Cell-associated human retinal pigment epithelium interleukin-8 and monocyte chemotactic protein-1: immunochemical and in-situ hybridization analyses.
Human retinal pigment epithelial (RPE) cells secrete chemokines, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in response to pro-inflammatory cytokines. In this study we (1) examined the efficiency of human RPE IL-8 and MCP-1 secretion, (2) determined the amount of neutrophil and monocyte chemotactic activity in human RPE cell conditioned media and cell extracts that is attributable to IL-8 and MCP-1, respectively, and (3) assessed the sensitivity of immunohistochemistry and in situ hybridization for detecting chemokine production by cytokine-stimulated human RPE cells. Conditioned media and extracts from human RPE cells stimulated with various physiologic concentrations of interleukin-1 beta (IL-1 beta) (0.2-20 ng ml-1), tumor necrosis factor (TNF-alpha) (0.2-20 ng ml-1) or interferon-gamma (IFN-gamma) (10-1000 U ml-1) were examined to compare secreted and cell associated levels of IL-8 and MCP-1 at various time points up to 24 hr. ELISA demonstrated that IL-8 and MCP-1 are both efficiently secreted by pro-inflammatory cytokine treated human RPE cells. Substantial dose- and time-dependent RPE secretion of IL-8 was observed following stimulation with IL-1 beta or TNF-alpha, but cell associated IL-8 was detectable only after high dose (20 ng ml-1) IL-1 beta stimulation and comprised less than 1% of the total IL-8 induced. Dose- and time-dependent RPE cell MCP-1 secretion was also observed following IL-1 beta > TNF-alpha > IFN-gamma stimulation, with an average of 4% of the total MCP-1 retained within RPE. Bioassays demonstrated neutrophil and monocyte chemotactic activity in conditioned media from stimulated RPE cells, but not in human RPE cell extracts. Inhibition of conditioned media-induced chemotaxis by specific anti-IL-8 or anti-MCP-1 antibodies demonstrated that IL-8 and MCP-1 were responsible for the majority of HRPE-derived neutrophil (> 60%) and monocyte (53-57%) chemotactic activity, respectively. Using in situ hybridization IL-8 mRNA was readily detected within IL-1 beta > TNF-alpha stimulated RPE cells and MCP-1 mRNA easily visualized within IL-1 beta > TNF-alpha > or IFN-gamma stimulated cells. Immunohistochemistry to detect IL-8 was positive only in RPE cells exposed to high dose IL-1 beta (20 ng ml-1) for 8 or 24 hr and was weak. Immunohistochemical staining for MCP-1 in RPE cells was more intense and was visualized within RPE cells stimulated with IL-beta, TNF-alpha, or IFN-gamma. This study demonstrates that: (1) RPE cells efficiently secrete IL-8 and MCP-1 upon stimulation with pro-inflammatory cytokines; (2) secreted IL-8 and MCP-1 account for the majority of human RPE neutrophil and monocyte chemotactic activity; (3) in situ hybridization readily detects IL-8 and MCP-1 mRNA in cytokine stimulated RPE cells; and (4) immunohistochemistry demonstrates cell-associated MCP-1 in cytokine stimulated RPE cells, but only minimal cell-associated IL-8.
Elner
V M
VM
Department of Ophthalmology (W. K. Kellogg Eye Center), University of Michigan, Ann Arbor 48105, USA.
Burnstine
M A
MA
Strieter
R M
RM
Kunkel
S L
SL
Elner
S G
SG
D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000032:analysis* / Q000235:genetics; D002634:Chemotaxis, Leukocyte; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000032:analysis* / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000276:immunology*; D012333:RNA, Messenger / Q000032:analysis; D013268:Stimulation, Chemical; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D018932:Chemokine CCL2; D017077:Culture Media, Conditioned; D007375:Interleukin-1; D016209:Interleukin-8; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma