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Dendritic cell homing to murine lung is not CCL5 or CCR5 dependent
$152
Abstracts
J ALLERGY CLIN IMMUNOL FEBRUARY 2003
30 Dendritic Cell Homing to Murine Lung Is Not CCL5 or CCR5 Dependent
32 theLPSNasal Stimulates T Cell Proliferation and Expression of IL-10 in Mucosa of Young Atopic Children but not in Atopic
M. H. Grayson 1, M. M. Rohlfing l, M. J. Holtzman2; qnternal Medicine; Division of Allergy and Immunology, Washington University School of Medicine, Saint Louis, MO, 2Internal Medicine; Division of Pulmonary and Critical Care Medicine, Washington University School of Medicine, Saint Louis, MO. RATIONALE: Dendritic cells (DC) are critical to the immune response, but to be effective they must be recruited from the bone marrow to the tissue site for encountering antigen. The migration of DC into peripheral tissues is likely mediated by CC chemokines, but the precise controls for this process still need to be defined. Here we assess the role of chemokine CCL5 (RANTES) and CCR5 (corresponding chemokine receptor) in dendritic cell homing to the lung. In particular, we compare tissue levels of DC and DC subsets recruited to the lungs of wild-type (WT) mice versus mice with targeted null mutations of the genes for CCR5 (CCR5-nuI1) or CCL5 (CCL5-null). METHODS: Lungs from WT, CCR5-nuI1, and CCL5-null mice were used to generate a single cell suspension using DNase-l, collagenase-I, and hyaluronidase. DC were identified by CD 1 lc expression and subsets were analyzed for MHC Class II, CDI lb, B220, CD4, and CD8 expression by flow cytometry. RESULTS: Lungs from WT mice contained slightly but not significantly more DC than lungs from CCR5- or CCL5-null mice (6.0 - 0.8%, 4.3 -+ 0.2%, 4.5 - 1.1% of total lung ceils, respectively; n=2 per condition). Analysis of subgroup markers showed that 64-78% of DC expressed MHC II, 40-53% expressed CDI lb, 22-28% expressed B220, and 8-14% expressed CD4 or CD8 with no significant differences among lungs from WT, CCR5-, and CCL5-null mice. CONCLUSIONS: CCL5 and CCR5 are not required for homeostatic DC migration to the murine lung under basal conditions, raising the possibility that these interactions may be more relevant during inflammatory conditions.
M. K. Tulic 1, J. J. Manoukian 2, D. H. Eidelman l, Q. Hamidl; IMeakinsChristie Laboratories, McGill University, Montreal, PQ, CANADA, 2Montreal Children's Hospital, Montreal, PQ, CANADA. RATIONALE: Previously we have shown that LPS can induce local Th l cytokine immunoreactivity and T cell proliferation in the nasal mucosa of non-atopic children. To determine if LPS can stimulate T cell immunoreactivity and cytokine production in atopic mucosa from young children and if so, is this response similar to the LPS response seen in atopic adults. METHODS: We cultured nasal mucosa from atopic children (n=8) or atopic adults (n=7) with or without allergen in presence or absence of LPS (0.1 /Jg/ml) for 24 hrs. T lymphocytes (CD3) were identified with immunocytochemistry and IL-10 mRNA expression using in situ hybridization. RESULTS: Constitutive expression of CD3+ cells and IL-10 mRNA was detected in unstimulated atopic adult and children mucosa. Allergen increased CD3-immunoreactivity and IL-10 mRNA expression in atopic adults (P<0.05) and children (P<0.05) compared to unstimulated mucosa. In atopic children, LPS caused 4.7-fold increase in the number of CD3positive cells and 6.5-fold increase in the number of IL-10 mRNA-positive cells (P<0.001). Similar increase was observed in atopic mucosa from children exposed to allergen and then stimulated with LPS (P<0.001). LPS had no effect on the number of CD3 or IL-10 mRNA-positive cells in adult mucosa. CONCLUSIONS: Our results suggest that LPS can induce local inflammation in atopic children. This inflammation however, is characterized by increased T lymphocytes and increased expression of immunomodulatory cytokine, IL-l0 which is not seen in adults. These findings underscore the capacity of the juvenile immune system to respond to bacterial products with a polarization of the immune response.
