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1.  Mast cell anaphylatoxin receptor expression can enhance IgE-dependent skin inflammation in mice 
Background
Mast cells express receptors for complement anaphylatoxins C3a and C5a (i.e., C3aR and C5aR), and C3a and C5a are generated during various IgE-dependent immediate hypersensitivity reactions in vivo. However, it is not clear to what extent mast cell expression of C3aR or C5aR influences C3a- or C5a-induced cutaneous responses or IgE-dependent mast cell activation and passive cutaneous anaphylaxis (PCA), in vivo.
Objective
Assess whether mouse skin mast cell expression of C3aR or C5aR influences: 1) the cells’ responsiveness to intra-dermal injections of C3a or C5a, or 2) the extent of IgE-dependent mast cell degranulation and PCA in vivo.
Methods
We measured the magnitude of cutaneous responses to i.d. injections of C3a or C5a, and the extent of IgE-dependent mast cell degranulation and PCA responses, in mice containing mast cells that did or did not express C3aR or C5aR.
Results
The majority of the skin swelling induced by i.d. injection of C3a or C5a required that mast cells at the site expressed C3aR or C5aR, respectively, and the extent of IgE-dependent degranulation of skin mast cells and of IgE-dependent PCA were significantly reduced when mast cells lacked either C3aR or C5aR. IgE-dependent PCA responses associated with local elevation of C3a occurred in antibody-deficient mice but not in mice deficient in FcεRIγ.
Conclusion
Expression of C3aR and C5aR by skin mast cells contributes importantly to the ability of C3a and C5a to induce skin swelling and can enhance mast cell degranulation and inflammation during IgE-dependent PCA in vivo.
doi:10.1016/j.jaci.2012.05.009
PMCID: PMC3597773  PMID: 22728083
Anaphylatoxin; anaphylaxis; C3a; C5a; complement; IgE; inflammation; mast cells
2.  IL1B Polymorphisms Modulate Cystic Fibrosis Lung Disease 
Pediatric pulmonology  2009;44(6):580-593.
Summary
Rationale: Variability in pulmonary disease severity is found in patients with cystic fibrosis (CF) who have identical mutations in the CF transmembrane conductance regulator (CFTR) gene. We hypothesized that one factor accounting for heterogeneity in pulmonary disease severity is variation in the family of genes affecting the biology of interleukin-1 (IL-1), which impacts acquisition and maintenance of Pseudomonas aeruginosa infection in animal models of chronic infection. Methods: We genotyped 58 single nucleotide polymorphisms (SNPs) in the IL-1 gene cluster in 808 CF subjects from the University of North Carolina and Case Western Reserve University (UNC/CWRU) joint cohort. All were homozygous for ΔF508, and categories of “severe” (cases) or “mild” (control subjects) lung disease were defined by the lowest or highest quartile of forced expired volume (FEV1) for age in the CF population. After adjustment for age and gender, genotypic data were tested for association with lung disease severity. Odds ratios (ORs) comparing severe versus mild CF were also calculated for each genotype (with the homozygote major allele as the reference group) for all 58 SNPs. From these analyses, nine SNPs with a moderate effect size, OR ≤ 0.5or > 1.5, were selected for further testing. To replicate the case-control study results, we genotyped the same nine SNPs in a second population of CF parent-offspring trios (recruited from Children’s Hospital Boston), in which the offspring had similar pulmonary phenotypes. For the trio analysis, both family-based and population-based associations were performed. Results: SNPs rs1143634 and rs1143639 in the IL1B gene demonstrated a consistent association with lung disease severity categories (P < 0.10) and longitudinal analysis of lung disease severity (P < 0.10) in CF in both the case-control and family-based studies. In females, there was a consistent association (false discovery rate adjusted joint P-value < 0.06 for both SNPs) in both the analysis of lung disease severity in the UNC/CWRU cohort and the family-based analysis of affection status. Conclusion: Our findings suggest that IL1β is a clinically relevant modulator of CF lung disease.
doi:10.1002/ppul.21026
PMCID: PMC3716579  PMID: 19431193
gene modifiers; cystic fibrosis; CFTR; IL-1 gene family
3.  Predictors of Mucoid Pseudomonas Colonization in Cystic Fibrosis Patients 
Pediatric pulmonology  2008;43(5):463-471.
