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

doi:10.1016/0161-5890(91)90157-F

Molecular Immunology

Volume 28, Issues 4–5, April–May 1991, Pages 437-447

https://doi.org/10.1016/0161-5890(91)90157-F Get rights and content

Abstract

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 ~ 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.

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The SMART PCR cDNA synthesis kit (Clontech, Palo Alto, CA) was used to generate high yields of full-length cDNA. Semiquantitative mRNA analysis using RT-PCR and Southern blot analysis was performed as described.3 The optimal number of PCR cycles was determined to be 20 to ensure logarithmic amplification.
It is generally believed that active invasion by cancer cells is essential to the metastatic process. In this report, we describe a murine mammary tumor (MCH66) model of metastasis that does not require invasion into the vascular wall of both the primary tumor and the target organ, in this case, the lung. The process involves intravasation of tumor nests surrounded by sinusoidal blood vessels, followed by intravascular tumor growth in the lung, without penetration of the vascular wall during the process. Comparative studies using a nonmetastatic MCH66 clone (MCH66C8) and another highly invasive metastatic cell line (MCH416) suggested that high angiogenic activity and sinusoidal remodeling of tumor blood vessels were prerequisites for MCH66 metastasis. Differential cDNA analysis identified several genes that were overexpressed by MCH66, including genes for the angiogenesis factor pleiotrophin, and extracellular matrix-associated molecules that may modulate the microenvironment toward neovascularization. Our analyses suggest that tumor angiogenesis plays a role in the induction of invasion-independent metastasis. This model should prove useful in screening and development of new therapeutic agents for cancer metastasis.
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To assess the potential for ingestion of yogurt to modulate immunity, its effects on basal gene expression of cytokines in systemic and mucosal sites were determined in mice. Yogurts were manufactured from pasteurized nonfat dry milk using five commercial starter cultures with or without Bifidobacterium sp. and Lactobacillus acidophilus. Treatment mice were fed the AIN-93G diet mixed 1:1 with unheated yogurt or heat-treated yogurt (wt/wt) for 2 and 4 weeks, and control mice were fed the AIN-93G diet mixed 1:1 (wt/wt) with nonfat dry milk. The viability of the various bacterial groups in unheated yogurts was maintained above 10⁶ CFU/g throughout the feeding period. The yogurt-feeding regimens did not significantly affect weight gain. Relative mRNA levels in spleen, mesenteric lymph nodes, or Peyer's patches for the cytokines interferon-γ, tumor necrosis factor-α, interleukin-2, -4, and -6, and the “housekeeping gene” β₂-microglobulin were determined by reverse transcriptase–polymerase chain reaction in conjunction with hybridization analysis. Prolonged feeding of some yogurts decreased expression of several cytokine mRNAs, the depression of tumor necrosis factor-α mRNA in the spleen being the most prominent effect. Heat-treated yogurts were more effective in altering cytokine mRNA expression than were unheated yogurts containing viable organisms. Generally, yogurts either had no effect or decreased specific cytokine mRNA in the test organs, regardless of whether they contained Bifidobacterium sp. and L. acidophilus. These results suggest that, in contrast with previous studies in vitro, some yogurt formulations may reduce rather than stimulate basal cytokine expression and that these effects are most prominent in the systemic compartment.
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Molecular Immunology

Abstract

References (29)

The common mediator of shock, cachexia, and tumor necrosis

Adv. Immun.

Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction

Analyt. Biochem.

Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells

J. biol. Chem.

Comparison of in vitro cell cytotoxic assays for tumor necrosis factor

J. Immun. Meth.

In vitro detection of lymphokines produced by in vivo activated lymphocytes

J. Immun. Meth.

Adrenalectomy sensitizes mice to the lethal effects of interleukin 1 and tumor necrosis factor

J. exp. Med.

Identity of tumor necrosis factor and the macrophage-secreted factor cachectin

Nature

In vivo expression and regulation of murine IL-2 receptors after antigen sensitization

J. Immun.

A comprehensive set of sequence analysis programs for the VAX

Nucl. Acid Res.

cDNA synthesis by cloned moloney murine leukemia virus reverse transcriptase lacking RNAse H activity

Focus

The effect of polyoma virus, serum factors, and dibutyryl cyclic AMP on dihydrofolate reductase synthesis, and the entry of quiescent cells into S phase

J. cell. Physiol.

Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenic cascade

Rev. Infect. Dis.

Tumor necrosis factor identified in multiple sclerosis brain

J. exp. Med.

Enhancement of human B cell proliferation and differentiation by tumor necrosis factor-alpha and interleukin 1

J. Immun.

Cited by (52)

The pharmacology study of a new recombinant TNF receptor-hyFc fusion protein

Immunotoxicity of epicutaneously applied anticoagulant rodenticide warfarin: Evaluation by contact hypersensitivity to DNCB in rats

An invasion-independent pathway of blood-borne metastasis: A new murine mammary tumor model

A fluorimeter-based RT-PCR method for the detection and quantitation of porcine cytokines

Effects of yogurt ingestion on mucosal and systemic cytokine gene expression in the mouse

Analysis of interleukin 12 protein production and mRNA expression in mice exposed topically to chemical allergens