Critical Role of Neutrophils for the Generation of Psoriasiform Skin Lesions in Flaky Skin Mice
2000; Elsevier BV; Volume: 114; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2000.00953.x
ISSN1523-1747
AutoresMargarete Schön, Robert Kubitza, Thomas Ruzicka, Michael P. Schön, Dirk Denzer,
Tópico(s)Psoriasis: Treatment and Pathogenesis
ResumoAlthough T cell dysregulation is thought to underlie the pathogenesis of psoriasis, prominent infiltration and microabscess formation by neutrophils is a distinctive hallmark feature of this common disorder. The exact role of neutrophils in the pathogenesis of psoriasiform alterations in vivo, however, is unknown. Similar to human psoriasis, flaky skin mice (fsn/fsn) revealed a prominent infiltrate of neutrophils, and microabscesses within the hyperproliferative epidermis were associated with de novo expression of intercellular adhesion molecule-1. Intraperitoneal injection with the neutrophil-depleting RB6-8C5 monoclonal antibody (anti-Ly-6G) resulted in a dramatic reduction of the epidermal thickness by 58% compared with isotype-treated animals (p < 0.001). In addition, epidermal microabscesses were conspicuously absent (p < 0.001), and cutaneous neutrophils and T cells, but not mast cells or dendritic cells, were markedly reduced in anti-Ly-6G-treated mice. Proinflammatory cytokines, including tumor necrosis factor α and interleukin-1, were also downregulated. Therapeutic effects occurred as early as 4 d after beginning of treatment. Wildtype skin was not affected.When the integrin αMβ2 (CD11b/CD18), which mediates neutrophil localization through binding to intercellular adhesion molecule-1, was blocked in vivo with the M1/70 monoclonal antibody, the epidermal thickness was reduced by 31% (p < 0.002), and neutrophil and T cell accumulation was diminished compared with control animals. Likewise, treatment of fsn/fsn mice with the MP1-22E9 monoclonal antibody neutralizing granulocyte macrophage-colony stimulating factor, a cytokine stimulating neutrophils by upregulating αMβ2, resulted in significant reduction of inflammation and acanthosis by 30% (p < 0.003). These results demonstrate a critical pathogenic role of neutrophils for hyperproliferative inflammatory lesions in fsn/fsn mice, suggesting that blocking neutrophil function may have therapeutic benefit in some human skin disorders. Although T cell dysregulation is thought to underlie the pathogenesis of psoriasis, prominent infiltration and microabscess formation by neutrophils is a distinctive hallmark feature of this common disorder. The exact role of neutrophils in the pathogenesis of psoriasiform alterations in vivo, however, is unknown. Similar to human psoriasis, flaky skin mice (fsn/fsn) revealed a prominent infiltrate of neutrophils, and microabscesses within the hyperproliferative epidermis were associated with de novo expression of intercellular adhesion molecule-1. Intraperitoneal injection with the neutrophil-depleting RB6-8C5 monoclonal antibody (anti-Ly-6G) resulted in a dramatic reduction of the epidermal thickness by 58% compared with isotype-treated animals (p < 0.001). In addition, epidermal microabscesses were conspicuously absent (p < 0.001), and cutaneous neutrophils and T cells, but not mast cells or dendritic cells, were markedly reduced in anti-Ly-6G-treated mice. Proinflammatory cytokines, including tumor necrosis factor α and interleukin-1, were also downregulated. Therapeutic effects occurred as early as 4 d after beginning of treatment. Wildtype skin was not affected. When the integrin αMβ2 (CD11b/CD18), which mediates neutrophil localization through binding to intercellular adhesion molecule-1, was blocked in vivo with the M1/70 monoclonal antibody, the epidermal thickness was reduced by 31% (p < 0.002), and neutrophil and T