Development of Anti-Mouse CC Chemokine Receptor 3 Monoclonal Antibodies for Flow Cytometry.

CC chemokine receptor 3 (CCR3), also known as CD193, belongs to class A of G protein-coupled receptors and is present in high levels in eosinophils, basophils, and airway epithelial cells. CCR3 is considered the therapeutic target for human immunodeficiency virus (HIV) infections and allergic diseases; therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR3 has been desired. This study aimed to establish a specific and sensitive mAb against mouse CCR3 (mCCR3) useful for flow cytometry analysis by employing the Cell-Based Immunization and Screening (CBIS) method. The generated anti-mCCR3 mAb, C3Mab-2 (rat IgG2b, kappa), was found to react with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells, according to flow cytometric analysis. Also, it reacted with P388 (mouse lymphoid neoplasm) or J774-1 (mouse macrophage-like) cells, which express endogenous mCCR3. Taken together, C3Mab-2, generated by the CBIS method, can be a valuable tool for detecting mCCR3 on the surface of mouse cells.

Platelets Independently Recruit into Asthmatic Lungs and Models of Allergic Inflammation via CCR3.

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti-mouse CD49b-PE (phycoerythrin) antibody. Platelet-endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.

Effects of lentivirus-mediated CCR3 RNA interference on the function of mast cells of allergic rhinitis in mice.

The CC chemokine receptor 3 (CCR3) expressed by eosinophils, mast cells and Th2 cells is closely related to allergic diseases. The objective of this study was to explore whether silencing of CCR3 with short hairpin RNAs (shRNAs) delivered by a lentiviral vector could impact the function of mast cells in a murine model of allergic rhinitis (AR) in vivo. The murine model of allergic rhinitis (AR) inducing by ovalbumin (OVA) was constructed, and the BALB/c mice were divided into normal control group, AR group, controlshRNA treated group and lentiviral CCR3-shRNA treated group. The recombinant lentivirus vectors which express a short hairpin RNA (shRNA) targeting the CCR3 were dropped into the nasal cavity of OVA-sensitized mice before the challenges. Real-time fluorescence quantitative PCR and western blotting were performed to observe inhibitory effect of CCR3 gene. Nasal symptoms of mice and OVA-specific IgE in each group were assessed. Concentrations of histamine, tryptase and Prostaglandin D2 (PGD2) in bone marrow, peripheral blood and nasal mucosa were analyzed. Furthermore, histological analysis and electron microscopy analysis were applied to detect the histology changes of nasal mucosa and the infiltration of mast cells in nasal mucosa. The results showed that administration of CCR3shRNA could effectively inhibit the expression of the CCR3 gene in bone marrow, peripheral blood and nasal mucosa, which reduced the nasal symptoms, the level of OVA-specific IgE, the inflammatory cells and mast cells infiltration into nasal cavity, and relieved the histopathological changes of nasal mucosa. In addition, intervention of CCR3shRNA could reduce the levels of the histamine, tryptase and PGD2 in bone marrow, peripheral blood and nasal mucosa. These results suggest that inhibition of CCR3 gene expression by shRNAs lentiviral vectors can effectively attenuate migration, infiltration and degranulation of mast cells in local tissues and alleviate the inflammation of allergic rhinitis mice.

Augmentation of the expression of the eotaxin receptor on duodenal neutrophils by IL-21.

Inflammation can occur via different mechanisms, such as via acute and chronic responses, on numerous occasions and function accordingly through various roles. There are more than five subsets of neutrophils; neutrophilic heterogeneity is modulated by the inflammatory condition. To understand the characteristics of inflammation, identification of atypical neutrophils is important. In this study, we found that the expression of eotaxin receptor (CD193) on atypical neutrophils in the duodenum is augmented in IL-21 isoform transgenic (Tg) mice. In a series of studies, we have established a Tg mouse strain to further investigate the functions of IL-21 in vivo. Interestingly, Tg mice immunized with ovalbumin (OVA) were more sensitive to OVA-induced systemic anaphylaxis as compared with wild type mice with duodenal and splenic gross congestion. Further analysis conducted in the duodenum of Tg mice revealed that only the number of neutrophils migrating into the duodenum was significantly increased prior to immunization. Previous studies have shown that the gastrointestinal compartment and the spleen constantly produce eotaxin, which regulates basal levels of tissue eosinophils. Therefore, we analyzed CD193 expression on neutrophils and eosinophils. As expected, its expression by duodenal neutrophils was upregulated in Tg mice. Furthermore, the addition of IL-21 into bone marrow cell culture increased the number of CD193+ neutrophils, which easily migrated into the duodenum. These observations suggested that CD193+ neutrophils increase in number under inflammatory conditions due to chronic IL-21 production.

