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PubMed2007

Modifying the beta,gamma leaving-group bridging oxygen alters nucleotide incorporation efficiency, fidelity, and the catalytic mechanism of DNA polymerase beta.

DNA polymerase catalysis and fidelity studies typically compare incorporation of "right" versus "wrong" nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the beta,gamma-bridging O with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) beta. T.G mismatches occur with fidelities similar to dGTP with the exception of the CH2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with Kds between 1 and 4 microM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brnsted correlations of log(kpol) versus leaving-group pKa for both right and wrong base incorporation reveal similar sensitivities (betalg approximately -0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pKas of CF2, O, and the sterically bulky CCl2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3'-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.

Base Sequence↗
PubMed2004

Carbon tetrachloride transformation on the surface of nanoscale biogenic magnetite particles.

Iron-reducing conditions in subsurface environments promote dechlorination reactions via both biotic and abiotic pathways, the latter often mediated via biologically activated minerals formed by dissimilatory iron-reducing bacteria (DIRB). Here we report the major products and pathways associated with the abiotic transformation of carbon tetrachloride (CT) by nanoscale biogenic magnetite/maghemite particles produced by the DIRB Geobacter metallireducens. Product formation and free radical/carbene trapping studies indicate that CT transformation occurs via three parallel pathways. The first pathway (hydrogenolysis) results in the formation of chloroform (45-50%) via a trichloromethyl free radical (*CCl3) and possibly a trichloromethyl carbanion (**CCl3-). The second and third pathways involve a dichlorocarbene intermediate (**CCl2), which either hydrolyzes to form CO (approximately 38%) (carbene hydrolysis), or undergoes further reduction to yield methane (8-10%) (carbene reduction). The mechanism of methane formation from **CCl2 is not known, but is speculated to involve a sequence of surface coordinated carbenoid and free radical complexes. The large fraction of relatively benign products formed by the carbene-mediated pathways suggests that magnetite/maghemite particles may have a beneficial application in the remediation of CT contaminated environments.

Carbon Tetrachloride↗
PubMed2003

Impairment of leukocyte trafficking in a murine pleuritis model by IL-4 and IL-10.

We have characterized leukocyte migration to the pleural cavity in a methylated-BSA (mBSA)-induced model of murine delayed-type hypersensitivity and evaluated the ability of IL-4 and IL-10 to modulate this response. Neutrophils, macrophages, T cells, and dendritic cells migrated to the pleural cavity in a time-dependent fashion following direct intrapleural antigen challenge, with neutrophils comprising the majority of exudate leukocytes in the cavity within the first 24 h and the number of mononuclear cells increasing at later times. Real-time quantitative PCR analysis of infiltrating leukocytes revealed a marked elevation of steady-state mRNA levels of IL-1beta and TNFalpha and the chemokines KC, MIP-2, CXCL9, CXCL10, CXCL11, CCL2, CCL3, and CCL4 at 6 h postchallenge, which diminished over time. In contrast, gammaIFN mRNA levels were maximal at 24 h and CCL5 expression was sustained throughout 72 h. ELISA analysis of pleural exudate fluid revealed significant elevations of KC and CCL2 protein levels at 6 h postantigen challenge and a peak increase in gammaIFN protein at 24 h, confirming our mRNA observations. Administration of recombinant murine IL-4 or IL-10 prior to challenge significantly blocked cell trafficking to the pleural cavity as well as peak levels of exudate gammaIFN, with IL-4 being more potent in impairing these responses. IL-4 administration also increased the proportion of naive T cells in the pleural cavity, as judged by CD62L and CD45RB expression. These results indicate that this in vivo model demonstrates a pattern of events associated with Th1-mediated leukocyte trafficking and underscore the potential utility of this in vivo model for evaluating therapeutic inhibitors of leukocyte trafficking.

Animals↗
PubMed2001

Chemokines as regulators of T cell differentiation.