Funding: NIH, NIAID
Funding: Ludwig Engel Fellowship, GSK/CIHR/CLA Fellowship
331 '""Innate Immune System in Human Breast Milk
333
N. Yannaras, M. Srivastava; Pediatrics, Metroheahh Medical Center, Cleveland, OH. The innate immune system is the body's nonspecific defense to foreign substances. Many antimicrobial proteins including defensins, Toll-like receptors (TLRs) and cathelicidins are involved in the recognition and elimination of pathogens. Breast milk is known to contain a host of antiinfectious factors that are important in transferring immunity to infants, although the innate immune system in breast milk has never been thoroughly researched. In previous studies, human ~-defensin- 1 (HBD- 1) was detected in breast milk. The rationale of the study is to look for the presence of other defensins, as well as TLRs and LL-37 (cathelicidin derived antimicrobial peptide), in breast milk. The methods included extraction, purification and quantification of RNA with subsequent use of semi-quantitative RT-PCR to detect defensin mRNA in 41 lactating mothers. In addition, we attempted the detection of TLRs and LL-37. Our results show that HBD-I (95%), HBD-2 (15%), HBD-3 (22%), HBD-4 (5%), human a-defensin-5 (HD-5) (68%), HD-6 (2%) are present at the transcriptional level in breast milk. All samples tested were positive for TLR2, TLR3, TLR4, TLR5, TLR7 and TLR10, 66% for TLRI, TLR6 and CDI4, 33% for TLR9, 0% for TLR8, 81% tbr LL-37 and 88% for HNP 1-3. In conclusion, the data obtained gives rise to the complex profile of the innate immune system in breast milk, which may provide protection to the developing digestive tract of the newborn and also to the maternal breast tissue. Furthermore, supplementation of infant formulas may serve as a potential strategy to increase immunity in bottle-fed infants.
F. Puggioni z, J. N. Francis 2, S. R. Durham2; IUniversity of Pavia, Department of Pulmonary Diseases, IRCCS Policlinico S. Matteo, Pavia, ITALY, 2Upper Respiratory Medicine, Imperial College, London, UNITED KINGDOM. RATIONALE: Monophosphoryl lipid A (MPL) is derived from the lypopolysaccharide (LPS) of Salmonella Minnesota R 595. MPL| has been used as an adjuvant in vaccines directed against tumor antigens, hepatitis B and is a component of a subcutaneous grass pollen vaccine (Pollinex 4/MPL| which was effective in hay fever, and associated with an increase in serum allergen specific IgG4 levels and a decrease in IgE levels (Drachenberg KY et al, Allergy 2001 ;56:498-505). However, little is known about the effects of MPL| on cellular responses to allergens. METHODS: We studied 10 adult atopic subjects suffering from hay fever (mean age: 32.8 yrs, 7 males, skin prick test to grass pollen mean wheal: 10 mm; RAST test mean value: 52 IU/ml). PBMCs were cultured for 6 days with Phleum Pratense extract (0, 2, 20 ~g/ml) (Allergy Therapeutics) and MPL| (0, 10 ~tg/ml). RESULTS: No differences in proliferative responses were observed. There was a significant increase in IFN-y production: allergen 20 [al/ml alone (median {interquartile range} = 28.7 lag/ml { 17.5, 94.9}) compared with allergen 20 lal/ml + MPL| 10 ~tg/ml: (1934.5 Ilg/ml {904, 3644.8}; p=0.002). There was a significant decrease in IL-5 production (4855 ~g/ml {2976.5, 8366} vs 2671.6 I-tg/ml {847.2, 6194.3}; p--0.01). IL-10 production did not change. Addition of neutralizing IL-12-antibody resulted in a 95% inhibition in allergen+MPL| IFN- 7 production. CONCLUSIONS: The combination of MPL| with grass pollen extract
Funding: Department of Pediatrics, Metrohealth Medical Center
Adults
Monophosphory, Lipid A: In Vitro Effects on Human T Cells Proliferation and Cytokines Production
Abstracts
J ALLERGY CLIN IMMUNOL FEBRUARY 2003
30 Dendritic Cell Homing to Murine Lung Is Not CCL5 or CCR5 Dependent