T helper type 1-related molecules as well as interleukin-15 are hyperexpressed in the skin lesions of patients with pyoderma gangrenosum.

Pyoderma gangrenosum (PG) is a rare, immune-mediated skin disease classified into the group of neutrophilic dermatoses. Although a number of studies confirmed the central role of innate immunity, only few studies have investigated the possible contributing role of acquired immunity. In particular, no reports concerning T helper type 1 (Th1) and Th2 cells are available as yet. Therefore, 15 patients with PG, five with Sweet's syndrome (SS) and nine skin specimens from healthy controls (HC) were investigated, evaluating the expression of Th1-related markers interleukin (IL)-12, interferon (IFN)-γ, C-X-C motif chemokine receptor 3 (CXCR3) and C-C motif chemokine receptor 5 (CCR5), of the Th2-related molecules IL-4, IL-5, IL-13 and CCR3, of the co-stimulatory axis CD40/CD40 ligand, of IL-15 and the natural killer (NK) cell marker CD56 in skin lesions by immunohistochemistry. Patients with PG and SS showed a higher expression of Th1 markers than HC. Conversely, IL-5- and CCR3-expressing cells were less numerous in PG skin lesions compared to SS (P = 0·0157 and < 0·0001, respectively). Both CD40 and CD40L were expressed more in PG than in SS and HC (P < 0·0001 for both). Finally, the number of IL-15+ and CD56+ cells was higher in the skin of patients with PG than in those of SS and HC (P < 0·0001 for both). Our results suggest that Th2 cells are down-regulated in PG. At the same time, over-expression of the co-stimulatory axis CD40/CD40L amplifies the impairment of the Th1/Th2 balance. Both these findings might explain the most aggressive behaviour of PG in comparison to SS. Moreover, over-expression of IL-15+ and CD56+ cells may suggest a possible role of NK cells in the pathogenesis of the disease.

Optimizing of the basophil activation test: Comparison of different basophil identification markers.

BACKGROUND: Flowcytometric identification of basophils is a prerequisite for measuring activation of basophils with IgE-dependent or IgE-independent stimuli. Aim of this study was to compare different marker combinations in a simultaneous multicolor flowcytometric measurement. METHODS: Ten patients with a grass pollen allergy and three controls were included in the study. Basophilic cells were gated by using anti-CCR3, anti-IgE, anti-CRTH2, anti-CD203c, and anti-CD3. Cells were activated by a monoclonal anti-FcεRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the allergen extract Phleum pratense. The activation marker anti-CD63 was used. RESULTS: The highest relative number of basophils was found with anti-CCR3+ cells, anti-IgE+ and anti-IgE+ /anti-CD203c+ cells, the lowest with CRTH2+/CD203c+/CD3- cells. A very good and good concordance of CCR3+ cells was seen with CCR3+/CD3- cells and CRTH2+/CD203c+/CD3- cells in all experiments. The contamination of the CCR3+ population with CD3+ cells and the contamination of the IgE+-population with CCR3- cells and CD203- cells were the lowest compared to all other marker combinations. CONCLUSIONS: As the highest relative number of basophils was identified by anti-CCR3 followed by the anti-IgE and anti-IgE/antiCD203c positive population in most cases, these markers can generally be recommended for identification of basophils. If a basophil population with very high purity is needed, anti-IgE should be chosen.

Murine antigen-induced inflammation--a model for studying induction, resolution and the adaptive phase of inflammation.