cell accumulation was diminished compared with control animals. Likewise, treatment of fsn/fsn mice with the MP1-22E9 monoclonal antibody neutralizing granulocyte macrophage-colony stimulating factor, a cytokine stimulating neutrophils by upregulating αMβ2, resulted in significant reduction of inflammation and acanthosis by 30% (p < 0.003). These results demonstrate a critical pathogenic role of neutrophils for hyperproliferative inflammatory lesions in fsn/fsn mice, suggesting that blocking neutrophil function may have therapeutic benefit in some human skin disorders. Neutrophilic granulocytes are prominently present within various chronic inflammatory skin disorders such as psoriasis, where neutrophil infiltration and focal accumulation within epidermal microabscesses (Munro's microabscesses) is a distinctive hallmark feature (Pinkus and Mehregan, 1966Pinkus H. Mehregan A.H. 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These include a number of neutrophil-activating polypeptide species such as interleukin-8 (IL-8), gro/MGSA (growth-related gene/melanoma growth stimulatory activity), or the complement split product C5ades arg (Schröder et al., 1987Schröder J.M. Mrowietz U. Morita E. Christophers E. Purification and partial biochemical characterisation of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin-1 activity.J Immunol. 1987; 139: 3474-3483PubMed Google Scholar,Schröder et al., 1988aSchröder J.M. Mrowietz U. Christophers E. Identification of different charged species of a human monocyte derived neutrophil activating peptide (MONAP).Biochem Biophys Res Commun. 1988; 152: 277-284Crossref PubMed Scopus (11) Google Scholar,1998b,van Damme et al., 1988van Damme J. van Beeuman J. Opdenakker G. 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Neutrophils can be stimulated by granulocyte macrophage-colony stimulating factor (GM-CSF) to rapidly upregulate surface expression of the αMβ2 integrin (CD11b/CD18) (Arnaout et al., 1986Arnaout M.A. Wang E.A. Clark S.C. Sieff C.A. Human recombinant granulocyte- macrophage colony-stimulating factor increases cell-to-cell adhesion and surface expression of adhesion-promoting surface glycoproteins on mature granulocytes.J Clin Invest. 1986; 78: 597-601Crossref PubMed Scopus (201) Google Scholar), which may result in extravasation and localization to ligand-bearing sites [e.g., intercellular adhesion molecule-1 (ICAM-1) expressing activated epidermal keratinocytes] (Gasson et al., 1984Gasson J.C. Weisbart R.H. Kaufman S.E. Clark S.C. Hewick R.M. Wong G.G. Golde D.W. Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils.Science. 1984; 226: 1339Crossref PubMed Scopus (323) Google Scholar;Clark and Kamen, 1987Clark S.C. Kamen R. The human hematopoietic colony-stimulating factors.Science. 1987; 236: 1229-1237Crossref PubMed Scopus (1172) Google Scholar). GM-CSF is expressed at elevated levels in psoriatic lesions, but it is unclear whether GM-CSF mediates inflammatory processes in psoriasis (Takematsu and Tagami, 1990Takematsu H. Tagami H. Granulocyte macrophage colony-stimulating factor in psoriasis.Dermatologica. 1990; 181: 16-20Crossref PubMed Scopus (27) Google Scholar). Neutrophils produce reactive oxygen intermediates and proteolytic enzymes, which are thought to affect growth and differentiation of keratinocytes, unmask some hidden antigens, or activate complement (Thelen et al., 1988Thelen M. Peveri P. Kernen P. von Tsarner V. Waltz A. Baggiolini M. Mechanisms of neutrophil activation by NAF, a novel monocyte-derived peptide agonist.FASEB J. 1988; 2: 2702-2706Crossref PubMed Scopus (220) Google Scholar;O'flaherty et al., 1991O'flaherty J.T. Rossi A.G. Redman J. Jacobsen D.P. 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Structural changes of human epidermis induced by human leukocyte-derived proteases.Exp Dermatol. 