Chemokines play well established roles as attractants of naïve and effector T cells. New studies indicate that chemokines also have roles in regulating T cell differentiation. Blocking Gi protein-coupled receptor signaling by pertussis toxin as well as deficiencies in G alpha 12, chemokine receptor 2 (CCR2), CCR5, chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1, or MCP-1), CCL3 (macrophage inflammatory protein 1 alpha, or MIP-1 alpha) and CCL5 (RANTES) have all been found to have effects on the magnitude and cytokine polarity of the T cell response. Here we focus on findings in the CCL2-CCR2 and CCL3-CCR5 ligand-receptor systems. The roles of these molecules in regulating T cell fate include possible indirect effects on antigen-presenting cells and direct effects on differentiating T cells. Models to account for the action of chemokines and G protein-coupled receptor signals in regulating T cell differentiation are discussed.

Animals↗
PubMed2006

Stimulation of purinergic receptors modulates chemokine expression in human keratinocytes.

ATP is abundantly released from stressed or damaged cells in response to mechanical stimulation, bacteria, or noxious agents. In this study, we have investigated the possible involvement of P2 receptors (receptor for extracellular nucleotides) in the expression and release of inflammatory mediators by human keratinocytes. Notably, extracellular ATP displayed a complex regulation of IFN-gamma-stimulated chemokine expression, with upregulation of CC chemokine ligand 2 (CCL2), CCL5 and CXC chemokine ligand 8 (CXCL8), and suppression of the receptor CXC chemokine receptor 3 (CXCR3), CXCL9, CXCL10, and CXCL11. The effect of ATP was mimicked by ADP and adenosine-5'-O-3-thiotriphosphate, whereas 2',3'-O-(4-benzoylbenzoyl) ATP (BzATP) downmodulated all chemokines investigated. UTP had no effect on IFN-gamma-stimulated chemokine secretion. The broad-spectrum P2 receptor antagonist suramin and the selective P2Y1 inhibitor adenosine 3'-phosphate 5'-phosphosulfate counteracted the effect of ATP on secretion of all the chemokines examined, whereas pyridoxal phosphate 6-azophenyl 2',4'-disulfonic acid and KN62 (1-[N,O-bis(5-isoquinoline sulfonyl)-N-methyl-L-tyrosyl] 4 phenylpiperazine) partially prevented the inhibitory effect of ATP on CXCL10 secretion, but on the other hand potentiated the ATP-stimulatory effect on CCL5, CCL2, and CXCL8 release. In lesional skin of psoriasis and atopic dermatitis patients, intense P2X7 reactivity was confined to the cell membrane of the basal layer, whereas diffuse P2Y1 immunostaining was found throughout the epidermis. Collectively, our data suggest that the orchestrated activation of distinct P2Y and P2X receptors modulates skin inflammation.

Adenosine Triphosphate↗
PubMed2007

Fimbrial lectins influence the chemokine repertoire in the urinary tract mucosa.

The defense against mucosal infections relies on chemokines that recruit inflammatory cells to the mucosa. This study examined if the chemokine response to uro-pathogenic Escherichia coli is influenced by fimbrial expression. The CXC (CXCL1, CXCL5, CXCL8, CXCL9, CXCL10) and CC chemokines (CCL2, CCL3, CCL5) were quantified after in vitro infection of uro-epithelial cells with a fimbriated E. coli pyelonephritis isolate, or with P or type 1 fimbriated transformants of an avirulent E. coli K-12 strain. The response profile was shown to vary with the fimbrial type. Type 1 fimbriated E. coli elicited mainly CXCL1 and CXCL8, whereas P fimbriated E. coli stimulated CCL2 and CCL5 and class II were more potent chemokine inducers than class III P fimbriae. Chemokines were also quantified in urine samples from 73 patients with febrile urinary tract infection, and analyzed as a function of disease severity and fimbrial expression by the strain infecting each patient. A complex CXC and CC chemokine response was detected in patient urine, with a significant influence of the fimbrial type. The results show that virulence factors like fimbriae may modify the mucosal chemokine response. This mechanism may allow the host to adjust the inflammatory cell infiltrate to fit the infecting strain.

Adhesins, Escherichia coli↗
PubMed2004

Vacuum ultraviolet-infrared photo-induced Rydberg ionization spectroscopy: C-H stretching frequencies for trans-2-butene and trichloroethene cations.