Murine zymosan-induced peritonitis is the model most frequently used to study resolution of inflammation. However, the antigen-induced peritonitis model may be better suited for studying resolution of inflammation and the adaptive phase that follows. The objective of this study was to provide an evaluation of the kinetics of cells and mediators during induction, resolution and the adaptive immune phases of a murine antigen-induced inflammation. Female C57BL/6 mice were immunized twice subcutaneously with mBSA and three weeks after the initial immunization they were injected intraperitoneally (i.p.) with mBSA, which induced peritonitis. Peritoneal cells were counted and expression of surface molecules and chemokine receptors analyzed with flow cytometry. Chemokine and cytokine concentrations in peritoneal fluid were determined by ELISA. Two neutrophil populations, differing in size and granularity and slightly in expression of surface molecules, were observed in the peritoneal cavity after induction of inflammation. Macrophages disappeared from the peritoneal cavity following i.p. administration of mBSA but appeared again as they differentiated from recruited monocytes and peaked in numbers at 48 h. At that time point, two distinct populations of macrophages were present in the peritoneal cavity; one with high expression of F4/80, also expressing the atypical chemokine receptor D6 as well as CCR7; the other expressing low levels of F4/80 and also expressing CD11c and CD138. Eosinophils appeared in the peritoneum 3h following i.p. administration of mBSA and peaked at 48 h. At that time point they had upregulated their expression of CCR3 but decreased their expression of CD11b. Peritoneal levels of CCL11 peaked at 6h and may have led to recruitment of the eosinophils. NK cells and T cells peaked at 48 h, whereas B cells peaked at 5 days, with the majority being B1 cells. Peritoneal concentrations of pro-inflammatory cytokines (IL-ß and IL-6) and chemokines (CCL2 and CCL3) peaked at 3h, whereas IL-1ra peaked at 6h, sTNF-R at 24h and sIL-6R and TGF-ß at 48 h. The results show kinetic alterations in cell populations and mediators in a murine model that may be an excellent model to study initiation and resolution of inflammation and the following adaptive phase.

Airway exposure to staphylococcal enterotoxin A potentiates allergen-induced bone marrow eosinophilia and trafficking to peripheral blood and airways.

Bone marrow (BM) eosinopoiesis is a common feature during allergen exposure in atopic individuals. Airway exposure to staphylococcal superantigens aggravates allergic airway disease and increases the output of BM eosinophils. However, the exact mechanisms regulating eosinophil mobilization and trafficking to the peripheral circulation and airways remain to be elucidated. Therefore, this study aimed to investigate the mechanisms determining the BM eosinopoiesis in allergic mice under exposure to staphylococcal enterotoxin A (SEA). Ovalbumin (OVA)-sensitized male BALB/C mice were intranasally exposed to SEA (1 µg), and at 4, 12, 24, and 48 h later animals were challenged with OVA (10 µg, twice a day). Measurement of IL-5, eotaxin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels, flow cytometry for CCR3(+), VLA4(+), and CCR3(+)VLA4(+), as well as adhesion assays to VCAM-1 were performed in BM. Prior airway exposure to SEA time dependently increased the BM eosinophil number in OVA-challenged mice. Eosinophils gradually disappear from peripheral blood, being recruited over time to the airways, where they achieve a maximal infiltration at 24 h. SEA exposure increased the levels of IL-5 and eotaxin (but not GM-CSF) in BM of OVA-challenged mice. Marked increases in CCR3(+) and CCR3(+)VLA4(+) expressions in BM eosinophils of OVA-challenged mice were observed, an effect largely reduced by prior exposure to SEA. Adhesion of BM eosinophils to VCAM-1 was increased in OVA-challenged mice, but prior SEA exposure abrogated this enhanced cell adhesion. Accumulation of BM eosinophils by airway SEA exposure takes place through IL-5- and CCR3-dependent mechanisms, along with downregulation of CCR3/VL4 and impaired cell adhesion to VCAM-1.

CCR3 and choroidal neovascularization.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly in industrialized countries. The "wet" AMD, characterized by the development of choroidal neovacularization (CNV), could result in rapid and severe loss of central vision. The critical role of vascular endothelial growth factor A (VEGF-A) in CNV development has been established and VEGF-A neutralization has become the standard care for wet AMD. Recently, CCR3 was reported to play an important role in CNV development and that CCR3 targeting was reported to be superior to VEGF-A targeting in CNV suppression. We investigated the role of CCR3 in CNV development using the Matrigel induced CNV and found that in both rats and mice, CNV was well-developed in the control eyes as well as in eyes treated with CCR3 antagonist SB328437 or CCR3 neutralizing antibodies. No statistically significant difference in CNV areas was found between the control and SB328437 or CCR3-ab treated eyes. Immunostaining showed no specific expression of CCR3 in or near CNV. In contrast, both VEGF-A neutralizing antibodies and rapamycin significantly suppressed CNV. These results indicate that CCR3 plays no significant role in CNV development and question the therapeutic approach of CCR3 targeting to suppress CNV. On the other hand, our data support the therapeutic strategies of VEGF-A and mTOR (mammalian target of rapamycin) targeting for CNV.

