 1992; 42: 378-383Crossref PubMed Scopus (675) Google Scholar). To date, all North American strains have belonged to the first group, B. burgdorferi sensu stricto. All three genospecies have been found in Europe, but most isolates there have been B. afzelii or B. garinii strains. These differences may account for certain regional variations in the clinical picture of the infection (Anthonissen et al., 1994Anthonissen F.M. De-Kesel M. Hoet P.P. Bigaignon G.H. Evidence for the involvement of different genospecies of Borrelia in the clinical outcome of Lyme disease in Belgium.Res Microbiol. 1994; 145: 327-331Crossref PubMed Scopus (66) Google Scholar;Picken et al., 1998Picken R.N. Strle F. Picken M.M. Ruzic-Sabljic E. Maraspin V. Lotric-Furlan S. Cimperman J. Identification of three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. garinii, and B. afzelii) among isolates from acrodermatitis chronica atrophicans lesions.J Invest Dermatol. 1998; 110: 211-214https://doi.org/10.1046/j.1523-1747.1998.00130.xCrossref PubMed Scopus (92) Google Scholar). Erythema migrans (EM), the characteristic expanding skin lesion of acute LB, is self-limited in weeks to months (Steere et al., 1983bSteere A.C. Bartenhagen N.H. Craft J.E. et al.The early clinical manifestations of Lyme disease.Ann Intern Med. 1983; 99: 76-82Crossref PubMed Scopus (497) Google Scholar;Åsbrink and Olsson, 1985Åsbrink E. Olsson I. Clinical manifestations of erythema chronicum migrans Afzelius in 161 patients.Acta Derm Venereol (Stockh). 1985; 65: 43-52PubMed Google Scholar;Åsbrink and Hovmark, 1988Åsbrink E. Hovmark A. Early and late cutaneous manifestations in Ixodes-borne borreliosis (erythema migrans borreliosis, Lyme borreliosis).Ann NY Acad Sci. 1988; 539: 4-15Crossref PubMed Scopus (178) Google Scholar). In contrast, acrodermatitis chronica atrophicans (ACA), the typical skin manifestation of late LB, is a chronic and usually progressive skin condition that does not resolve spontaneously. It starts with an inflammatory phase and progresses slowly to an atrophic phase (Åsbrink and Hovmark, 1988Åsbrink E. Hovmark A. Early and late cutaneous manifestations in Ixodes-borne borreliosis (erythema migrans borreliosis, Lyme borreliosis).Ann NY Acad Sci. 1988; 539: 4-15Crossref PubMed Scopus (178) Google Scholar;Weber et al., 1993Weber K. Pfister H.W. Reimers C.D. Clinical overview.in: Weber K. Burgdorfer W. Aspects of Lyme Borreliosis. Springer, Berlin-Heidelberg-New York1993: 93-104Crossref Google Scholar). In a previous study in Austria, 1Zöchling N, Müllegger RR, Schlüpen EM et al. Molecular subtyping of Borrelia burgdorferi (Bb) in lesional skin from Stryrian (Austrian) patients with various manifestations of dermatoborreliosis (DB). J Invest Dermatol 104:687 1995 (abstr.)1Zöchling N, Müllegger RR, Schlüpen EM et al. Molecular subtyping of Borrelia burgdorferi (Bb) in lesional skin from Stryrian (Austrian) patients with various manifestations of dermatoborreliosis (DB). J Invest Dermatol 104:687 1995 (abstr.) as in all of Europe, B. afzelii is most often recovered from EM lesions, and ACA is almost always due to infection with this borrelial species (Wienecke et al., 1994Wienecke R. Zöchling N. Neubert U. Schlüpen E.M. Meurer M. Volkenandt M. Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronic atrophicans.J Invest Dermatol. 1994; 103: 19-22Abstract Full Text PDF PubMed Google Scholar;Picken et al., 1998Picken R.N. Strle F. Picken M.M. Ruzic-Sabljic E. Maraspin V. Lotric-Furlan S. Cimperman J. Identification of three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. garinii, and B. afzelii) among isolates from acrodermatitis chronica atrophicans lesions.J Invest Dermatol. 