Summary
Rationale: Chronic mucoid Pseudomonas aeruginosa within the airway in cystic fibrosis (CF) patients can determine prognosis. Understanding the risk factors of mucoid P. aeruginosa acquisition may change how we deliver care. This study aims to evaluate whether presence of risk factors reported to predict disease severity including gender, CFTR genotype, bacterial organisms in airway cultures, and serum levels of vitamins A and E, albumin, C-reactive protein, alpha 1-antitrypsin, and immunoglobulins increased the risk of mucoid P. aeruginosa acquisition. Methods: Primary endpoint was age at first transition from negative to positive culture for mucoid P. aeruginosa. Cox proportional hazards regression with time-dependent covariates examined development of mucoid P. aeruginosa infection and its association with longitudinally measured serum biomarkers, pulmonary function, and culture results for other organisms. Results: Median ages at CF diagnosis and at first culture were 0.55 and 5.7 years, respectively. Median number of cultures/patient was 17. Of the 323 subjects, 150 developed mucoid P. aeruginosa during a median 8.1 years’ follow-up. In multivariate analysis, gender (relative hazard [RH] 0.55 for male vs. female, P=0.001), number of DF508 alleles (RH 1.66 for1 or 2 vs. 0, P=0.04), FEV1 % (RH 1.16 for 10% decrease, P=0.008), and most recent Staphylococcus aureus status (RH 0.24 for positive vs. negative, P< 0.0001) remained statistically significant. Conclusion: Female gender, number of DF508 alleles, decreased lung function, and lack of S. aureus on recent sputum culture are important risk factors for early detection of mucoid P. aeruginosa.
doi:10.1002/ppul.20794
PMCID: PMC3693457  PMID: 18361452
cystic fibrosis; Pseudomonas colonization
4.  C5a Receptor Deficiency Alters Energy Utilization and Fat Storage 
PLoS ONE  2013;8(5):e62531.
Objective
To investigate the impact of whole body C5a receptor (C5aR) deficiency on energy metabolism and fat storage.
Design
Male wildtype (WT) and C5aR knockout (C5aRKO) mice were fed a low fat (CHOW) or a high fat high sucrose diet-induced obesity (DIO) diet for 14 weeks. Body weight and food intake were measured weekly. Indirect calorimetry, dietary fatload clearance, insulin and glucose tolerance tests were also evaluated. Liver, muscle and adipose tissue mRNA gene expression were measured by RT-PCR.
Results
At week one and 12, C5aRKO mice on DIO had increased oxygen consumption. After 12 weeks, although food intake was comparable, C5aRKO mice had lower body weight (−7% CHOW, −12% DIO) as well as smaller gonadal (−38% CHOW, −36% DIO) and inguinal (−29% CHOW, −30% DIO) fat pads than their WT counterparts. Conversely, in WT mice, C5aR was upregulated in DIO vs CHOW diets in gonadal adipose tissue, muscle and liver, while C5L2 mRNA expression was lower in C5aRKO on both diet. Furthermore, blood analysis showed lower plasma triglyceride and non-esterified fatty acid levels in both C5aRKO groups, with faster postprandial triglyceride clearance after a fatload. Additionally, C5aRKO mice showed lower CD36 expression in gonadal and muscle on both diets, while DGAT1 expression was higher in gonadal (CHOW) and liver (CHOW and DIO) and PPARγ was increased in muscle and liver.
Conclusion
These observations point towards a role (either direct or indirect) for C5aR in energy expenditure and fat storage, suggesting a dual role for C5aR in metabolism as well as in immunity.
doi:10.1371/journal.pone.0062531
PMCID: PMC3646841  PMID: 23667486
5.  Th17 cytokines are critical for RSV associated airway hyperreponsiveness through regulation by complement C3a and tachykinins1 
Respiratory syncytial virus (RSV) infection is associated with serious lung disease in infants and immunocompromised individuals and is linked to development of asthma. In mice, acute RSV infection causes airway hyperresponsiveness (AHR), inflammation, and mucus hypersecretion. Infected cells induce complement activation, producing the anaphylatoxin C3a. Here we show RSV infected wild type mice produce Th17 cytokines, a response not previously associated with viral infections. Mice deficient in the C3aR (C3aR1−/−) fail to develop AHR following acute RSV infection, and production of Th17 cytokines was significantly attenuated. Tachykinin production has also been implicated in RSV pathophysiology, and tachykinin receptor null mice (TACR1−/−) were similarly protected from developing AHR. These animals were also deficient in production of Th17 cytokines. Tachykinin release was absent in C3aR1−/− mice, while C3a levels were unchanged in TACR1−/− animals. Thus, our data reveal a crucial sequence following acute RSV infection where initial C3a production causes tachykinin release, followed by activation of the IL-17A pathway. Deficiency of either receptor affords protection from AHR, identifying two potential therapeutic targets.
doi:10.4049/jimmunol.1101789
PMCID: PMC3186836  PMID: 21918196
respiratory syncytial virus; C3a anaphylatoxin; complement; inflammation; IL17A; airway hyperresponsiveness; tachykinins; substance P; hemokinin-1
6.  Effect of Inhibition of Angiotensin-Converting Enzyme and/or Neutral Endopeptidase on Neuropathy in High-Fat-Fed C57Bl/6J Mice 
Journal of Obesity  2012;2012:326806.