Local proliferation and mobilization of CCR3(+) CD34(+) eosinophil-lineage-committed cells in the lung.

Emerging evidence suggests that haematopoietic CD34(+) progenitor cells migrate from bone marrow (BM) to sites of allergen exposure where they can undergo further proliferation and final maturation, potentially augmenting the degree of tissue inflammation. In the current study we used a well-characterized mouse model of allergen-induced airway inflammation to determine the role of CCR3 receptor-ligand interactions in the migration and function of CD34(+) cells. Allergen exposure significantly increased BM, blood and airway CD34(+) CCR3(+) cells as well as airway CD34(+) CCR3(+) stem cell antigen-1-positive (Sca-1(+) ) and CD34(+) CD45(+) interleukin-5 receptor-α-positive (IL-5Rα(+) ) cells. A portion of the newly produced CD34(+) CCR3(+), Sca-1(+) CCR3(+) and IL-5Ralpha(+) lung cells showed a significant proliferative capacity in response to allergen when compared with saline-treated animals. In addition, in vitro colony formation of lung CD34(+) cells was increased by IL-5 or eotaxin-2 whereas eotaxin-2 had no effect on BM CD34(+) cells. Furthermore, both eotaxin-1 and eotaxin-2 induced migration of BM and blood CD34(+) CCR3(+) cells in vitro. These data suggest that the CCR3/eotaxin pathway is involved in the regulation of allergen-driven in situ haematopoiesis and the accumulation/mobilization of eosinophil-lineage-committed progenitor cells in the lung. Hence, targeting both IL-5 and CCR3-mediated signalling pathways may be required to control the inflammation associated with allergen-induced asthma.

Therapeutic effects of Baicalein on atopic dermatitis-like skin lesions of NC/Nga mice induced by dermatophagoides pteronyssinus.

The present study was conducted to investigate the effects of Baicalein (BE), which is hydrolyzed product of Baicalin (BA), on atopic dermatitis (AD). AD was induced in NC/Nga mice by DPE treatment. BE hydrogels treatment reduced the levels of skin severity scores. BE hydrogels treatment also decreased inflammatory cytokines such as TNF-alpha, IL-6, and its level in the serum. BE hydrogels treatment elevated IFN-gamma level in the spleenocyte culture supernatant. Cell numbers in the skin positive to CD3+/CD69+, CCR3+, CD11b+/Gr-1+, B220+/IgE+ all of which were up-regulated in AD-induced mice were decreased and returned to normal levels. Histological examination showed that infiltration levels of immune cells in the skin of AD-induced NC/Nga mice were much improved by BE hydrogels treatment. These results thus suggest that BE can regulate molecular mediators and immune cells that are functionally associated with atopic dermatitis induced in NC/Nga mice, and may play an important role in recovering AD symptoms.

A new basophil activation test using CD63 and CCR3 in allergy to antibiotics.

BACKGROUND: Flow cytometric basophil activation tests (BAT) have been developed as cellular tests for in vitro diagnosis of IgE-mediated reactions. Different markers and techniques have been used after stimulation with various allergens. OBJECTIVE: It was the aim of the present study to compare an established BAT (Flow-CAST) with a newly developed basophil activation protocol using CD63 and CCR3 (Flow2 CAST) in patients with type-I allergy to antibiotics. MATERIALS AND METHODS: Twenty-four patients with a history of type-I allergy to antibiotics were examined. A careful allergy history was taken, and skin tests and determination of specific IgE antibodies were performed. Two different BAT using CD63 expression but different protocols were carried out after stimulation with different concentrations of antibiotics. Fifteen healthy subjects without a history of antibiotic allergy were studied as controls. RESULTS: The Flow2 CAST showed a higher sensitivity than the Flow-CAST (55% vs. 53%) with regard to patients' history. Specificity was 80% both for the Flow2 CAST and for the Flow-CAST with regard to controls with negative history and negative RAST. CONCLUSION: These results show the value of two different BAT as cellular tests in the in vitro diagnosis of patients with antibiotic allergy with equal specificity and a slightly higher sensitivity for the Flow2 CAST.