1998; 110: 211-214https://doi.org/10.1046/j.1523-1747.1998.00130.xCrossref PubMed Scopus (92) Google Scholar;Strle et al., 1999Strle F. Nadelman R.B. Cimperman J. et al.Comparison of culture-confirmed erythema migrans caused by Borrelia burgdorferi sensu stricto in New York State and by Borrelia afzelii in Slovenia.Ann Intern Med. 1999; 130: 32-36Crossref PubMed Scopus (169) Google Scholar). A variety of transient, nonspecific signs and symptoms may occur with European EM, including elevated temperature, lymphadenopathy, arthralgias, myalgias, or fatigue, but they usually do not suggest spread of the spirochete to specific organs (Weber and Neubert, 1986Weber K. Neubert U. Clinical features of early erythema migrans disease and related disorders.Zbl Bakt Hyg A. 1986; 263: 209-228PubMed Google Scholar;Åsbrink and Hovmark, 1988Åsbrink E. Hovmark A. Early and late cutaneous manifestations in Ixodes-borne borreliosis (erythema migrans borreliosis, Lyme borreliosis).Ann NY Acad Sci. 1988; 539: 4-15Crossref PubMed Scopus (178) Google Scholar). In contrast, in the U.S.A., where EM is caused by B. burgdorferi sensu stricto infection, faster spreading of skin lesions is frequently seen (Strle et al., 1999Strle F. Nadelman R.B. Cimperman J. et al.Comparison of culture-confirmed erythema migrans caused by Borrelia burgdorferi sensu stricto in New York State and by Borrelia afzelii in Slovenia.Ann Intern Med. 1999; 130: 32-36Crossref PubMed Scopus (169) Google Scholar), often accompanied by signs and symptoms, such as headache and neck stiffness or multiple skin lesions, that suggest hematogenous dissemination of the spirochete to other organ systems (Steere et al., 1983bSteere A.C. Bartenhagen N.H. Craft J.E. et al.The early clinical manifestations of Lyme disease.Ann Intern Med. 1983; 99: 76-82Crossref PubMed Scopus (497) Google Scholar;Berger, 1984Berger B.W. Erythema chronicum migrans of Lyme disease.Arch Dermatol. 1984; 120: 1017-1021Crossref PubMed Scopus (89) Google Scholar;Strle et al., 1999Strle F. Nadelman R.B. Cimperman J. et al.Comparison of culture-confirmed erythema migrans caused by Borrelia burgdorferi sensu stricto in New York State and by Borrelia afzelii in Slovenia.Ann Intern Med. 1999; 130: 32-36Crossref PubMed Scopus (169) Google Scholar). In Europe, the most common extracutaneous complications of ACA are arthropathy and peripheral neuropathy restricted to the affected limb (Åsbrink and Hovmark, 1988Åsbrink E. Hovmark A. Early and late cutaneous manifestations in Ixodes-borne borreliosis (erythema migrans borreliosis, Lyme borreliosis).Ann NY Acad Sci. 1988; 539: 4-15Crossref PubMed Scopus (178) Google Scholar), whereas in the U.S.A. ACA is almost never seen. Cytokines, which are important regulators and effectors of the immune response, influence the outcome of infectious and autoimmune diseases. The importance of these responses in the outcome of B. burgdorferi infection has been shown to have a genetically determined component in inbred strains of mice. When infected experimentally with B. burgdorferi, C3H mice develop severe arthritis, whereas BALB/c mice develop only mild arthritis (Barthold et al., 1990Barthold S.W. Beck D.S. Hansen G.M. Terwilliger G.A. Moody K.D. Lyme borreliosis in selected strains and ages of laboratory mice.J Infect Dis. 1990; 162: 133-138Crossref PubMed Scopus (316) Google Scholar;Schaible et al., 1991Schaible U.E. Kramer M.D. Wallich R. Tran T. Simon M.M. Experimental Borrelia burgdorferi infection in inbred mouse strains: antibody response and association of H-2 genes with resistance and susceptibility to development of arthritis.Eur J Immunol. 1991; 21: 2397-2405Crossref PubMed Scopus (78) Google Scholar;Yang et al., 1994Yang L. Weis J.H. Eichwald E. Kolbert C.P. Persing D.H. Weis J.J. Heritable susceptibility to severe Borrelia burgdorferi-induced arthritis is dominant and is associated with persistence of large numbers of spirochetes in tissues.Infect Immun. 1994; 62: 492-500PubMed Google Scholar). These strain-related differences are associated with polarized cytokine patterns and differences in spirochete burden in infected tissues. When infected with the spirochete, C3H mice, which have a high spirochete burden (Yang et al., 1994Yang L. Weis J.H. Eichwald E. Kolbert C.P. Persing D.H. Weis J.J. Heritable susceptibility to severe Borrelia burgdorferi-induced arthritis is dominant and is associated with persistence of large numbers of spirochetes in tissues.Infect Immun. 