We have demonstrated that treating diet-induced obese (DIO) mice with the vasopeptidase inhibitor ilepatril improved neural function. Vasopeptidase inhibitors block angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) activity. We propose that increased activity of ACE and NEP contributes to pathophysiology of DIO. To address this issue C57Bl/6J mice or mice deficient in NEP were fed a high-fat diet and treated with ilepatril, enalapril, ACE inhibitor, or candoxatril, NEP inhibitor, using both prevention and intervention protocols. Endpoints included glucose utilization and neural function determination. In the prevention study glucose tolerance was impaired in DIO C57Bl/6J mice and improved with ilepatril or enalapril. Sensory nerve conduction velocity, thermal nociception, and intraepidermal nerve fiber density were impaired in DIO C57Bl/6J mice and improved with ilepatril or candoxatril. In the intervention study only enalapril improved glucose tolerance. Sensory nerve conduction velocity and intraepidermal nerve fiber density were improved by all three treatments, whereas thermal nociception was improved by ilepatril or candoxatril. In NEP-deficient mice DIO impaired glucose utilization and this was improved with enalapril. Nerve function was not impaired by DIO in NEP-deficient mice. These studies suggest that ACE and NEP play a role in pathophysiology associated with DIO.
doi:10.1155/2012/326806
PMCID: PMC3465928  PMID: 23056927
7.  Memory T cells migrate to and reject vascularized cardiac allografts independent of the chemokine receptor CXCR31 
Transplantation  2011;91(8):827-832.
Background
Memory T cells migrate to and reject transplanted organs without the need for priming in secondary lymphoid tissues, but the mechanisms by which they do so are not known. Here we tested whether CXCR3, implicated in the homing of effector T cells to sites of infection, is critical for memory T cell migration to vascularized allografts.
Methods
CD4 and CD8 memory T cells were sorted from alloimmunized CXCR3−/− and wildtype B6 mice and co-transferred to congenic B6 recipients of BALB/c heart allografts. Graft-infiltrating T cells were quantitated 20 and 72 hours later by flow cytometry. Migration and allograft survival were also studied in splenectomized alymphoplastic (aly/aly) recipients, which lack secondary lymphoid tissues.
Results
We found that polyclonal and antigen-specific memory T cells express high levels of CXCR3. No difference in migration of wildtype vs CXCR3−/− CD4 and CD8 memory T cells to allografts could be detected in either wildtype or aly/aly hosts. In the latter, wildtype and CXCR3−/−memory T cells precipitated acute rejection at similar rates. Blocking CCR5, a chemokine receptor also upregulated on memory T cells, did not delay graft rejection mediated by CXCR3−/− memory T cells.
Conclusions
CXCR3 is not critical for the migration of memory T cells to vascularized organ allografts. Blocking either CXCR3 or CXCR3 and CCR5 does not delay acute rejection mediated by memory T cells. These findings suggest that the mechanisms of memory T cell homing to transplanted organs may be distinct from those required for their migration to sites of infection.
doi:10.1097/TP.0b013e31820f0856
PMCID: PMC3073028  PMID: 21285915
T lymphocyte; chemokine receptor; transplantation; rejection
8.  Vasopeptidase Inhibitor Ilepatril (AVE7688) Prevents Obesity- and Diabetes-induced Neuropathy in C57Bl/6J Mice 
Neuropharmacology  2010;60(2-3):259-266.
Previously we demonstrated that inhibition of neutral endopeptidase (NEP), a protease that degrades vaso- and neuro-active peptides, and angiotensin converting enzyme (ACE) with a vasopeptidase inhibitor improves vascular and neural function in diabetic rat models. The purpose of this study was to determine whether inhibition of NEP and ACE or deletion of NEP provides protection from nerve impairment caused by diabetes or diet induced obesity (DIO). To determine the role of NEP and ACE inhibition in neuropathy related to insulin-deficient diabetes or DIO we used C57Bl/6J mice treated with AVE7688, a vasopeptidase inhibitor, or NEP deficient (−/−) mice. Mice at 12 weeks of age were fed a high fat diet for 12 weeks or were diabetic for duration of 12 weeks following a single injection of high dose streptozotocin. Both a prevention and intervention protocol was used for AVE7688 treatment. Glucose utilization was impaired in DIO C57Bl/6J and NEP −/− mice. However, treating DIO C57Bl/6J or NEP −/− mice with AVE7688 improved glucose tolerance. Thermal hypoalgesia and nerve conduction slowing were present in both streptozotocin-diabetic and DIO C57Bl/6J mice but not in AVE7688 treated C57Bl/6J mice or NEP −/− mice exposed to either streptozotocin-induced diabetes or a high fat diet. Intraepidermal nerve fiber (IENF) profiles were decreased in the hindpaw of C57Bl/6J diabetic or DIO mice and this improved when the mice were treated with AVE7688. IENF profiles were not decreased in diabetic or DIO NEP (−/−) mice. These studies suggest that NEP plays a role in regulating nerve function in insulin-deficient diabetes and DIO.