The function of CCR3 on mouse bone marrow-derived mast cells in vitro.

The mechanisms governing the population of tissues by mast cells are not fully understood, but several studies using human mast cells have suggested that expression of the chemokine receptor CCR3 and migration to its ligands may be important. In CCR3-deficient mice, a change in mast cell tissue distribution in the airways following allergen challenge was reported compared with wild-type mice. In addition, there is evidence that CCR3 is important in mast cell maturation in mouse. In this study, bone marrow-derived mast cells (BMMCs) were cultured and CCR3 expression and the migratory response to CCR3 ligands were characterized. In addition, BMMCs were cultured from wild-type and CCR3-deficient mice and their phenotype and migratory responses were compared. CCR3 messenger RNA was detectable in BMMCs, but this was not significantly increased after activation by immunoglobulin E (IgE). CCR3 protein was not detected on BMMCs during maturation and expression could not be enhanced after IgE activation. Resting and IgE-activated immature and mature BMMCs did not migrate in response to the CCR3 ligands eotaxin- 1 and eotaxin-2. Comparing wild-type and CCR3-deficient BMMCs, there were no differences in mast cell phenotype or ability to migrate to the mast cell chemoattractants leukotriene B4 and stem cell factor. The results of this study show that CCR3 may not mediate mast cell migration in mouse BMMCs in vitro. These observations need to be considered in relation to the findings of CCR3 deficiency on mast cells in vivo.

[The expression of periphery blood leucocyte CCR3 and CCR5 in the children with Epstein-Barr virus associated infectious mononucleosis].

OBJECTIVE: To explore the expression of periphery blood leucocyte CCR3 and CCR5 and to comprehend T helper cell in the Children with Epstein-Barr virus associated infectious mononucleosis. METHODS: We defined the children according to the diagnosis criterion through Paul-Bunnell test inspecting the children's periphery blood unusual lymphocyte and detecting their anti-EBV-CA-IgM, anti-EBV-CA-IgG and anti-EBV-NA-IgG by ELISA and counted the ratio of CCR3 + and CCR5 + cells in lymphocytes with flow cytometry. RESULTS: The ratio of unusual lymphocyte in IM was higher than that of the healthy control group (P < 0.05). The ratio of CCR3 + cells in IM group was higher than that of the healthy control group (P < 0.05). The ratio of CCR5 + cells in IM group was significantly lower than that of the healthy control group. CCR3 + had direct interrelation with fever continued time and the ratio of unusual lymphocyte. There was a negative interrelation between CCR5 and fever continued time (P < 0.05). CONCLUSIONS: Children infectious of IM expressed higher level of CCR3 + and lower level of CCR5 + and there was a tendency of Th2 polarization with over production of T helper cell divide imbalance. CCR3 + and CCR5 + may be important targets to judge the degree of seriousness of IM.

CCR3: Shedding new light on a dark problem?

A recent work by Ambati et al. represents a bold step towards a more effective diagnosis and treatment of age-related macular degeneration, with the new evidence showing that CCR3, a chemokine receptor, is an early marker of and potential therapeutic target for choroidal neovascularization development. In the wake of such a novel and significant finding, additional illumination to confirm and consolidate the promise shown by CCR3 will soon follow.

Toll-like receptors, chemokine receptors and death receptor ligands responses in SARS coronavirus infected human monocyte derived dendritic cells.

BACKGROUND: The SARS outbreak in 2003 provides a unique opportunity for the study of human responses to a novel virus. We have previously reported that dendritic cells (DCs) might be involved in the immune escape mechanisms for SARS-CoV. In this study, we focussed on the gene expression of toll-like receptors (TLRs), chemokine receptors (CCRs) and death receptor ligands in SARS-CoV infected DCs. We also compared adult and cord blood (CB) DCs to find a possible explanation for the age-dependent severity of SARS. RESULTS: Our results demonstrates that SARS-CoV did not modulate TLR-1 to TLR-10 gene expression but significantly induced the expression of CCR-1, CCR-3, and CCR-5. There was also strong induction of TNF-related apoptosis-inducing ligand (TRAIL), but not Fas ligand gene expression in SARS-CoV infected DCs. Interestingly, the expressions of most genes studied were higher in CB DCs than adult DCs. CONCLUSION: The upregulation of chemokines and CCRs may facilitate DC migration from the infection site to the lymph nodes, whereas the increase of TRAIL may induce lymphocyte apoptosis. These findings may explain the increased lung infiltrations and lymphoid depletion in SARS patients. Further explorations of the biological significance of these findings are warranted.

