Archived Comments for:
CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use
RT-PCR does not distinguish IL-4 mRNA from the IL-4R antagonist IL-4delta2
Eric Glare, Alfred Hospital
31 March 2005
Boeuf et al. [1] have utilised RNA standards and real-time PCR to quantify a range of cytokine mRNA including Interleukin-4 (IL-4). However, the authors do not mention that the IL-4 gene contains 4 exons separated by 3 introns and produces two alternatively spliced mRNA. IL-4 mRNA is composed of all 4 exons spliced together whilst IL-4delta2 mRNA is missing exon 2 [2]. The deletion of exon 2 does not result in a frameshift and IL-4delta2 mRNA encodes a truncated IL-4 protein. Studies with recombinant human IL-4delta2 indicate that IL-4delta2 is a naturally occurring receptor antagonist of IL-4 [3, 4].
An alignment search with the IL-4 primer sequences used by the authors against the public genomic database [5] reveal that the primer pair have matches with both IL-4 (Accession number NM_000589) and IL-4delta2 (Accession number NM_172348) cDNA sequences with predicted PCR products of 101 and 53 bp respectively.
Quantitative RT-PCR for IL-4 was conducted on PBMC with and without stimulation (see figure 4 [1]). IL-4delta2 has frequently been described in RNA from PBMC [6, 7] and may be co-ordinately transcribed with IL-4 so that subsequent expression of IL-4delta2 may terminate the IL-4 immune response [8]. Consequently it is likely that the authors are actually reporting total IL-4 gene expression and not just IL-4 mRNA levels.
As the authors comment, immune responses are the result of the cytokine milieu rather than the action of any one cytokine. Given that IL-4delta2 is a receptor antagonist of IL-4, separate quantification of IL-4 and IL-4delta2 mRNA expression should be included in this concept.
Eric M Glare
Melbourne, Australia
1. Boeuf P, Vigan-Womas I, Jublot D, Loizon S, Barale J, Akanmori B, Mercereau-Puijalon O, Charlotte Behr: CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use. BMC Immunology 2005, 6:5.
2. Klein SC, Golverdingen JG, Bouwens AG, Tilanus MG, de Weger RA: An alternatively spliced interleukin 4 form in lymphoid cells. Immunogenetics 1995, 41(1):57.
3. Atamas SP, Choi J, Yurovsky VV, White B: An alternative splice variant of human IL-4, IL-4 delta 2, inhibits IL-4-stimulated T cell proliferation. Journal of Immunology 1996, 156(2):435-41.
4. Arinobu Y, Atamas SP, Otsuka T, Niiro H, Yamaoka K, Mitsuyasu H, Niho Y, Hamasaki N, White B, Izuhara K: Antagonistic effects of an alternative splice variant of human IL-4, IL- 4delta2, on IL-4 activities in human monocytes and B cells. Cell Immunol 1999, 191(2):161-7.
5. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402.
6. Sorg RV, Enczmann J, Sorg UR, Schneider EM, Wernet P: Identification of an alternatively spliced transcript of human interleukin-4 lacking the sequence encoded by exon 2. Experimental Hematology 1993, 21(4):560-3.
7. Glare EM, Divjak M, Rolland JM, Walters EH: Asthmatic airway biopsy specimens are more likely to express the IL-4 alternative splice variant IL-4delta2. J Allergy Clin Immunol 1999, 104(5):978-82.
8. Glare EM, Divjak M, Bailey MJ, Walters EH: The usefulness of competitive PCR: airway gene expression of IL-5, IL-4, IL-4delta2, IL-2, and IFN-gamma in asthma. Thorax 2001, 56(7):541-8.
RT-PCR does not distinguish IL-4 mRNA from the IL-4R antagonist IL-4delta2
31 March 2005
Boeuf et al. [1] have utilised RNA standards and real-time PCR to quantify a range of cytokine mRNA including Interleukin-4 (IL-4). However, the authors do not mention that the IL-4 gene contains 4 exons separated by 3 introns and produces two alternatively spliced mRNA. IL-4 mRNA is composed of all 4 exons spliced together whilst IL-4delta2 mRNA is missing exon 2 [2]. The deletion of exon 2 does not result in a frameshift and IL-4delta2 mRNA encodes a truncated IL-4 protein. Studies with recombinant human IL-4delta2 indicate that IL-4delta2 is a naturally occurring receptor antagonist of IL-4 [3, 4].
An alignment search with the IL-4 primer sequences used by the authors against the public genomic database [5] reveal that the primer pair have matches with both IL-4 (Accession number NM_000589) and IL-4delta2 (Accession number NM_172348) cDNA sequences with predicted PCR products of 101 and 53 bp respectively.
Quantitative RT-PCR for IL-4 was conducted on PBMC with and without stimulation (see figure 4 [1]). IL-4delta2 has frequently been described in RNA from PBMC [6, 7] and may be co-ordinately transcribed with IL-4 so that subsequent expression of IL-4delta2 may terminate the IL-4 immune response [8]. Consequently it is likely that the authors are actually reporting total IL-4 gene expression and not just IL-4 mRNA levels.
As the authors comment, immune responses are the result of the cytokine milieu rather than the action of any one cytokine. Given that IL-4delta2 is a receptor antagonist of IL-4, separate quantification of IL-4 and IL-4delta2 mRNA expression should be included in this concept.
Eric M Glare
Melbourne, Australia
1. Boeuf P, Vigan-Womas I, Jublot D, Loizon S, Barale J, Akanmori B, Mercereau-Puijalon O, Charlotte Behr: CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use. BMC Immunology 2005, 6:5.
2. Klein SC, Golverdingen JG, Bouwens AG, Tilanus MG, de Weger RA: An alternatively spliced interleukin 4 form in lymphoid cells. Immunogenetics 1995, 41(1):57.
3. Atamas SP, Choi J, Yurovsky VV, White B: An alternative splice variant of human IL-4, IL-4 delta 2, inhibits IL-4-stimulated T cell proliferation. Journal of Immunology 1996, 156(2):435-41.
4. Arinobu Y, Atamas SP, Otsuka T, Niiro H, Yamaoka K, Mitsuyasu H, Niho Y, Hamasaki N, White B, Izuhara K: Antagonistic effects of an alternative splice variant of human IL-4, IL- 4delta2, on IL-4 activities in human monocytes and B cells. Cell Immunol 1999, 191(2):161-7.
5. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402.
6. Sorg RV, Enczmann J, Sorg UR, Schneider EM, Wernet P: Identification of an alternatively spliced transcript of human interleukin-4 lacking the sequence encoded by exon 2. Experimental Hematology 1993, 21(4):560-3.
7. Glare EM, Divjak M, Rolland JM, Walters EH: Asthmatic airway biopsy specimens are more likely to express the IL-4 alternative splice variant IL-4delta2. J Allergy Clin Immunol 1999, 104(5):978-82.
8. Glare EM, Divjak M, Bailey MJ, Walters EH: The usefulness of competitive PCR: airway gene expression of IL-5, IL-4, IL-4delta2, IL-2, and IFN-gamma in asthma. Thorax 2001, 56(7):541-8.
Competing interests
None declared