1999; 8: 46-52Crossref PubMed Scopus (12) Google Scholar). In addition, a decrease of SKALP/elafin activity may contribute to pathogenic neutrophil functions in some forms of psoriasis (Kuijpers et al., 1996Kuijpers A.L. Zeeuwen P.L. de Jongh G.J. van de Kerkhof P.C. Alkernade H.A. Schalkwijk J. Skin-derived antileukoproteinase (SKALP) is decreased in pustular forms of psoriasis. A clue to the pathogenesis of pustule formation.Arch Dermatol Res. 1996; 288: 641-647Crossref PubMed Google Scholar). This is possibly augmented by decreased activity of protease inhibitors, such as α1-proteinase inhibitor (Barszcz et al., 1988Barszcz D. Zarebska Z. Glinska-Ferenz M. Jablonska S. Tigalonowa M. Glinski W. Alpha1-proteinase inhibitor in psoriasis: reduced activity in symptom-free patients and during flare.Arch Dermatol Res. 1988; 280: 198-206Crossref PubMed Scopus (9) Google Scholar). Circumstantial evidence that neutrophils are involved in altered keratinocyte differentiation in psoriasis comes from the observation that Munro's microabscesses are located exclusively in areas of parakeratosis (van de Kerkhof and Lammers, 1987van de Kerkhof P.C.M. Lammers A.M. Intraepidermal accumulation of polymorphonuclear leukocytes in chronic stable plaque psoriasis.Dermatologica. 1987; 174: 224-227Crossref PubMed Scopus (23) Google Scholar). As neutrophils are present in very early lesions of psoriasis (van de Kerkhof and Chang, 1989van de Kerkhof P.C.M. Chang A. Migration of polymorphonuclear leukocytes in psoriasis.Skin Pharmacol. 1989; 2: 138-154Crossref PubMed Scopus (18) Google Scholar) and can stimulate DNA synthesis of keratinocytes in vitro through lipid mediators (Ristow, 1982Ristow H.J. Stimulation of DNA synthesis in cultured mouse epidermal cells by human peripheral leukocytes.J Invest Dermatol. 1982; 79: 408-411Crossref PubMed Scopus (11) Google Scholar;Kragballe et al., 1985Kragballe K. Desjarlais L. Vorhees J.J. Leukotrienes B4, C4 and D4 stimulate DNA synthesis in cultured human epidermal keratinocytes.Br J Dermatol. 1985; 113: 43-52Crossref PubMed Scopus (147) Google Scholar), it has been thought that neutrophils play an important pathogenic role in the psoriatic disease process (Nibbering et al., 1993Nibbering P.H. Thio B. Zomerdijk T.P. Bezemer A.C. Beijersbergen R.L. van Furth R. Effects of monomethylfumarate on human granulocytes.J Invest Dermatol. 1993; 101: 37-42Abstract Full Text PDF PubMed Google Scholar). There is no in vivo evidence to support this concept, however. To assess the in vivo function of neutrophils for the generation of hyperproliferative inflammatory skin alterations, we utilized the spontaneous mouse mutation flaky skin (fsn/fsn) (Sundberg et al., 1994Sundberg J.P. Boggess D. Schultz L.D. Beamer W.G. The Flaky Skin (fsn) mutation, chromosome?.in: Sundberg J.D. Handbook of Mouse Mutations with Skin and Hair Abnormalities. CRC Press, Boca Raton, FL1994: 253-268Google Scholar,Sundberg et al., 1997Sundberg J.P. France M. Boggess D. Sundberg B.A. Jenson A.B. Beamer W.G. Schultz L.D. Development and progression of psoriasiform dermatitis and systemic lesions in the flaky skin (fsn) mouse mutation.Pathobiology. 1997; 65: 261-286Crossref Scopus (44) Google Scholar;Pelsue et al., 1998Pelsue S.C. Schweitzer P.A. Schweitzer I.B. et al.Lymphadenopathy, elevated serum IgE levels, autoimmunity, and mast cell accumulation in flaky skin mutant mice.Eur J Immunol. 