We have demonstrated the two-color vacuum ultraviolet (VUV)-infrared (IR) photoinduced Rydberg ionization (PIRI) experiment. Trichloroethene (ClCH=CCl2) and trans-2-butene (trans-CH3CH=CHCH3) were prepared in Rydberg states in the range of effective principal quantum number n* approximately 7-93 by VUV excitation prior to IR-induced autoionization. The observed VUV-IR-PIRI spectra are found to be independent of n*, suggesting that the electron Rydberg orbital is conserved, i.e., the Rydberg electron is behaving as a spectator during the excitation process. The observed IR active C-H stretching vibrational frequencies nu12+ = 3072+/-5 cm(-1) for ClCH=CCl2+ and nu23+ =2908+/-3 cm(-1), nu25+ =2990+/-10 cm(-1) and nu30+ =3022+/-10 cm(-1) for trans-CH3CH=CHCH3+ are compared with predictions based on ab initio quantum-chemical procedures and density functional calculations.

Journal Article↗
PubMed2004

Electron-impact ionization of CCl4 and CCl2F2.

Absolute partial and total cross sections for electron-impact ionization of CCl4 and CCl2F2 are reported for electron energies from threshold to 1000 eV. The product ions are mass analyzed using a time-of-flight mass spectrometer and detected with a position-sensitive detector whose output demonstrates that all product ion species are collected with equal efficiency irrespective of their initial kinetic energies. Data are presented for production of CCl3(+), CCl2(+), CCl+, C+, Cl2(+), and CCl3(2+) from CCl4; and for production of CCl(2)F+, CClF2(+), CClF(+), (CCl+ + CF2(+)), Cl+, CF+, F+, and C+ from CCl2F2. Data are also reported for formation of (CCl2(+),Cl+) and (CCl+, Cl+) ion pairs from CCl4. The total cross section for each target is obtained as the sum of the partial cross sections. The overall uncertainty in the absolute cross sections for most of the singly charged ions is +/- 5-7 %. The present partial cross sections for lighter fragment ions are found to be considerably greater than had been previously reported but the most recent total cross section measurements agree well with those reported here. Neither the binary-encounter-Bethe theory nor the Deutsch-Mark theory reproduces the experimental cross sections correctly for both targets.

Journal Article↗
PubMed2005

Induction of a proinflammatory program in normal human thyrocytes by the RET/PTC1 oncogene.

Rearrangements of the RET receptor tyrosine kinase gene generating RET/PTC oncogenes are specific to papillary thyroid carcinoma (PTC), the most frequent thyroid tumor. Here, we show that the RET/PTC1 oncogene, when exogenously expressed in primary normal human thyrocytes, induces the expression of a large set of genes involved in inflammation and tumor invasion, including those encoding chemokines (CCL2, CCL20, CXCL8, and CXCL12), chemokine receptors (CXCR4), cytokines (IL1B, CSF-1, GM-CSF, and G-CSF), matrix-degrading enzymes (metalloproteases and urokinase-type plasminogen activator and its receptor), and adhesion molecules (L-selectin). This effect is strictly dependent on the presence of the RET/PTC1 Tyr-451 (corresponding to RET Tyr-1062 multidocking site). Selected relevant genes (CCL20, CCL2, CXCL8, CXCR4, L-selectin, GM-CSF, IL1B, MMP9, UPA, and SPP1/OPN) were found up-regulated also in clinical samples of PTC, particularly those characterized by RET/PTC activation, local extrathyroid spread, and lymph node metastases, when compared with normal thyroid tissue or follicular thyroid carcinoma. These results, demonstrating that the RET/PTC1 oncogene activates a proinflammatory program, provide a direct link between a transforming human oncogene, inflammation, and malignant behavior.

Blotting, Western↗
PubMed2002

Identification of biologically active chemokine isoforms from ascitic fluid and elevated levels of CCL18/pulmonary and activation-regulated chemokine in ovarian carcinoma.

Chemokines are important in leukocyte homeostasis, inflammation, angiogenesis, and metastasis. Here, the molecular diversity of chemokines present in ovarian carcinoma was studied by purifying the proteins to homogeneity from ascitic fluid. Biologically active intact CCL2 and processed CXCL8, CCL3, and CCL18 isoforms were recovered. CCL7 and CCL20 were also purified, but their levels were 10-fold lower compared with CXCL8, CCL2, and CCL3 and even 100-fold lower than the amounts of CCL18 isolated. In ascitic fluids from patients with ovarian carcinoma (n = 12), significantly higher levels of CXCL8 and CCL18 (2.0 versus 0.7 ng/ml (p = 0.01) and 120 versus 44 ng/ml (p = 0.0002), respectively) were detected compared with those in nonovarian carcinoma patients (n = 12). In contrast to CXCL8, CCL18 was not inducible in carcinoma cell lines. Immunostaining showed CCL18 expression in tumor-infiltrating cells with monocyte/macrophage morphology but not in the ovarian carcinoma cells. Our data demonstrate that biochemically heterogenous but biologically active forms of several chemokines are present at different concentrations in ovarian carcinoma ascitic fluid. This points to a delicate balance of chemokines in epithelial ovarian cancer and to a potentially major role for CXCL8 and CCL18 in this tumor.