doi:10.1016/j.neuropharm.2010.09.008
PMCID: PMC3014404  PMID: 20849865
diabetic neuropathy; neutral endopeptidase; vasopeptidase inhibitor; streptozotocin; diet-induced obesity; pain
9.  Chemokine receptor CXCR3 promotes growth of glioma 
Carcinogenesis  2010;32(2):129-137.
Human glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of GBM indicates a need to identify new therapeutic targets. In this study, we examined the role of CXCR3 in glioma progression using the GL261 murine model of malignant glioma. Intracranial GL261 tumors express CXCL9 and CXCL10 in vivo. Glioma-bearing CXCR3-deficient mice had significantly shorter median survival time and reduced numbers of tumor-infiltrated natural killer and natural killer T cells as compared with tumor-bearing wild-type (WT) mice. In contrast, pharmacological antagonism of CXCR3 with NBI-74330 prolonged median survival times of both tumor-bearing WT and CXCR3-deficient mice when compared with vehicle-treated groups. NBI-74330 treatment did not impact tumor infiltration of lymphocytes and microglia. A small percentage of GL261 cells were identified as CXCR3+, which was similar to the expression of CXCR3 in several grade IV human glioma cell lines (A172, T98G, U87, U118 and U138). When cultured as gliomaspheres (GS), the human and murine lines increased CXCR3 expression; CXCR3 expression was also found in a primary human GBM-derived GS. Additionally, CXCR3 isoform A was expressed by all lines, whereas CXCR3-B was detected in T98G-, U118- and U138-GS cells. CXCL9 or CXCL10 induced in vitro glioma cell growth in GL261- and U87-GS as well as inhibited cell loss in U138-GS cells and this effect was antagonized by NBI-74330. The results suggest that CXCR3 antagonism exerts a direct anti-glioma effect and this receptor may be a potential therapeutic target for treating human GBM.
doi:10.1093/carcin/bgq224
PMCID: PMC3026840  PMID: 21051441
10.  C5a receptor enables participation of mast cells in immune complex arthritis independent of Fc γ receptor modulation 
Arthritis and rheumatism  2010;62(11):3322-3333.
OBJECTIVE
Mast cells are tissue-resident immune sentinels implicated in the pathogenesis of inflammatory joint disease. We hypothesized that complement fragments could be key activators of synovial mast cells in autoimmune arthritis.
METHODS
In vivo studies employed murine K/BxN arthritis, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR−/− and control mast cells were engrafted into mast cell-deficient W/Wv mice to examine the requirement for this receptor in arthritis. C5aR-dependent activation of mast cells was investigated in C5aR−/− animals and in murine and human mast cell cultures.
RESULTS
Murine synovial mast cells express functional C5aR. Unlike their wild-type counterparts, C5aR−/− mast cells adoptively transferred into W/Wv mice were incompetent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR−/− mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG-mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate expression of Fc γ receptors (FcγR) or otherwise alter activation threshold. In human mast cells, C5a promoted production of the neutrophil chemotaxin interleukin 8, and recruitment of neutrophils at 24h after serum administration was impaired in C5aR−/− mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis.
CONCLUSION
Stimulation via C5aR is required to unleash the pro-inflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism distinct from C5a-modulated FcγR expression.
doi:10.1002/art.27659
PMCID: PMC2970731  PMID: 20662064
11.  Prolongation of Cardiac and Islet Allograft Survival by a Blocking Hamster Anti-Mouse CXCR3 Monoclonal Antibody 
Transplantation  2008;86(1):137-147.
Background
Acute allograft rejection requires a multifaceted immune response involving trafficking of immune cells into the transplant and expression of effector cell functions leading to graft destruction. The chemokine receptor CXCR3 and its ligands, CXCL9, CXCL10 and CXCL11, constitute an important pathway for effector cell recruitment post-transplant. However, analysis of CXCR3 expression and function has been hampered by a general lack of availability of a neutralizing anti-CXCR3 monoclonal antibody (mAb) for use in experimental models.
