Measurement of protein cytokines in tissue extracts by enzyme-linked immunosorbent assays: application to lipopolysaccharide-induced differential milieu of cytokines.
OBJECTIVE
Determination of protein cytokines in local tissues would help to evaluate their local role in health, sickness behavior and immune-mediated diseases. Therefore, developing a simple quantitative method of protein cytokines in tissues/organs is highly important.


METHODS
Mouse tissues were collected following intraperitoneal administration of endotoxin-free PBS or lipopolysaccharide. A mild detergent, 0.1% Igepal, was added in a buffer to enhance cytokines extraction. The tissues were then disrupted, homogenized, centrifuged and the supernatants were collected and assayed using solid-phase immunoassays.


RESULTS
The presence of 0.1% Igepal extracted significantly more TNF-alpha from liver (322%: p<0.01), brain (358%: p<0.05), lungs (1600%: p<0.01), and more IL-10 from liver (220%: p<0.001), brain (4650%: p<0.001) than PBS alone. On the other hand, using 0.1% Igepal did not increase IFN-gamma extraction from liver, spleen, brain, lungs, skin and kidneys more than PBS alone. Furthermore, i.p. administration of LPS induced a differential milieu of cytokines. LPS increased significantly the production of TNF-alpha, IFN-gamma, and IL-10 from liver (521%, 123%, 72%: p<0.01, 0.04, 0.04), brain (470%, 122%, 280%: p< 0.01, 0.03, 0.01), peritoneal lavage (p<0.001) and blood (p<0.001). However, the pattern of increase was different for the above cytokines in spleen, skin, lungs and kidneys.


CONCLUSIONS
The extraction of protein cytokines from tissues was superior with addition of mild detergent. Furthermore, our results showed a differential cytokines response to LPS with respect to tissue and cytokine type. This method should provide an important tool for studying local protein cytokines in behavioral pattern, sickness behavior, and immune-mediated diseases as well as to determine local therapeutic efficacy of immunomodulatory drugs.
Matalka
Khalid Z
KZ
Faculty of Pharmacy and Medical Technology, University of Petra, Amman, Jordan. kzm@uop.edu.jo
Tutunji
Maha F
MF
Abu-Baker
Mona
M
Abu Baker
Yousef
Y
D000818:Animals; D001923:Brain Chemistry / Q000276:immunology; D002457:Cell Extracts / Q000737:chemistry / Q000276:immunology; D016207:Cytokines / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification*; D003902:Detergents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007371:Interferon-gamma / Q000032:analysis / Q000097:blood / Q000302:isolation & purification; D016753:Interleukin-10 / Q000032:analysis / Q000097:blood / Q000302:isolation & purification; D007668:Kidney / Q000737:chemistry / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008099:Liver / Q000737:chemistry / Q000276:immunology; D008168:Lung / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D017830:Octoxynol; D009928:Organ Specificity; D011092:Polyethylene Glycols; D012867:Skin / Q000737:chemistry / Q000276:immunology; D013154:Spleen / Q000737:chemistry / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism
D002457:Cell Extracts; D016207:Cytokines; D003902:Detergents; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D011092:Polyethylene Glycols; D007371:Interferon-gamma; D017830:Octoxynol; C010615:Nonidet P-40