1994; 62: 492-500PubMed Google Scholar), produce gradually increasing amounts of interferon-γ (IFN-γ) with low to absent interleukin-4 (IL-4) levels, a Th1 pattern of cytokine production (Keane-Myers and Nickell, 1995Keane-Myers A. Nickell S.P. Role of IL-4 and IFN-γ in modulation of immunity to Borrelia burgdorferi in mice.J Immunol. 1995; 155: 2020-2028PubMed Google Scholar;Matyniak and Reiner, 1995Matyniak J.E. Reiner S.L. T-helper phenotype and genetic susceptibility in experimental Lyme disease.J Exp Med. 1995; 181: 1251-1254Crossref PubMed Scopus (151) Google Scholar;Kang et al., 1997Kang I. Barthold S.W. Persing D.H. Bockenstedt L.K. T-helper-cell cytokines in the early evolution of murine Lyme arthritis.Infect Immun. 1997; 65: 3107-3111PubMed Google Scholar). BALB/c mice initially have high levels of IFN-γ, which probably accounts for their low spirochete burden, but they switch to the production of IL-4, a Th2 cytokine pattern (Keane-Myers and Nickell, 1995Keane-Myers A. Nickell S.P. Role of IL-4 and IFN-γ in modulation of immunity to Borrelia burgdorferi in mice.J Immunol. 1995; 155: 2020-2028PubMed Google Scholar;Matyniak and Reiner, 1995Matyniak J.E. Reiner S.L. T-helper phenotype and genetic susceptibility in experimental Lyme disease.J Exp Med. 1995; 181: 1251-1254Crossref PubMed Scopus (151) Google Scholar;Kang et al., 1997Kang I. Barthold S.W. Persing D.H. Bockenstedt L.K. T-helper-cell cytokines in the early evolution of murine Lyme arthritis.Infect Immun. 1997; 65: 3107-3111PubMed Google Scholar), which is important in the resolution of joint inflammation. In patients with Lyme arthritis, cultured peripheral blood or synovial fluid mononuclear cells produced high amounts of IFN-γ and tumor necrosis factor α (TNF-α), but little or no IL-4 (Yssel et al., 1991Yssel H. Shanafelt M.C. Soderberg C. Schneider P.V. Anzola J. Peltz G. Borrelia burgdorferi activates a T helper type 1-like T-cell subset in Lyme arthritis.J Exp Med. 1991; 174: 593-601Crossref PubMed Scopus (233) Google Scholar;Yin et al., 1997Yin Z. Braun J. Neure L. et al.T-cell cytokine pattern in the joints of patients with Lyme arthritis and its regulation by cytokines and anticytokines.Arthritis Rheum. 1997; 40: 69-79Crossref PubMed Scopus (92) Google Scholar;Gross et al., 1998Gross D.M. Steere A.C. Huber B.T. T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis.J Immunol. 1998; 160: 1022-1028PubMed Google Scholar;Chen et al., 1999Chen J. Field J.A. Glickstein L. Molloy P. Huber B.T. Steere A.C. Association of antibiotic treatment-resistant Lyme arthritis with T cell responses to dominant epitopes of outer-surface protein A of Borrelia burgdorferi.Arthritis Rheum. 1999; 42: 1813-1822Crossref PubMed Scopus (82) Google Scholar). The reactivity of T cells and the production of pro-inflammatory cytokines were greater in synovial fluid than in peripheral blood (Gross et al., 1998Gross D.M. Steere A.C. Huber B.T. T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis.J Immunol. 1998; 160: 1022-1028PubMed Google Scholar). Moreover, the severity of arthritis correlated directly with the ratio of Th1 to Th2 cells in synovial fluid, such that the higher the number of Th1 cells, the more severe the joint swelling (Gross et al., 1998Gross D.M. Steere A.C. Huber B.T. T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis.J Immunol. 1998; 160: 1022-1028PubMed Google Scholar). In the synovial membrane of Lyme arthritis patients, mRNA expression of pro-inflammatory cytokines, including IFN-γ, TNF-α, and IL-1β, was predominant, but mRNA for anti-inflammatory cytokines was found as well (Yin et al., 1997Yin Z. Braun J. Neure L. et al.T-cell cytokine pattern in the joints of patients with Lyme arthritis and its regulation by cytokines and anticytokines.Arthritis Rheum. 1997; 40: 69-79Crossref PubMed Scopus (92) Google Scholar;Harjacek et al., 2000Harjacek M. Diaz-Cano S. Alman B.A. Coburn J. Ruthazer R. Wolfe H. Steere A.C. Prominent expression of mRNA for proinflammatory cytokines in synovium in patients with juvenile rheumatoid arthritis or chronic Lyme arthritis.J Rheumatol. 