Methods
We report the generation, characterization and use of CXCR3-173, a new hamster mAb specific for mouse CXCR3 that recognizes CXCR3 on cells from wild-type but not CXCR3-/- mice.
Results
Using CXCR3-173 mAb, we demonstrate CXCR3 expression on primary memory phenotype CD4+ and CD8+ T cells, naturally occurring CD4+CD25+ Foxp3+ regulatory T cells, natural killer T (NKT) cells, and ~25% of NK cells. CXCR3-173 blocked chemotaxis in vitro in response to CXCL10 or CXCL11 but not CXCL9. When injected into mice, this mAb significantly prolonged both cardiac and islet allograft survival. When combined with a subtherapeutic regimen of rapamycin, CXCR3-173 mAb induced long-term (>100 d) survival of cardiac and islet allografts. The in vivo effects of CXCR3-173 mAb were not associated with effector lymphocyte depletion.
Conclusion
These data highlight the utility of CXCR3-173 mAb in developing immunotherapeutic approaches to inhibit transplant rejection and potentially other immune-mediated diseases in murine models.
doi:10.1097/TP.0b013e31817b8e4b
PMCID: PMC3140461  PMID: 18622291
transplant rejection; chemokine receptor; immunotherapy
12.  Deficiency of CXCR2, but not other chemokine receptors, attenuates a murine model of autoantibody-mediated arthritis 
Arthritis and rheumatism  2010;62(7):1921-1932.
Objective
Chemokines coordinate leukocyte trafficking in homeostasis and during immune responses. Prior studies of their role in arthritis have employed animal models with both an initial adaptive immune response and an inflammatory effector phase. This study focused on chemokines and their receptors in the effector phase of arthritis using the K/BxN serum-transfer model.
Methods
A time-course microarray analysis of serum-transferred arthritis was performed, examining ankle, synovial fluid and peripheral blood. Upregulation of chemokines was confirmed by quantitative RT-PCR. The functional relevance of chemokine induction was assessed by transferring serum into mice deficient in CCR1–CCR7, CCR9, CXCR2, CXCR3, CXCR5, CX3CR1, CCL2 or CCL3. Further mechanistic analysis of CXCR2 involved treatment of arthritic mice with a CXCR2 antagonist, bone-marrow transfers with CXCR2+/− and CXCR2−/− donors and recipients, flow cytometry of synovial cells, and competition experiments measuring enrichment of CXCR2-expressing neutrophils in arthritic joints of mice with mixed CXCR2+/+ and CXCR2−/− bone-marrow.
Results
Gene-expression profiling revealed upregulation of the CXCR2 ligands CXCL1, CXCL2 and CXCL5 in the joint in parallel with disease activity. CXCR2−/− mice had attenuated disease relative to CXCR2+/− littermates, as did mice receiving the CXCR2 inhibitor, while deficiency of other chemokine receptors did not affect arthritis severity. CXCR2 was required only on hematopoietic cells and was widely expressed on synovial neutrophils. CXCR2-expressing neutrophils were preferentially recruited to arthritic joints in the presence of CXCR2-deficient neutrophils.
Conclusion
CXCR2 (but not other chemokine receptors) is critical for the development of autoantibody-mediated arthritis, exhibiting a cell-autonomous role in neutrophil recruitment to inflamed joints.
doi:10.1002/art.27470
PMCID: PMC2994550  PMID: 20506316
13.  CD46 Protects against Chronic Obstructive Pulmonary Disease 
PLoS ONE  2011;6(5):e18785.
Background
Chronic obstructive pulmonary disease and emphysema develops in 15% of ex-smokers despite sustained quitting, while 10% are free of emphysema or severe lung obstruction. The cause of the incapacity of the immune system to clear the inflammation in the first group remains unclear.
Methods and Findings
We searched genes that were protecting ex-smokers without emphysema, using microarrays on portions of human lungs surgically removed; we found that loss of lung function in patients with chronic obstructive pulmonary disease and emphysema was associated with a lower expression of CD46 and verified this finding by qRT-PCR and flow cytometry. Also, there was a significant association among decreased CD46+ cells with decreased CD4+T cells, apoptosis mediator CD95 and increased CD8+T cells that were protecting patients without emphysema or severe chronic obstructive pulmonary disease. CD46 not only regulates the production of T regulatory cells, which suppresses CD8+T cell proliferation, but also the complement cascade by degradation of C3b. These results were replicated in the murine smoking model, which showed increased C5a (produced by C3b) that suppressed IL12 mediated bias to T helper 1 cells and elastin co-precipitation with C3b, suggesting that elastin could be presented as an antigen. Thus, using ELISA from elastin peptides, we verified that 43% of the patients with severe early onset of chronic obstructive pulmonary disease tested positive for IgG to elastin in their serum compared to healthy controls.