1998; 28: 1379-1388Crossref PubMed Google Scholar), as these animals showed both hyperproliferative inflammatory skin changes and accumulation and epidermal microabscess formation by neutrophils. Using these mice in antibody treatment studies, we present data showing that depletion of neutrophils results in dramatic alleviation of pathologic features in this hyperproliferative inflammatory disorder and that blocking either GM-CSF or the αMβ2 integrin (CD11b/CD18) has similar effects, albeit to a lesser extent. These data provide direct evidence that neutrophils are critically involved in the pathogenesis of psoriasiform lesions in vivo. Breeding pairs of CBy.A fsn/J mice (Jackson Laboratory, Bar Harbor, ME) were maintained in a specific-pathogen-free environment in a barrier facility. They were kept with a 12 h daily lighting, 50%-70% relative humidity, and at a temperature of 19–23°C in type-3 cages. Mice received autoclaved food (#1314, Altromin, Germany) and water (adjusted with HCl to pH 2.5–3.0 to prevent growth of microorganisms). As the genetic defect resulting in the flaky skin phenotype is unknown and as homozygous mutant mice were not fertile (Sundberg et al., 1994Sundberg J.P. Boggess D. Schultz L.D. Beamer W.G. The Flaky Skin (fsn) mutation, chromosome?.in: Sundberg J.D. Handbook of Mouse Mutations with Skin and Hair Abnormalities. CRC Press, Boca Raton, FL1994: 253-268Google Scholar), the offspring of heterozygous mice were used for all experiments. In the CBy.A congenic background, erythrosquamous skin lesions first appeared at the age of 5–6 wk, allowing the separation of homozygous mutant mice (hereafter designated fsn/fsn) from their wildtype or heterozygous littermates (hereafter designated +/?). Mice were used for histologic or immunohistochemical studies at the age of 6, 9, 11, 13, or 17 wk. From each of these time points, five fsn/fsn mice were compared with five phenotypically normal mice (littermates in most cases). For treatment studies, mice were used between 12 and 16 wk of age, after it had been established that the phenotype remained stable within this time frame. Mice were injected intra- peritoneally with 50, 100, 150, or 200 μg of the RB6-8C5 monoclonal antibody (MoAb) in 200 μl phosphate-buffered saline (PBS). Mice received one, two, three, or four injections in 2 d intervals. Mice were euthanized and tissues were collected 1 d after the final antibody administration. To study the effects of anti-αM or anti-GM-CSF MoAbs, mice were injected with 200 μl PBS containing 200 μg of MoAbs M1/70 or MP1-22E9, respectively. Control groups received a mixture of 200 μg R35-95 MoAb (rat IgG2a) and 200 μg SFR3-DR5 MoAb (rat IgG2b) in 200 μl PBS. Five +/? and five fsn/fsn mice were included in each treatment group. Depletion of neutrophils reported previously (Tepper et al., 1992Tepper R.I. Coffman R.L. Leder P. An eosinophil-dependent mechanism for the antitumor effect of interleukin-4.Science. 1992; 257: 548-551Crossref PubMed Scopus (463) Google Scholar) was confirmed by differential white blood counts and hematoxylin and eosin staining of paraffin sections. Penetration of MoAbs into the skin was confirmed by direct immunostaining of cryostat-cut sections using a biotinylated antirat antibody. Hybridoma cell lines were maintained in RPMI 1640 supplemented with 10% Ig-depleted fetal bovine serum, 10−5 M β-mercaptoethanol, 1% nonessential amino acids, 1% L-glutamine, 1% penicillin/streptomycin/amphotericin, and 15 mM HEPES buffer, and were cultured at 37°C with 5% CO2. MoAbs were purified from culture supernatant by affinity chromatography using a Protein G-sepharose column (Pharmacia Biotech). Bound MoAb was eluted using 0.5 M acetic acid, neutralized with 1:20 by volume of 1 M tri(hydroxymethyl)-aminomethane, pH 8.0, and dialyzed against PBS overnight. The concentration of MoAbs was determined fluorometrically (Bradford assay), and for in vivo studies MoAbs were diluted at 1 mg per ml in PBS. Immunostaining of cryostat-cut sections of mouse skin confirmed the specificity of the purified MoAbs. The protein G-sepharose columns were regenerated by elution with 1 M acetic acid, pH 2.5. Hybridomas