32 theLPSNasal Stimulates T Cell Proliferation and Expression of IL-10 in Mucosa of Young Atopic Children but not in Atopic
M. H. Grayson 1, M. M. Rohlfing l, M. J. Holtzman2; qnternal Medicine; Division of Allergy and Immunology, Washington University School of Medicine, Saint Louis, MO, 2Internal Medicine; Division of Pulmonary and Critical Care Medicine, Washington University School of Medicine, Saint Louis, MO. RATIONALE: Dendritic cells (DC) are critical to the immune response, but to be effective they must be recruited from the bone marrow to the tissue site for encountering antigen. The migration of DC into peripheral tissues is likely mediated by CC chemokines, but the precise controls for this process still need to be defined. Here we assess the role of chemokine CCL5 (RANTES) and CCR5 (corresponding chemokine receptor) in dendritic cell homing to the lung. In particular, we compare tissue levels of DC and DC subsets recruited to the lungs of wild-type (WT) mice versus mice with targeted null mutations of the genes for CCR5 (CCR5-nuI1) or CCL5 (CCL5-null). METHODS: Lungs from WT, CCR5-nuI1, and CCL5-null mice were used to generate a single cell suspension using DNase-l, collagenase-I, and hyaluronidase. DC were identified by CD 1 lc expression and subsets were analyzed for MHC Class II, CDI lb, B220, CD4, and CD8 expression by flow cytometry. RESULTS: Lungs from WT mice contained slightly but not significantly more DC than lungs from CCR5- or CCL5-null mice (6.0 - 0.8%, 4.3 -+ 0.2%, 4.5 - 1.1% of total lung ceils, respectively; n=2 per condition). Analysis of subgroup markers showed that 64-78% of DC expressed MHC II, 40-53% expressed CDI lb, 22-28% expressed B220, and 8-14% expressed CD4 or CD8 with no significant differences among lungs from WT, CCR5-, and CCL5-null mice. CONCLUSIONS: CCL5 and CCR5 are not required for homeostatic DC migration to the murine lung under basal conditions, raising the possibility that these interactions may be more relevant during inflammatory conditions.
M. K. Tulic 1, J. J. Manoukian 2, D. H. Eidelman l, Q. Hamidl; IMeakinsChristie Laboratories, McGill University, Montreal, PQ, CANADA, 2Montreal Children's Hospital, Montreal, PQ, CANADA. RATIONALE: Previously we have shown that LPS can induce local Th l cytokine immunoreactivity and T cell proliferation in the nasal mucosa of non-atopic children. To determine if LPS can stimulate T cell immunoreactivity and cytokine production in atopic mucosa from young children and if so, is this response similar to the LPS response seen in atopic adults. METHODS: We cultured nasal mucosa from atopic children (n=8) or atopic adults (n=7) with or without allergen in presence or absence of LPS (0.1 /Jg/ml) for 24 hrs. T lymphocytes (CD3) were identified with immunocytochemistry and IL-10 mRNA expression using in situ hybridization. RESULTS: Constitutive expression of CD3+ cells and IL-10 mRNA was detected in unstimulated atopic adult and children mucosa. Allergen increased CD3-immunoreactivity and IL-10 mRNA expression in atopic adults (P<0.05) and children (P<0.05) compared to unstimulated mucosa. In atopic children, LPS caused 4.7-fold increase in the number of CD3positive cells and 6.5-fold increase in the number of IL-10 mRNA-positive cells (P<0.001). Similar increase was observed in atopic mucosa from children exposed to allergen and then stimulated with LPS (P<0.001). LPS had no effect on the number of CD3 or IL-10 mRNA-positive cells in adult mucosa. CONCLUSIONS: Our results suggest that LPS can induce local inflammation in atopic children. This inflammation however, is characterized by increased T lymphocytes and increased expression of immunomodulatory cytokine, IL-l0 which is not seen in adults. These findings underscore the capacity of the juvenile immune system to respond to bacterial products with a polarization of the immune response.