Conclusions
These data suggest that higher expression of CD46 in the lungs of ex-smoker protects them from emphysema and chronic obstructive pulmonary disease by clearing the inflammation impeding the proliferation of CD8+ T cells and necrosis, achieved by production of T regulatory cells and degradation of C3b; restraining the complement cascade favors apoptosis over necrosis, protecting them from autoimmunity and chronic inflammation.
doi:10.1371/journal.pone.0018785
PMCID: PMC3089601  PMID: 21573156
14.  C5a receptor-deficient dendritic cells promote induction of Treg and Th17 
European journal of immunology  2010;40(3):710-721.
C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in dendritic cells (DC) affects the development of regulatory T cell (Treg) and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen-derived DC results in increased production of TGF-β leading to de novo differentiation of Foxp3+ Treg within 12h after co-incubation with CD4+ T cells from DO11.10/RAG2-/- mice. Stimulation of C5aR-/- DC with ovalbumin and TLR2 ligand Pam3CSK4 increased TGF-β production and induced high levels of IL-6 and IL-23 but only minor amounts of IL-12 leading to differentiation of Th cells producing IL-17A and IL-21. Th17 differentiation was also found in vivo after adoptive transfer of CD4+ Th cell into C5aR-/- mice immunized with OVA and Pam3CSK4. The altered cytokine production of C5aR-/- DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17 cell differentiation through regulation of DC function.
doi:10.1002/eji.200939333
PMCID: PMC3040298  PMID: 20017191
C5a; complement; dendritic cell; T cells; IL-17A
15.  Mast Cell Protease 5 Mediates Ischemia-Reperfusion Injury of Mouse Skeletal Muscle1 
Ischemia with subsequent reperfusion (IR) injury is a significant clinical problem that occurs after physical and surgical trauma, myocardial infarction, and organ transplantation. IR injury of mouse skeletal muscle depends on the presence of both natural IgM and an intact C pathway. Disruption of the skeletal muscle architecture and permeability also requires mast cell (MC) participation, as revealed by the fact that IR injury is markedly reduced in c-kit defective, MC-deficient mouse strains. In this study, we sought to identify the pathobiologic MC products expressed in IR injury using transgenic mouse strains with normal MC development, except for the lack of a particular MC-derived mediator. Histologic analysis of skeletal muscle from BALB/c and C57BL/6 mice revealed a strong positive correlation (R2 = 0.85) between the extent of IR injury and the level of MC degranulation. Linkage between C activation and MC degranulation was demonstrated in mice lacking C4, in which only limited MC degranulation and muscle injury were apparent. No reduction in injury was observed in transgenic mice lacking leukotriene C4 synthase, hemopoietic PGD2 synthase, N-deacetylase/N-sulfotransferase-2 (enzyme involved in heparin biosynthesis), or mouse MC protease (mMCP) 1. In contrast, muscle injury was significantly attenuated in mMCP-5-null mice. The MCs that reside in skeletal muscle contain abundant amounts of mMCP-5 which is the serine protease that is most similar in sequence to human MC chymase. We now report a cytotoxic activity associated with a MC-specific protease and demonstrate that mMCP-5 is critical for irreversible IR injury of skeletal muscle.
PMCID: PMC2951006  PMID: 15905575
16.  International Union of Pharmacology LXXIII: Nomenclature for the Formyl Peptide Receptor (FPR) Family 
Pharmacological reviews  2009;61(2):119-161.
Formyl peptide receptors (FPRs) are a small group of 7-transmembrane domain, G protein-coupled receptors that are expressed mainly by mammalian phagocytic leukocytes and known to be important in host defense and inflammation. The three human FPRs (FPR1, FPR2/ALX and FPR3) share significant sequence homology and are encoded by clustered genes. Collectively, these receptors bind an extraordinarily numerous and structurally diverse group of agonistic ligands, including N-formyl and non-formal peptides of different composition, that chemoattract and activate phagocytes. N-formyl peptides, which are encoded in nature only by bacterial and mitochondrial genes and result from obligatory initiation of bacterial and mitochondrial protein synthesis with N-formylmethionine, is the only ligand class common to all three human receptors. Surprisingly, the endogenous anti-inflammatory peptide annexin 1 and its N-terminal fragments also bind human FPR1 and FPR2/ALX, and the anti-inflammatory eicosanoid lipoxin A4 is an agonist at FPR2/ALX. In comparison, fewer agonists have been identified for FPR3, the third member in this receptor family. Strucural and functional studies of the FPRs have produced important information for understanding the general pharmacological principles governing all leukocyte chemoattractant receptors. This article aims to provide an overview of the discovery and pharmacological characterization of FPRs, to introduce an IUPHAR recommended nomenclature, and to discuss unmet challenges, including the mechanisms used by these receptors to bind diverse ligands and mediate different biological functions.
doi:10.1124/pr.109.001578
PMCID: PMC2745437  PMID: 19498085
17.  CTL mobilization to virus-infected tissue requires CD4+ T cell help 
Nature  2009;462(7272):510-513.