CCR3 is a target for age-related macular degeneration diagnosis and therapy.

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 neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular 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 because 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 in clinical use at present, and, unlike VEGF-A blockade, is 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.

IgE-FcepsilonRI interactions determine HIV coreceptor usage and susceptibility to infection during ontogeny of mast cells.

Progenitor mast cells (prMCs), derived from CD34(+) precursors are CD4(+)/CCR5(+)/CXCR4(+) and susceptible to CCR5(R5)-tropic virus but only marginally susceptible to CXCR4(X4)-tropic HIV. As infected prMCs mature within extravascular compartments, they become both latently infected and HIV-infection resistant, and thus capable of establishing an inducible reservoir of CCR5-tropic infectious clones. In this report we provide the first evidence that IgE-FcepsilonRI interactions, occurring during a unique period of mast cell (MC) ontogeny, enhance prMC susceptibility to X4 and R5X4 virus. IgE-FcepsilonRI interactions significantly increased expression of CXCR4 mRNA ( approximately 400- to 1800-fold), enhanced prMC susceptibility to X4 and R5X4 virus ( approximately 3000- to 16,000-fold), but had no significant effect on CD4, CCR3, or CCR5 expression, susceptibility to R5 virus, or degranulation. Enhanced susceptibility to infection with X4 virus occurred during the first 3-5 wk of MC ontogeny and was completely inhibited by CXCR4-specific peptide antagonists and omalizumab, a drug that inhibits IgE-FcepsilonRI interactions. IgE-FcepsilonRI coaggregation mediated by HIVgp120 or Schistosoma mansoni soluble egg Ag accelerated maximal CXCR4 expression and susceptibility to X4 virus by prMCs. Our findings suggest that for HIV-positive individuals with atopic or helminthic diseases, elevated IgE levels could potentially influence the composition of CXCR4-tropic and R5X4-tropic variants archived within the long-lived tissue MC reservoir created during infection.

Rejection of intradermally injected syngeneic tumor cells from mice by specific elimination of tumor-associated macrophages with liposome-encapsulated dichloromethylene diphosphonate, followed by induction of CD11b(+)/CCR3(-)/Gr-1(-) cells cytotoxic against the tumor cells.

Tumor cell expansion relies on nutrient supply, and oxygen limitation is central in controlling neovascularization and tumor spread. Monocytes infiltrate into tumors from the circulation along defined chemotactic gradients, differentiate into tumor-associated macrophages (TAMs), and then accumulate in the hypoxic areas. Elevated TAM density in some regions or overall TAM numbers are correlated with increased tumor angiogenesis and a reduced host survival in the case of various types of tumors. To evaluate the role of TAMs in tumor growth, we here specifically eliminated TAMs by in vivo application of dichloromethylene diphosphonate (DMDP)-containing liposomes to mice bearing various types of tumors (e.g., B16 melanoma, KLN205 squamous cell carcinoma, and 3LL Lewis lung cancer), all of which grew in the dermis of syngeneic mouse skin. When DMDP-liposomes were injected into four spots to surround the tumor on day 0 or 5 after tumor injection and every third day thereafter, both the induction of TAMs and the tumor growth were suppressed in a dose-dependent and injection number-dependent manner; and unexpectedly, the tumor cells were rejected by 12 injections of three times-diluted DMDP-liposomes. The absence of TAMs in turn induced the invasion of inflammatory cells into or around the tumors; and the major population of effector cells cytotoxic against the target tumor cells were CD11b(+) monocytic macrophages, but not CCR3(+) eosinophils or Gr-1(+) neutrophils. These results indicate that both the absence of TAMs and invasion of CD11b(+) monocytic macrophages resulted in the tumor rejection.