Funding: NIH, NIAID
Funding: Ludwig Engel Fellowship, GSK/CIHR/CLA Fellowship
331 '""Innate Immune System in Human Breast Milk
333
N. Yannaras, M. Srivastava; Pediatrics, Metroheahh Medical Center, Cleveland, OH. The innate immune system is the body's nonspecific defense to foreign substances. Many antimicrobial proteins including defensins, Toll-like receptors (TLRs) and cathelicidins are involved in the recognition and elimination of pathogens. Breast milk is known to contain a host of antiinfectious factors that are important in transferring immunity to infants, although the innate immune system in breast milk has never been thoroughly researched. In previous studies, human ~-defensin- 1 (HBD- 1) was detected in breast milk. The rationale of the study is to look for the presence of other defensins, as well as TLRs and LL-37 (cathelicidin derived antimicrobial peptide), in breast milk. The methods included extraction, purification and quantification of RNA with subsequent use of semi-quantitative RT-PCR to detect defensin mRNA in 41 lactating mothers. In addition, we attempted the detection of TLRs and LL-37. Our results show that HBD-I (95%), HBD-2 (15%), HBD-3 (22%), HBD-4 (5%), human a-defensin-5 (HD-5) (68%), HD-6 (2%) are present at the transcriptional level in breast milk. All samples tested were positive for TLR2, TLR3, TLR4, TLR5, TLR7 and TLR10, 66% for TLRI, TLR6 and CDI4, 33% for TLR9, 0% for TLR8, 81% tbr LL-37 and 88% for HNP 1-3. In conclusion, the data obtained gives rise to the complex profile of the innate immune system in breast milk, which may provide protection to the developing digestive tract of the newborn and also to the maternal breast tissue. Furthermore, supplementation of infant formulas may serve as a potential strategy to increase immunity in bottle-fed infants.
F. Puggioni z, J. N. Francis 2, S. R. Durham2; IUniversity of Pavia, Department of Pulmonary Diseases, IRCCS Policlinico S. Matteo, Pavia, ITALY, 2Upper Respiratory Medicine, Imperial College, London, UNITED KINGDOM. RATIONALE: Monophosphoryl lipid A (MPL) is derived from the lypopolysaccharide (LPS) of Salmonella Minnesota R 595. MPL| has been used as an adjuvant in vaccines directed against tumor antigens, hepatitis B and is a component of a subcutaneous grass pollen vaccine (Pollinex 4/MPL| which was effective in hay fever, and associated with an increase in serum allergen specific IgG4 levels and a decrease in IgE levels (Drachenberg KY et al, Allergy 2001 ;56:498-505). However, little is known about the effects of MPL| on cellular responses to allergens. METHODS: We studied 10 adult atopic subjects suffering from hay fever (mean age: 32.8 yrs, 7 males, skin prick test to grass pollen mean wheal: 10 mm; RAST test mean value: 52 IU/ml). PBMCs were cultured for 6 days with Phleum Pratense extract (0, 2, 20 ~g/ml) (Allergy Therapeutics) and MPL| (0, 10 ~tg/ml). RESULTS: No differences in proliferative responses were observed. There was a significant increase in IFN-y production: allergen 20 [al/ml alone (median {interquartile range} = 28.7 lag/ml { 17.5, 94.9}) compared with allergen 20 lal/ml + MPL| 10 ~tg/ml: (1934.5 Ilg/ml {904, 3644.8}; p=0.002). There was a significant decrease in IL-5 production (4855 ~g/ml {2976.5, 8366} vs 2671.6 I-tg/ml {847.2, 6194.3}; p--0.01). IL-10 production did not change. Addition of neutralizing IL-12-antibody resulted in a 95% inhibition in allergen+MPL| IFN- 7 production. CONCLUSIONS: The combination of MPL| with grass pollen extract
Funding: Department of Pediatrics, Metrohealth Medical Center
Adults
Monophosphory, Lipid A: In Vitro Effects on Human T Cells Proliferation and Cytokines Production