Quantitation of cytokine mRNA levels utilizing the reverse transcriptase-polymerase chain reaction following primary antigen-specific sensitization in vivo--I. Verification of linearity, reproducibility and specificity.
The role of cytokines in vivo has been difficult to assess. This difficulty is due, in part, to the limited number of producer cells and the strict regulation of cytokine production. In order to address this situation, we have developed assays which allow us to quantitate both protein production and steady state mRNA levels from specific in vivo sites. In this report, we present data utilizing these assays on cells obtained from draining LN following specific sensitization with antigen in vivo. In order to determine the relative quantities of cytokine mRNA, we modified the reverse transcriptase-polymerase chain reaction which had been previously described. The modified assay is (1) linear over a large concn range of input template (2) demonstrates a high degree of reproducibility (SE approximately 13%) and (3) is very sensitive. Utilizing this assay, we have measured a constitutive mRNA (DHFR), quantitated both the presence of lymphokine mRNA (IL-2) and the induction of cytokine mRNA (TNF alpha). In this report we have examined the kinetics of TNF alpha mRNA expression and have demonstrated that following epicutaneous sensitization with picryl chloride, there is rapid induction (within 24 hr) of TNF alpha mRNA in the draining LN and that the levels of mRNA remain detectable through d7. In addition, we determined the time course of production of TNF protein by the draining LN cells and found that it was similar to that of the mRNA levels. A potential pathologic role for immune response generated TNF alpha is also discussed. We believe these experiments demonstrate that cytokine production following antigen-specific sensitization in vivo can be analyzed at both the cellular and molecular level. The data suggests that this approach can be used to study cytokine regulation in vivo.
Mohler
K M
KM
Department of Immunology, Lilly Research Laboratories, Indianapolis, IN 46285.
Butler
L D
LD
D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000235:genetics*; D015870:Gene Expression; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000235:genetics; D008198:Lymph Nodes / Q000166:cytology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000502:physiology*; D051379:Mice; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
D016207:Cytokines; D007376:Interleukin-2; D009841:Oligonucleotides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D013762:Tetrahydrofolate Dehydrogenase; D012194:RNA-Directed DNA Polymerase