producing the following MoAbs were cultured: M1/70 [anti-CD11b (αM-integrin), rat IgG2b; ATCC, Rockville, MD], 2E6 [anti-CD18 (β2-integrin), hamster IgG; ATCC], YN1/1.7.4 [anti-CD54 (ICAM-1), rat IgG2a; ATCC], N22 [anti major histocompatibility complex (MHC) class II, hamster IgG; ATCC], and RB6-8C5 (anti-Ly-6G, rat IgG; kindly provided by R. Coffman, DNAX Research Institute, Palo Alto, CA). The following MoAbs were purchased: R59-40 (rat IgG1 control; Pharmingen, San Diego, CA), R35-95 (rat IgG2a control, Pharmingen), R35-38 (rat IgG2b control, Pharmingen), UC8-4B3 (hamster IgG control, Pharmingen), 500A2 (anti-CD3ε, hamster IgG, Pharmingen), MP1-22E9 (anti-GM-CSF, rat IgG2a, Pharmingen). Biotinylated goat antihamster serum and mouse-adsorbed rabbit antirat serum were purchased from Vector Laboratories (Burlingame, CA). Histologic pro-cedures were performed using 5–6 μm sections of paraffin-embedded tissue. Hematoxylin and eosin or Giemsa staining of the sections was performed according to standard protocols. Chloroacetate esterase staining was performed as described previously (Yam et al., 1971Yam L.T. Li C.Y. Crosby W.H. Cytochemical identification of monocytes and granulocytes.Am J Clin Pathol. 1971; 55: 283-290Crossref PubMed Scopus (2029) Google Scholar). Briefly, 1 g new fuchsin (Sigma, St Louis, MO) was dissolved in 25 ml 2 N HCl, and an equal volume of freshly prepared 4% NaNO2 was added. Then, 0.05 ml of the new fuchsin solution and 1 mg naphthol-AS-D-chloroacetate (Sigma) dissolved in 0.5 ml N,N′-dimethyl-formamide (Sigma) was added to 9.5 ml phosphate buffer (0.15 M, pH 7.6). Tissue sections were incubated with the final solution for 10 min, and counterstained with hematoxylin and LiCO3. For immunohistochemistry, 7 μm cryostat-cut sections were stained by the ABC-immunoperoxidase method (Vector). Briefly, air-dried sections were fixed in acetone for 10 min (except for cytokine detection which required a short fixation of 25–30 s) and incubated with buffer containing 10% fetal bovine serum, 10% normal goat serum, 5% normal rabbit serum, and 1% normal horse serum for 30 min. Sections were then incubated with 10 μg per ml of the primary antibody for 1 h, followed by 0.3% H2O2 in PBS for 20 min to block endogenous peroxidase. According to the primary antibody used, slides were incubated with biotinylated secondary antibody, followed by the avidin–peroxidase complex according to the manufacturer's instructions (Vector) for 45 min. Slides were then submerged in 3-amino-9-ethylcarbazole substrate solution in 0.1 M acetate buffer (pH 5.2). Color development was stopped by 10% formalin (pH 5.2) for 10 min. Subsequently, slides were counterstained with hematoxylin and LiCO3. Photomicrographs of stained sections from each mouse were scanned using the Nikon Coolscan II® software. Epidermal thickness was assessed at 10 different points in each hematoxylin and eosin stained section, and the average thickness was calculated. Infiltrating leukocytes were evaluated as cells per mm skin. Data are represented as mean ± SD (n = 5). Statistical significance was assessed by the paired two-tailed Student's t test, and p < 0.05 was considered significant. The offspring of heterozygous intercrosses were monitored clinically for the occurrence of erythrosquamous skin lesions. The first psoriasiform skin lesions were observed at 5–6 wk of age and were characterized clinically by the development of erythema and loose whitish scales. These alterations were most pronounced on the dorsal skin, worsened until adulthood, and spread over most of the body surface. No further increase in clinical severity was apparent after 11–13 wk of age. In contrast to other rodent models with hyperproliferative inflammatory skin disorders (Hammer et al., 1990Hammer R.E. Maika S.D. Richardson J.A. Tang J.P. Taurog J.D. Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human b2m: an animal model of HLA-B27-associated human disorders.Cell. 1990; 63: 1099-1112Abstract Full Text PDF PubMed Scopus (788) Google Scholar;Carroll et al., 1995Carroll J.M. Romero M.R. Watt F.M. Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.Cell. 1995; 83: 957-968Abstract Full Text PDF PubMed Scopus (277) Google Scholar;Schön et al., 1997Schön M.P. Detmar M. Parker C.M. Murine psoriasis-like disorder induced by naive CD4+ T-cells.Nature Med. 1997; 3: 183-188Crossref PubMed Scopus (138) Google Scholar), the ears of fsn/fsn mice were largely spared. Dorsal skin from fsn/fsn and +/? mice were harvested at 6, 9, 11, 13, and 17 wk of age to histopathologically assess the morphology of the skin lesions and determine the distribution of neutrophils (n = 5 for each time point and genotype). As expected (Sundberg et al., 1993Sundberg J.G. Boggess D. Sundberg B.A. Beamer W.G. Shultz L.D. Epidermal dendritic cell populations in the flaky skin mouse mutant.Immunol Invest. 1993; 22: 389-401Crossref PubMed Scopus (32) Google Scholar,Sundberg et al., 1994Sundberg J.P. Boggess D. Schultz L.D. Beamer W.G. The Flaky Skin (fsn) mutation, chromosome?.in: Sundberg J.D. Handbook of Mouse Mutations with Skin and Hair Abnormalities. CRC Press, Boca Raton, FL1994: 253-268Google Scholar), there was a 7.5 fold thickening of the viable epidermal layers (acanthosis) in homozygous mutant mice compared with +/? littermates [0.603 mm (SD = 0.03) vs 0.080 mm (SD = 0.009), p < 0.0001) (Figure 1a). This was associated with profound hyperkeratosis (increased thickness of the stratum corneum). In addition, dermal blood vessels were increased in number and size, and there was a dense mixed inflammatory infiltrate composed of mononuclear cells, macrophages, mast cells, and abundant neutrophilic granulocytes. Neutrophils were seen within the vasculature adjacent to endothelial cells, in the perivascular area, and diffusely within the dermis. Notably, neutrophils focally accumulated within the hyperproliferative epidermis and formed intraepidermal microabscesses similar to Munro's microabscesses seen in human psoriasis (Figure 1a). Occasionally, areas of parakeratosis (nuclei in the stratum corneum) indicating altered keratinocyte differentiation were observed, and these were located in some cases next to neutrophil accumulations or epidermal microabscesses. Neutrophil infiltration was readily seen at 6 wk of age. The number of epidermal microabscesses appeared to increase between 6 wk (2.9 per mm skin, SD = 0.7) and 13 wk of age (5.4 per mm skin, SD = 0.8), although this difference did not reach statistical significance (p = 0.078). No dermal neutrophils or epidermal microabscesses were seen in the skin of +/? mice (Figure 1a). Psoriasiform distribution of neutrophils in cutaneous lesions of fsn/fsn mice was confirmed by immunohistochemical staining with the RB6-8C5 MoAb, which exclusively stained polymorphonuclear cells within the dermis and epidermis of fsn/fsn mice (Figure 1b, left panel). These cells were not present within the skin of their normal littermates (Figure 1b, right panel) indicating that this antigen is a selective marker to target neutrophils infiltrating the skin of fsn/fsn mice. As neutrophil infiltration and micro- abscess formation in fsn/fsn mice were similar to those seen in human psoriasis, we sought to deplete neutrophils to directly assess their role in the pathogenesis and maintenance of the murine hyperproliferative inflammatory lesions in vivo. +/? and fsn/fsn mice were injected intraperitoneally four times in 2 d intervals with 200 μg of the neutrophil-depleting anti-Ly-6G MoAb RB6-8C5 (Tepper et al., 1992Tepper R.I. Coffman R.L. Leder P. An eosinophil-dependent mechanism for the antitumor effect of interleukin-4.Science. 