Ascites↗
PubMed2005

Simultaneous blockade of NFkappaB, JNK, and p38 MAPK by a kinase-inactive mutant of the protein kinase TAK1 sensitizes cells to apoptosis and affects a distinct spectrum of tumor necrosis factor [corrected] target genes.

The inflammatory response is characterized by the induction (or repression) of hundreds of genes. The activity of many of these genes is controlled by MAPKs and the IkappaB kinase-NFkappaB pathway. To reveal the effects of blocking these pathways simultaneously, fibroblasts were infected with retroviruses encoding TAK1K63W, an inactive mutant of the protein kinase TAK1. Expression of this protein inhibited tumor necrosis factor (TNF)-induced activation of NFkappaB, JNK, and p38 MAPK and sensitized the cells to TNF-induced apoptosis. 23 different microarray experiments were used to analyze the expression of >7000 genes in these cells. We identified 518 genes that were regulated by TNF in both TAK1K63W-expressing cells and control cells, 37 genes induced by TNF only when TAK1K63W was present, and 48 TNF-induced genes that were suppressed by TAK1K63W. The TNF-inducible genes that were most strongly suppressed by TAK1K63W, ccl2, ccl7, ccl5, cxcl1, cxcl5, cxcl10, saa3, and slpi also had much lower basal levels of expression, indicating that TAK1 also played a role in their normal expression. Chromatin immunoprecipitation studies on four of these genes suggested that inactivation of TAK1 activity led to direct suppression of expression at the transcriptional level because of impaired recruitment of RNA polymerase II to their promoters. ccl2 induction by TNF or interleukin-1 was also suppressed in cells that expressed TAK1 antisense RNA or that were genetically deficient in JNK1/2 or p65 NFkappaB. These data suggest that regulation of the expression of a selected group of inflammation-related genes is funneled through TAK1, making it a potentially useful target for more specific anti-inflammatory drug development.

Animals↗
PubMed2004

Expression of chemokines by human fetal microglia after treatment with the human immunodeficiency virus type 1 protein Tat.

Chemokines are important mediators of inflammation. It has been demonstrated that there is an increase in chemokine expression in both the sera and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1). The HIV-1 viral protein, Tat, a transcriptional regulator, has been detected in the central nervous system (CNS) of infected individuals, and has been demonstrated to induce chemokines from various cells within the brain. The authors now show that the interaction of human microglia, the resident phagocytes of the brain, with Tat leads to dramatic increases in the secretion of the chemokines CCL2, CXCL8, CXCL10, CCL3, CCL4, and CCL5. Treatment of microglia with Tat plus specific inhibitors of signal transduction pathways demonstrated that the induction of each chemokine is regulated differently. Tat-induced expression of CCL2 and CCL4 was mediated by the activation of the extracellular regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI3K) pathway, whereas the induction of CXCL8 and CCL3 was mediated only by the p38 MAPK pathway. Tat-induced CXCL10 expression was mediated, to some extent, by activation of the ERK1/2 MAPK pathway, phosphatidylinositol 3-kinase pathway, and the p38 MAPK pathway, whereas CCL5 expression was not mediated by any pathway tested. Western blot analysis demonstrated phosphorylation of ERK 1/2 and Akt upon stimulation of microglia with Tat. These data suggest that a soluble HIV-1 viral protein can alter the chemokine balance in the brain, which can then lead to an influx of inflammatory cells and contribute to the neuropathogenesis of HIV-1 infection.

AIDS Dementia Complex↗
PubMed2002

Choline-binding protein A of Streptococcus pneumoniae elicits chemokine production and expression of intercellular adhesion molecule 1 (CD54) by human alveolar epithelial cells.

