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Cathelin-Related Antimicrobial Peptide (CRAMP)

Mapping in Human Lung Cancer & Rat/Mouse Lung Tissue

Mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like 1/mouse formyl peptide receptor-like 2 as the receptor and acts as an immune adjuvant.

Mammalian antimicrobial proteins, such as defensins and cathelicidin, have stimulating effects on host leukocytes. Cathelin-related antimicrobial peptide (CRAMP), the orthologue of human cathelicidin/LL-37, is the sole identified murine cathelicidin. CRAMP has been shown to have both antimicrobial and angiogenic activities. However, whether CRAMP, like human cathelicidin/LL-37, also exhibits a direct effect on the migration and function of leukocytes is not known. We have observed that CRAMP, like LL-37, was chemotactic for human monocytes, neutrophils, macrophages, and mouse peripheral blood leukocytes. CRAMP also induced calcium mobilization and the activation of MAPK in monocytes. CRAMP-induced calcium flux in monocytes was desensitized by MMK-1, an agonistic ligand specific for formyl peptide receptor-like-1 (FPRL1), and vice versa, suggesting the use of FPRL1 by CRAMP as a receptor. Furthermore, CRAMP induced the chemotaxis of human embryonic kidney 293 cells transfected with either FPRL1 or mouse formyl peptide receptor-2, the mouse homologue of FPRL1, but not by untransfected parental human embryonic kidney 293 cells, confirming the use of FPRL1/mouse formyl peptide receptor-2 by CRAMP. Injection of CRAMP into mouse air pouches resulted in the recruitment predominantly of neutrophils and monocytes, indicating that CRAMP acts as a chemotactic factor in vivo. Finally, simultaneous administration of OVA with CRAMP to mice promoted both humoral and cellular Ag-specific immune responses. Thus, CRAMP functions as both a chemoattractant for phagocytic leukocytes and an enhancer of adaptive immune response.

Kurosaka et al. J Immunol. 2005 May 15;174(10):6257-65.

Stimulation and cross-desensitization of Ca2+ flux in monocytes by CRAMP

Stimulation and cross-desensitization of Ca2+ flux in monocytes by CRAMP. Ca2+ flux of fura 2-loaded human monocytes in response to MMK-1 (A) and CRAMP (B) was measured by recording the ratio of emission at 510 after simultaneous excitation at 340 and 380. Cross-desensitization of Ca2+ flux was determined by sequential addition of MMK-1 and CRAMP at the indicated concentrations and for the indicated time periods (C) or vice versa (D). Kurosaka K,, et al. J Immunol. 2005 May 15;174(10):6257-65.

CRAMP usage of FPRL1 or mFPR2 to mediate chemotaxis.

CRAMP usage of FPRL1 or mFPR2 to mediate chemotaxis. The migration of FPRL1/HEK293 (A) and mFPR2/HEK293 (B) cells in response to CRAMP or W peptide (positive control) was examined, and the average cell migration (mean ± SD) of triplicate wells was determined. CRAMP did not induce the migration of parental HEK293 cells or HEK293 cells expressing several other chemokine receptors (data not shown).

Kurosaka et al. J Immunol. 2005 May 15;174(10):6257-65.

 

Mapping in lung Cancer Tissue by CRAMP (140-173) mouse Antibody (H-072-15)

  

  

  

Protocol for antibody staining

Tissue Sample

Human lung cancer tissues, Rat & Mouse lung tissues

Fixative

10% formalin

Embedding

Paraffin

Negative Control

No primary antibody

Pretreatment

N/A

Blocking

3% H2O2, 2% Normal Goat Serum

Primary Antibody

rabbit anti-CRAMP (Mouse) antibody (Cat. No.: H-072-16)

Optimal Dilution

1: 500

Secondary Antibody

Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min

Amplification

Streptavidin-HRP (Vector), 1:400, 30 min

Detection System

HRP

Substrate

DAB (Sigma), 3 min

Counterstained

Hematoxylin, 30 sec

 

Mapping in Rat/Mouse Lung Tissue (H-072-15)

%072-15%


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