Approaches to determine expression of inflammatory cytokines

Abstract

There is an increasing awareness of the role of inflammation in cancer. Immune responses can limit the growth of some tumors, but paradoxically, may promote the growth of others. Cytokines are critical mediators of both the innate and the adaptive immune responses. In this chapter, we will describe several methods for the detection of inflammatory cytokines. First, we will describe a protocol for quantification of cytokine mRNA by real-time quantitative PCR. In addition, we will describe detection of cytokine proteins by ELISA as well as by novel cytokine bead arrays. Finally, a method will be described for in situ detection of cytokine production by immunohistochemistry.

Fig. 1

Real-time PCR plots. (A) Amplification plot. Fluorescence increase is shown as a function of cycle number. Delta Rn represents the normalized reporter (Rn) fluorescence intensities after subtraction of baseline intensities. Baseline cycles are those before a steady upward trend in fluorescence occurs. Generally, the baseline cycles are determined by a program software and need not be adjusted. This threshold is set automatically by program software, but sometimes, manual adjustments can improve data quality. The threshold must be set within the exponential phase of the curve, as that is the phase of the reaction when components are not limiting and close to a doubling of product occurs with each cycle. For this reason, it is easiest to plot the Delta Rn on a logarithmic scale, since this magnifies the exponential phase compared to a linear view. In this example, the threshold could be set a bit lower, such that it would include trace number 3. However, threshold 1 is set too low, because it fails to exclude nonspecific signal generated at the first four cycles of the reaction, when no specific product is present yet. Trace number 4 cannot reliably be included in the analysis, therefore. On the other hand, threshold 3 is set too high, as it no longer falls within the exponential phase. Reliable amplification curves all run parallel to one another and have similar shapes. Trace number 1 looks reliable, but trace number 2 cannot be trusted, because of its deviant shape. (B) Standard curve. The cycle at which a sample crossed the threshold (threshold cycle, Ct) is plotted against the starting concentration (on a logarithmic scale-Log CO). The slope of the standard curve (closed squares) is between 3.1 and 3.5, indicating almost perfect exponential kinetics of the PCR reaction and the reliability of the curve fit (R²) is high. The standard curve could still be improved by excluding standard sample number 2, which is a clear outlier. Measurements of most experimental samples (unknowns, closed circles) can be trusted, except for sample number 2, which falls outside the standard curve.

Fig. 2

Skin confocal micrographs of a cytokine-like peptide (Aβ) and immune cell-surface markers after transcutaneous immunization. To characterize dermal immune responses to Aβ plus cholera toxin (Aβ/CT) transcutaneous (t.c.) immunization, skin tissues were prepared from nontransgenic C57BL/6 mice t.c.-immunized for 18 h with PBS (control, Top), CT alone (Middle), or Aβ/CT (Bottom) as indicated and then analyzed by laser scanning confocal microscopy with the indicated antibodies (antibody 4G8 was used to reveal Aβ). Note the presence of CD207 + CD11c+ Langerhans cells in Aβ-positive regions in the Aβ/CT t.c.-immunized group. DAPI (blue signal) was used as a nuclear counterstain in merged images shown to the right. (Scale bar: 50 μm.). This figure and figure legend was reproduced with permission from Proceedings of the National Academy of Sciences of the United States of America.