Recruitment of neutrophils into alveolar air spaces is an early event in the pathogenesis of pneumonia due to Streptococcus pneumoniae. This results from chemokines released by activated endothelial and epithelial cells and alveolar macrophages. Culture supernatants of 6 wild-type strains of S. pneumoniae, shown to contain choline-binding protein A (CbpA; clades A and B), induced release of chemokine CXCL8 from the human alveolar epithelial cell line A549, whereas a CbpA deletion mutant elicited significantly reduced CXCL8 release, compared with that of its isogenic parent (P<.01). Recombinant CbpA up-regulated expression of messenger RNA of CXCL8 and CCL2 but not of XCL1, CXCL10, CCL1, CCL3, CCL4, or CCL5 in A549 cells and induced increased secretion of CXCL8, CCL2, CXCL1, and CXCL5 in a dose- and time-dependent manner. CbpA also increased the expression of intercellular adhesion molecule 1 (CD54) by A549 cells. Thus, CbpA of S. pneumoniae induces the transcription and release of proinflammatory molecules by human alveolar epithelial cells.

Antigens, Bacterial↗
PubMed1989

Inactivation of O6-alkylguanine-DNA alkyltransferase in HeLa cells by cisplatin.

Inactivation of O6-alkylguanine-DNA alkyltransferase (O6-AGT) in HeLa CCL2 cells by cisplatin was studied. HeLa CCL2 cells treated with cisplatin showed a dose-dependent decline in O6-AGT activity. After cisplatin was removed and replaced with fresh medium, the transferase level began to rise slowly. By 72 h slightly more than 80% of the activity was recovered. It seems that the activity of the alkyltransferase can be inactivated by platinated DNA adducts. The data suggest that the O6-platinum-guanine formation and the O6-alkyltransferase depletion are not responsible for cytotoxicity but may result in a base substitution mutation in mammalian cells.

Cell Survival↗
PubMed2004

Inflammatory status and cartilage regenerative potential of synovial fibroblasts from patients with osteoarthritis and chondropathy.

OBJECTIVES: To evaluate the inflammatory status and the cartilage regenerative potential of pathological synovial fibroblasts from patients with osteoarthritis (OA) compared with non-inflamed synovium (NS)-derived cells from patients with chondropathy. METHODS: The inflammatory cell phenotype was investigated based on the constitutive and inducible surface expression and secretion of various effector molecules using flow cytometry or ELISA assays. The capacity of cells to produce cartilage-like extracellular matrix was assessed using acid Alcian blue staining and type II collagen immunostaining after treatment with transforming growth factor beta1 (TGF-beta1). RESULTS: OA and NS fibroblasts consistently expressed CD29, CD44, CD49e, CD54, CD90 and CD106. Expression of high-affinity receptors for IL-4, IL-15, CXCL8 and CXCL12 was also detected but only intracellularly. All types of fibroblasts spontaneously released abundant amounts of CXCL12, CCL2, IL-6 and tissue inhibitor of metalloproteinase 1, while the production of IL-11, TGF-beta1, matrix metalloproteinase 1 (MMP-1) and MMP-9 was detected at moderate levels. Several other secreted factors remained undetectable. No statistically significant differences were noted between the two groups of fibroblasts. Treatment with the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) up-regulated the same set of surface and secreted molecules, including CD54, CD106, membrane IL-15, CCL2 and CCL5. Under TGF-beta1 treatment and adipogenic culture conditions, both OA and NS fibroblasts displayed chondrogenic and adipocytic activities that were reduced in OA compared with NS cells. CONCLUSIONS: OA synovial fibroblasts did not display a distinct activated inflammatory phenotype compared with NS cells. However, they did differ in their reduced ability to produce cartilage-like matrix. This difference may be an additional important factor contributing to OA pathogenesis.

Adipocytes↗
PubMed2005

VIP down-regulates TLR4 expression and TLR4-mediated chemokine production in human rheumatoid synovial fibroblasts.