CD4+ T helper cells are well known for their role in providing critical signals during priming of cytotoxic CD8+ T lymphocyte (CTL) responses in vivo. T help is required for the generation of primary CTL responses as well as in promoting protective CD8+ memory T cell development1. However, the role of CD4 help in the control of CTL responses at the effector stage is unknown. Here, we show that fully helped effector CTLs are not themselves self-sufficient for entry into the infected tissue, but rely on the CD4+ T cells to provide the necessary cue. CD4+ T helper cells control the migration of CTL indirectly through the secretion of IFN-γ and induction of local chemokine secretion in the infected tissue. Our results reveal a previously unappreciated role of CD4 help in mobilizing effector CTL to the peripheral sites of infection where they help to eliminate infected cells.
doi:10.1038/nature08511
PMCID: PMC2789415  PMID: 19898495
18.  The Regulation of Liver Cell Survival by Complement1 
Complement effectors are known to contribute to host cell injury in several inflammatory diseases. Contrary to this paradigm, in this study utilizing surgical liver resection (partial hepatectomy) in various complement-deficient mice as a model, we have demonstrated that complement anaphylatoxins C3a and C5a are required for the survival of liver cells during regeneration. The mechanisms of these cytoprotective functions of complement were related to the regulation of IL-6 and TNF production or release after liver resection. Disturbances in the cytokine milieu, induced by a loss of complement activity, were found to alter prosurvival signaling, including the IL-6/STAT3 and PI3K/Akt/mammalian target of rapamycin pathways. In conclusion, this study documents functions of complement proteins as prosurvival factors that, through their interactions with cytokines, inhibit apoptotic signaling in proliferating cells of epithelial origin.
doi:10.4049/jimmunol.0804179
PMCID: PMC2732128  PMID: 19380788
19.  The Roles of Streptozotocin Neurotoxicity and Neutral Endopeptidase in Murine Experimental Diabetic Neuropathy 
Experimental Diabetes Research  2010;2009:431980.
We demonstrated that inhibition of neutral endopeptidase (NEP), a protease that degrades vaso- and neuroactive peptides, improves vascular and neural function in diabetic animal models. In this study we explored the role of NEP in neuropathy related to either insulin-deficient diabetes or diet-induced obesity using NEP deficient (−/−) mice. Initial studies showed that streptozotocin, in the absence of subsequent hyperglycemia, did not induce nerve conduction slowing or paw thermal hypoalgesia. Glucose disposal was impaired in both C57Bl/6 and NEP −/− mice fed a high fat diet. Thermal hypoalgesia and nerve conduction slowing were present in both streptozotocin-diabetic and high fat fed C57Bl/6 mice but not in NEP −/− mice exposed to either streptozotocin-induced diabetes or a high fat diet. These studies suggest that streptozotocin does not induce neurotoxicity in mice and that NEP plays a role in regulating nerve function in insulin-deficient diabetes and diet-induced obesity.
doi:10.1155/2009/431980
PMCID: PMC2817866  PMID: 20148083
20.  CCR3 is a therapeutic and diagnostic target for neovascular age-related macular degeneration 
Nature  2009;460(7252):225-230.
Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularization (CNV). We report that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in CNV endothelial cells in humans with AMD, and that, despite the expression of its ligands eotaxin-1, -2, and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation as it occurred in mice lacking eosinophils or mast cells and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor-A (VEGF-A) neutralization, which is currently in clinical use, and, unlike VEGF-A blockade, not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.
doi:10.1038/nature08151
PMCID: PMC2712122  PMID: 19525930
21.  An Indispensable Role for the Chemokine Receptor CCR10 in IgA Antibody Secreting Cell Accumulation1 
The differential expression of chemokines and chemokine receptors, by tissues and leukocytes respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA antibody secreting cells to mucosal surfaces such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. Here we describe the generation of a CCR10 deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10 deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.
PMCID: PMC2748758  PMID: 18941222
22.  Functional roles for C5a receptors in sepsis 
Nature medicine  2008;14(5):551-557.