1992; 257: 548-551Crossref PubMed Scopus (463) Google Scholar). Control mice received an isotype-matched antibody. Penetration of the MoAbs into the skin of the recipient mice was confirmed by immunohistochemistry using a goat antirat antibody to detect the injected MoAbs within the skin (not shown). Neutrophil depletion reported previously (Tepper et al., 1992Tepper R.I. Coffman R.L. Leder P. An eosinophil-dependent mechanism for the antitumor effect of interleukin-4.Science. 1992; 257: 548-551Crossref PubMed Scopus (463) Google Scholar) was confirmed by differential white blood counts (data not shown). When the skin of the treated mice was examined histologically, no apparent effect of the anti-Ly-6G treatment was seen in +/? mice (Figure 2 and Table 1). In contrast, epidermal thickness was dramatically reduced by 58% in anti-Ly-6G-treated fsn/fsn mice compared with control fsn/fsn mice [0.251 mm (SD = 0.04) vs 0.603 mm (SD = 0.03), p < 0.001, Figure 2 and Table 1]. This was accompanied by a markedly diminished neutrophil infiltrate, and epidermal microabscesses were almost completely absent in anti-Ly-6G-treated fsn/fsn mice compared with control mice [0.2 (SD = 0.03) vs 5.3 (SD = 0.61) abscesses per mm skin, p < 0.001, Figure 2].Table 1Effect of antibody treatment on histopathologic parameters in the skin of fsn/fsn mice (n = 5 in each group) and +/? mice (n = 5)ap < 0.05Genotype Treatmentfsn/fsn isotypefsn/fsn anti-Ly-6Gfsn/fsn anti-αMfsn/fsn anti-GM-CSF+/? isotype+/? anti-Ly-6G+/? anti-αM+/? anti-GM-CSFEpidermal thickness (mm)0.603 ± 0.030.251 ± 0.04**p < 0.05, comparing with mice of the same genotype treated with an isotype-matched control MoAb.0.416 ± 0.04*p < 0.050.422 ± 0.03**p < 0.05, comparing with mice of the same genotype treated with an isotype-matched control MoAb.0.080 ± 0.0090.080 ± 0.0060.080 ± 0.0060.081 ± 0.007CD3+ T cells per mm108.4 ± 19.338.0 ± 6.7**p < 0.05, comparing with mice of the same genotype treated with an isotype-matched control MoAb.61.2 ± 4.1*p < 0.0555.2 ± 6.8*p < 0.0511.5 ± 0.812.1 ± 2.310.8 ± 1.211.9 ± 1.1MHC II+ epidermal dendritic cells per mm47.2 ± 2.444.4 ± 5.146.8 ± 1.845.1 ± 3.824.4 ± 0.922.9 ± 1.424.6 ± 1.722.2 ±1.5Mast cells per mm96.2 ± 16.097.2 ± 8.097.6 ± 3.395.9 ± 12.39.6 ± 0.79.4 ± 1.010.3 ± 0.69.6 ± 0.7a p < 0.05* p < 0.05** p < 0.05, comparing with mice of the same genotype treated with an isotype-matched control MoAb. Open table in a new tab In subsequent series of experiments, the treatment was repeated using 50, 100, 150, or 200 μg of MoAbs and mice were injected one, two, three, or four times with these concentrations in 2 d intervals. It was found that psoriasiform features were alleviated using doses as low as 100 μg of the RB6-8C5 MoAb. As assessed by reduction of the epidermal thickness, therapeutic effects became apparent after 3–4 d, and no further changes occurred when mice were observed for more than 6 d (data not shown). To assess the effect of neutrophil depletion in fsn/fsn mice in more detail, the distribution of CD3+ T lymphocytes, MHC class II+ epidermal dendritic cells (Langerhans cells), and mast cells was assessed by immuno- histochemistry, Giemsa staining and chloroacetate esterase staining. It was found that in fsn/fsn mice treated with anti-Ly-6G MoAb, the number of cutaneous CD3+ T cells was reduced significantly by 65% compared with mice treated with an isotype control MoAb [108.4 cells per mm (SD = 19.3) vs 38.0 cells per mm (SD = 6.7), p < 0.005, Figure 3(a,b) and Table 1]. In contrast, immunohistochemical staining using an anti-MHC class II MoAb revealed that injection with the anti-Ly-6G MoAb did not diminish the number of epidermal dendritic cells [47.
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