2000; 27: 497-503PubMed Google Scholar). In neuroborreliosis, cultured mononuclear cells from the peripheral blood and cerebrospinal fluid produced primarily IFN-γ, whereas IL-4 production was strikingly low (Wang et al., 1995Wang W.Z. Fredrikson S. Sun J.B. Link H. Lyme neuroborreliosis: evidence for persistent up-regulation of Borrelia burgdorferi-reactive cells secreting interferon-γ.Scand J Immunol. 1995; 42: 694-700Crossref PubMed Scopus (28) Google Scholar;Oksi et al., 1996Oksi J. Savolainen J. Pène J. Bousquet J. Laippala P. Viljanen M. Decreased interleukin-4 and increased gamma interferon production by peripheral blood mononuclear cells of patients with Lyme borreliosis.Infect Immun. 1996; 64: 3620-3623PubMed Google Scholar;Ekerfelt et al., 1997Ekerfelt C. Ernerudh J. Bunikis J. et al.Compartmentalization of antigen specific cytokine responses to the central nervous sytem in CNS borreliosis: secretion of IFN-γ predominates over IL-4 secretion in response to outer surface proteins of Lyme disease Borrelia spirochetes.J Neuroimmunol. 1997; 79: 155-162https://doi.org/10.1016/s0165-5728(97)00118-5Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Secretion of IFN-γ was most pronounced in cerebrospinal fluid (Wang et al., 1995Wang W.Z. Fredrikson S. Sun J.B. Link H. Lyme neuroborreliosis: evidence for persistent up-regulation of Borrelia burgdorferi-reactive cells secreting interferon-γ.Scand J Immunol. 1995; 42: 694-700Crossref PubMed Scopus (28) Google Scholar;Ekerfelt et al., 1997Ekerfelt C. Ernerudh J. Bunikis J. et al.Compartmentalization of antigen specific cytokine responses to the central nervous sytem in CNS borreliosis: secretion of IFN-γ predominates over IL-4 secretion in response to outer surface proteins of Lyme disease Borrelia spirochetes.J Neuroimmunol. 1997; 79: 155-162https://doi.org/10.1016/s0165-5728(97)00118-5Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Cytokine profiles have not yet been determined in patients with skin manifestations of LB, however, including in those with EM or ACA. In an effort to understand pathogenic factors that lead to different outcomes in dermatoborrelioses, we used in situ hybridization (ISH) with cytokine-specific riboprobes to analyze cytokine responses in patients with EM, a self-limited skin lesion, compared to those with ACA, a chronic, progressive lesion that does not resolve spontaneously. In 1997–8, we carried out a retrospective study of cytokine profiles of lesional skin in patients with EM or ACA. The study subjects consisted of 42 patients with EM (median age 43.5 y; 29 males and 13 females) and 27 patients with ACA (median age 63 y; seven males and 20 females) seen between June 1993 and December 1996 at the Department of Dermatology in Graz, Austria, for whom biopsy specimens and complete clinical data were available. Patients who had been treated with antibiotics prior to sampling or patients with conditions (e.g., atopy) or medications (e.g., nonsteroidal anti-inflammatory drugs or corticosteroids) that could potentially influence the status of the immune system were not included. EM was defined clinically as an expanding, sharply demarcated erythema, sometimes with partial central clearing, of at least 5 cm in diameter (Stanek et al., 1996Stanek G. O'Connell S. Cimmino M. et al.European Union concerted action on risk assessment in Lyme borreliosis: clinical case definitions for Lyme borreliosis.Wien Klin Wochenschr. 1996; 108: 741-747PubMed Google Scholar). Of the 42 study patients with EM, 16 (38%) had IgM and/or IgG antibody responses to B. burgdorferi, using purified, native flagella of B. afzelii, strain DK-1, as the test antigen (Dako Lyme Borreliosis ELISA Kit; Dako, Glostrup, Denmark) (Hansen et al., 1988Hansen K. Hindersson P. Pedersen N.S. Measurement of antibodies to the Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease.J Clin Microbiol. 1988; 26: 338-346PubMed Google Scholar). In addition, in 14 of 29 patients tested (48%), B. burgdorferi DNA was detected in lesional skin, using nested polymerase chain reaction (PCR) with two oligonucleotide primer pairs, as previously described (Wienecke et al., 1993Wienecke R. Neubert U. Volkenandt R. Molecular detection of Borrelia burgdorferi in formalin-fixed, paraffin-embedded lesions of Lyme disease.J Cutan Pathol. 