The function of the C5a receptors, C5ar (encoded by C5ar) and C5l2 (encoded by Gpr77), especially of C5l2, which was originally termed a ‘default receptor’, remains a controversial topic. Here we investigated the role of each receptor in the setting of cecal ligation and puncture-induced sepsis by using antibody-induced blockade of C5a receptors and knockout mice. In ‘mid-grade’ sepsis (30-40% survival), blockade or absence of either C5ar or C5l2 greatly improved survival and attenuated the buildup of proinflammatory mediators in plasma. In vivo appearance or in vitro release of high mobility group box 1 protein (HMGB1) required C5l2 but not C5ar. In ‘high-grade’ sepsis (100% lethality), the only protective condition was the combined blockade of C5l2 and C5ar. These data suggest that C5ar and C5l2 contribute synergistically to the harmful consequences in sepsis and that C5l2 is required for the release of HMGB1. Thus, contrary to earlier speculation, C5l2 is a functional receptor rather than merely a default receptor.
doi:10.1038/nm1753
PMCID: PMC2753858  PMID: 18454156
23.  CCR5 plays a key role in the early memory CD8+ T cell response to respiratory virus infections 
Immunity  2008;29(1):101-113.
Summary
Innate recognition of invading pathogens in peripheral tissues results in the recruitment of circulating memory CD8+ T cells to sites of localized inflammation during the early phase of a recall response. However, the mechanisms that control the rapid recruitment of these cells to peripheral sites are poorly understood, particularly in relation to influenza and parainfluenza infections of the respiratory tract. In this study, we demonstrate a crucial role for CCR5 in the accelerated recruitment of memory CD8+ T cells to the lung airways during virus challenge. Most importantly, CCR5 deficiency resulted in decreased recruitment of memory T cells expressing key effector molecules and impaired control of virus replication during the initial stages of a secondary response. These data highlight the critical importance of early memory T cell recruitment for the efficacy of cellular immunity in the lung.
doi:10.1016/j.immuni.2008.05.011
PMCID: PMC2519120  PMID: 18617426
24.  Accelerated Prion Replication in, but Prolonged Survival Times of, Prion-Infected CXCR3−/− Mice▿  
Journal of Virology  2008;82(24):12464-12471.
Prion diseases have a significant inflammatory component. Glia activation, which is associated with increased production of cytokines and chemokines, may play an important role in disease development. Among the chemokines upregulated highly and early upregulated during scrapie infections are ligands of CXCR3. To gain more insight into the role of CXCR3 in a prion model, CXCR3-deficient (CXCR3−/−) mice were infected intracerebrally with scrapie strain 139A and characterized in comparison to similarly infected wild-type controls. CXCR3−/− mice showed significantly prolonged survival times of up to 30 days on average. Surprisingly, however, they displayed accelerated accumulation of misfolded proteinase K-resistant prion protein PrPSc and 20 times higher infectious prion titers than wild-type mice at the asymptomatic stage of the disease, indicating that these PrP isoforms may not be critical determinants of survival times. As demonstrated by immunohistochemistry, Western blotting, and gene expression analysis, CXCR3-deficient animals develop an excessive astrocytosis. However, microglia activation is reduced. Quantitative analysis of gliosis-associated gene expression alterations demonstrated reduced mRNA levels for a number of proinflammatory factors in CXCR3−/− compared to wild-type mice, indicating a weaker inflammatory response in the knockout mice. Taken together, this murine prion model identifies CXCR3 as disease-modifying host factor and indicates that inflammatory glial responses may act in concert with PrPSc in disease development. Moreover, the results indicate that targeting CXCR3 for treatment of prion infections could prolong survival times, but the results also raise the concern that impairment of microglial migration by ablation or inhibition of CXCR3 could result in increased accumulation of misfolded PrPSc.
doi:10.1128/JVI.01371-08
PMCID: PMC2593330  PMID: 18842729
25.  Modulation of the anti-tumor immune response by complement 
Nature immunology  2008;9(11):1225-1235.
The involvement of complement activation products in promoting tumor growth has not yet been recognized. Here we show that generation of complement C5a in the tumor microenvironment enhanced tumor growth by suppressing the anti-tumor CD8+ T cell-mediated response. This suppression was associated with the recruitment of myeloid-derived suppressor cells (MDSCs) into tumors and augmentation of their T cell-directed suppressive capabilities. Amplification of MDSC suppressive capacity by C5a occurred through regulation of the production of reactive oxygen and nitrogen species. Pharmacological blockade of C5a receptor significantly impaired tumor growth to a degree comparable to the effect produced by the anti-cancer drug Taxol. Thus, this study demonstrates a therapeutic role for complement inhibition in the treatment of cancer.
doi:10.1038/ni.1655
PMCID: PMC2678913  PMID: 18820683

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