OBJECTIVES: Vasoactive intestinal peptide (VIP) has demonstrated therapeutic effects in arthritis by inhibiting both innate and acquired immune responses. We investigated the potential effects of VIP in the regulation of Toll-like receptor (TLR) expression and function in synovial fibroblasts from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Cultured fibroblast-like synoviocytes (FLS) were obtained from patients with RA and OA. The effects of VIP on basal or TNF-alpha or lipopolysaccharide (LPS)-induced TLR2, TLR4 and MyD88 expression and its effects on TLR4-mediated CCL2 and CXCL8 chemokine production were studied by reverse transcription-polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay. RESULTS: TLR2, TLR4 and MyD88 mRNA expression was increased in RA FLS compared with OA FLS. The largest increase was observed for TLR4 and there was also overexpression at the protein level in RA FLS. TLR4 and MyD88 mRNA and proteins were induced by LPS and TNF-alpha in RA FLS. VIP down-regulated the induced but not the constitutive expression of TLR4 and MyD88 in RA FLS. VIP treatment decreased CCL2 and CXCL8 chemokine production in response to TLR4 activation with LPS in RA FLS. CONCLUSIONS: We demonstrate that VIP down-regulates LPS and TNF-alpha activation of TLR4 expression and the TLR4 functional response in terms of proinflammatory chemokine production. These studies suggest that the pleiotropic anti-inflammatory actions of VIP involve inhibitory effects on TLR4 expression and signalling.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Bacterial cytochromes c biogenesis.

We report the primary sequence analyses of two loci, hel and ccl, whose gene products are required specifically for the biogenesis of c-type cytochromes in the Gram-negative photosynthetic bacterium Rhodobacter capsulatus. Genetic and molecular analyses show that the hel locus contains at least four genes, helA, helB, helC, and orf52, and the ccl locus contains two genes, ccl1 and ccl2, that are essential for cytochromes c biogenesis. HelA is homologous to a class of proteins called ABC transporters and helA, helB, and helC are proposed to encode an export complex. Cytochrome c2-alkaline phosphatase gene fusions were used to show that apocytochrome c2 synthesis and secretion are not affected by the hel and ccl defects. Ccl1 and Ccl2 possess typical signal sequences to direct them to the periplasm. The periplasmic orientation of Ccl1 was confirmed using a Ccl1-alkaline phosphatase gene fusion. The Ccl1-alkaline phosphatase gene fusion analysis also demonstrated that Ccl1 does not require hel genes for its synthesis and secretion. Ccl1 is homologous to proteins encoded by chloroplast and mitochondrial genes, suggesting analogous functions in these organelles. Taken together, these results support the hypothesis that the hel-encoded proteins are required for the export of heme to the periplasm where it is subsequently ligated to the c-type apocytochromes.

Alkaline Phosphatase↗
PubMed2006

A canine model of cutaneous late-phase reactions: prednisolone inhibition of cellular and cytokine responses.

Immunoglobulin E (IgE)-mediated late-phase reactions can be induced in atopic humans by intradermal injection of relevant allergens or anti-IgE antibodies. The histology of these reactions resembles that of naturally occurring atopic dermatitis. Strikingly similar responses can be induced in dogs, suggesting that a canine model could prove valuable for preclinical investigation of drugs targeting late-phase reactions. This study was designed to characterize the cellular, cytokine and chemokine responses after intradermal anti-IgE injection in untreated and prednisolone-treated dogs. Normal beagles were untreated or treated with prednisolone before intradermal injection of polyclonal rabbit anti-canine IgE or normal rabbit IgG. Biopsies were taken before injection and 6, 24 and 48 hr after injection. Samples were evaluated by histological and immunohistochemical staining, as well as by real-time quantitative polymerase chain reaction analysis. Dermal eosinophil and neutrophil numbers increased dramatically within 6 hr after injection of rabbit anti-canine IgE, and remained moderately elevated at 48 hr. The numbers of CD1c(+) and CD3(+) mononuclear cells were also increased at 6 hr. The real-time quantitative polymerase chain reaction demonstrated marked increases in mRNA expression for interleukin-13 (IL-13), CCL2, CCL5 and CCL17. Levels of mRNA for IL-2, IL-4, IL-6 and IFN-gamma did not change within the limits of detection. Prednisolone administration suppressed the influx of neutrophils, eosinophils, CD1c(+) and CD3(+) cells, as well as expression of IL-13, CCL2, CCL5 and CCL17. These data document the cytokine and chemokine responses to anti-IgE injection in canine skin, and they demonstrate the ability of the model to characterize the anti-inflammatory effects of a known therapeutic agent.

Animals↗