1993; 20: 385-388Crossref PubMed Scopus (61) Google Scholar). Altogether, 34 of 42 patients (81%) with EM had serologic and/or PCR evidence of borrelial infection. The remaining eight patients with negative laboratory results had the characteristic clinical appearance of EM. Speciation of B. burgdorferi sensu lato subtypes was not done in the PCR-positive EM patients. ACA was defined clinically as a doughy bluish-red swelling, with or without signs of atrophy, on the extensor surfaces of the extremities (Stanek et al., 1996Stanek G. O'Connell S. Cimmino M. et al.European Union concerted action on risk assessment in Lyme borreliosis: clinical case definitions for Lyme borreliosis.Wien Klin Wochenschr. 1996; 108: 741-747PubMed Google Scholar). All 27 study patients (100%) with ACA had a positive serologic test result for IgG antibodies to B. burgdorferi. PCR testing was not done in these patients. Four millimeter punch biopsy specimens were taken from all patients from the active border of EM lesions or from the area of ACA lesions with the most prominent signs of inflammation. For comparison, we used 4 mm punch biopsy specimens obtained from seven EM patients from clinically uninvolved skin in the area opposite the site of the EM lesion. All samples were placed immediately in 4% formaldehyde/phosphate-buffered saline (PBS) and fixed overnight, dehydrated in graded ethanol, and embedded in paraffin. From each sample, serial sections of 4 μm were cut for histopathology and serial sections of 5 μm for immunohistochemistry (IHC) and ISH. In all patients, serial sections were stained with hematoxylin and eosin for histopathologic examination. In a subset of patients with EM or ACA in whom enough tissue was available, the expression of leukocyte differentiation antigens CD3 (T cells), CD20 (B cells), and CD68 (macrophages) was determined, using the avidin-biotin-peroxidase complex method (Vectastain ABC Elite Kit; Vector Laboratories, Burlingame, CA). For IHC, tissue sections mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) were deparaffinized with xylene and rehydrated through a decreasing ethanol series. Enhancement of immunoreactivity was achieved by microwave heating of the sections in 10 mM sodium citrate (pH 6.0). For CD3 and CD68, additional pretreatment with proteinase K (5 μg per ml) for 20 min was performed, followed by immersion in 4% paraformaldehyde/PBS. Non-specific binding was blocked using normal horse serum diluted 1:100 in PBS for 20 min. Sections were then incubated with a primary polyclonal rabbit antibody to CD3 (1:10 dilution in 2% bovine serum albumin/PBS) or monoclonal mouse antibodies to CD20 (1:100) or CD68 (1:100) for 30 min (Dako USA, Carpinteria, CA). Binding was detected with the universal biotinylated horse antimouse/rabbit IgG secondary antibody (Vector) diluted 1:50 in PBS containing normal horse serum for 30 min. The avidin-biotin-horseradish peroxidase complex (1:250 dilution) was then applied for 30 min. Sections were developed for 5 min with 3,3′-diaminobenzidine tetrahydrochloride as chromogenic substrate in H2O2, counterstained with hematoxylin, and mounted in an aqueous medium. As negative controls, nonspecific rabbit or mouse IgG antibodies (Vector) were used instead of the primary antibodies. As positive controls, the respective primary antibodies were applied to tissue sections of inflamed tonsils (a gift from Dr. P. Kwan, Department of Pathology, Tufts University, Boston, MA). All controls yielded the expected negative or positive results. Evaluation of IHC was performed by one blind observer using a Zeiss light microscope (Carl Zeiss, Thornwood, NY). The entire tissue section was examined under 400× magnification. The percentage of cells positive for a respective leukocyte marker was estimated relative to the total number of infiltrating cells. Positivity was graded on a scale from 0 to 3 as follows: 0, all infiltrating cells negative; 1, up to a third of all cells positive; 2, between one-third and two-thirds of all cells positive; 3, more than two-thirds of all cells positive. In each patient, the expression of mRNA for various pro-inflammatory (TNF-α, IL-1β, IL-6, IFN-γ, IL-2) and anti-inflammatory (IL-4, IL-10) cytokines was determined in skin biopsy samples by ISH with cytokine-specific riboprobes. To generate the specific antisense and sense probes, coding region cDNA fragments, which were kindly provided by Dr. P. Bucy (University of Alabama, Birmingham, AL), were subcloned into pBluescript vectors (Stratagene, La Jolla, CA). The cDNA fragments correspond to the following nucleotides of the respective human full-length cytokine sequences: TNF-α, 344–917 (574 bp); IL-1β, 445–1055 (611 bp); IL-6, 19–1128 (1110 bp); IFN-γ, 220–1193 (974 bp); IL-2, 145–800 (656 bp); IL-4, 64–518 (455 bp); and IL10, 411–1410 (1000 bp). GenBank accession numbers for these sequences are as follows: TNF-α, M10988; IL-1β, M15330; IL-6, M14584; IFN-γ, X13274; IL-2, V00564; IL-4, M13982; IL10, M57627. The sequences of the probes were confirmed (personal communication, Dr. P. Bucy, University of Alabama). For replication, cDNA-containing plasmids were transformed into competent Escherichia coli DH10B cells (Life Technologies, Gaithersburg, MD). Plasmid DNA was then purified from transformants (Wizard Plus Minipreps DNA Purification System; Promega, Madison, WI), digested with restriction endonucleases, and fractionated by agarose gel electrophoresis to screen for the presence of the appropriate insert. Following linearization of the plasmids with the appropriate restriction enzymes, transcription reactions were performed using T3or T7polymerases in the presence of digoxigenin-labeled UTP (SP6/T7 DIG RNA Labeling Kit; Boehringer Mannheim, Indianapolis, IN) to obtain antisense and sense riboprobes. The size of the transcribed RNA was tested by agarose gel electrophoresis. Labeling efficiency was determined on dot blots by comparing serial dilutions of the digoxigenin-labeled probe with a prelabeled control using chemiluminescent detection with antidigoxigenin Fab fragments conjugated to alkaline phosphatase, according to the manufacturer's protocol (Boehringer). ISH was performed with modifications of previously described methods (Lan et al., 1996Lan H.Y. Mu W. Ng Y.Y. Nikolic-Paterson D.J. Atkins R.C. A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology.J Histochem Cytochem. 1996; 44: 281-287Crossref PubMed Scopus (89) Google Scholar). Serial tissue sections were mounted on silane-coated Superfrost Plus slides (Fisher) and dried for 2 h at 60°C. Sections were deparaffinized with xylene and rehydrated through graded ethanol. Following treatment with 0.1 M glycine and permeabilization with 0.3% Triton X-100, the slides were placed in 10 mM sodium citrate (pH 6.0) and brought to the boil three times in a microwave oven (600 W). Sections were then incubated with proteinase K (5 μg per ml) for 20 min and immersed in 4% paraformaldehyde/PBS for 5 min, followed by ethanol dehydration. Thereafter, 25 μl of hybridization solution (40% formamide, 2 × sodium citrate/chloride buffer, 1 × Denhardt's solution, 10% dextran sulfate, 0.01 M dithiothreitol, 1 mg yeast tRNA per ml, and 1 mg sheared, denatured salmon sperm DNA per ml) containing 5 ng of the respective heat-denatured antisense or sense riboprobe were applied to each tissue section and covered with a cover slip. Hybridization was carried out overnight at 42°C in a microheating system with a moist environment (TruTemp; Robbins Scientific, Sunnyvale, CA). All steps were carried out with precautions to prevent RNAse degradation, including use of separate glassware and autoclaved solutions prepared in diethylpyrocarbonate-treated water. After hybridization, sections were washed under stringent conditions including treatment with 20 μg per ml RNAse A (Boehringer). For detection of hybridized probes, sections were blocked with normal horse serum and
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