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Technical Briefs |
Dept. of Med., Univ. of Wisconsin-Madison, 600 Highland Ave., Madison, WI 53792, and Geriatric Res., Education and Clin. Center, William S. Middleton Memorial VA Hosp., 2500 Overlook Terrace, Madison, WI 53705
a address correspondence to this author, at Middleton Veterans Hospital: fax 608-262-7648, e-mail agudmund{at}facstaff.wisc.edu
Circadian and ultradian variation is a prominent feature of most endogenous biodynamic processes and has been extensively studied in neuroendocrine systems (1). Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in many age-related diseases, including postmenopausal osteoporosis (2)(3). Estrogen has been shown to directly inhibit IL-6 production (4)(5). Very little information on the circadian rhythms and no information on ultradian rhythms of IL-6 or other pro-inflammatory cytokines exists. Recent research has demonstrated a strong reciprocal interaction between IL-6 and the hypothalamic–pituitary–adrenal (HPA) axis (6)(7). Specifically, recombinant IL-6 has been shown to stimulate the HPA axis in humans (8). The recent availability of highly sensitive assays for measuring IL-6 provides the opportunity to study the rhythmic pattern of this cytokine. A study in men, in which samples were collected every 3 h by direct venipuncture, showed a large circadian variation in circulating IL-6 with a peak at 0100 and a nadir at 1000 (9). More frequent sampling is required to elucidate ultradian fluctuations, which typically have periods of 60 to 90 min (10).
For practical reasons, indwelling venous catheters rather than direct venipunctures have been used for studying frequently sampled time series. We designed a study, using an indwelling venous catheter for continuous integrated 15-min sample collection, to establish the normal variations of circulating IL-6 over 24 h in healthy postmenopausal women, and to assess the effect of estrogen replacement therapy on these patterns. In studying the first subject, it became apparent that serum IL-6 reached higher values than expected. Here, we demonstrate that an indwelling peripheral venous catheter leads to local tissue production of IL-6. We also demonstrate that this local production of IL-6 does not affect endogenous cortisol concentrations.
The study protocol was approved by the University of Wisconsin Human Subjects Committee, and the subject gave written informed consent. The study was conducted at the General Clinical Research Center at the University of Wisconsin, Madison.
The subject in this study was a healthy 59-year-old woman who was 6 years postmenopause. A thromboresistant blood withdrawal and tubing set (DakMed, Buffalo, NY) was inserted in a peripheral vein at 0800. Integrated blood samples were collected at 15-min intervals over 25 h by continuous withdrawal with a peristaltic pump (DakMed). Clotted blood samples were centrifuged at 1520g for 10 min, then separated and frozen at -70 °C until IL-6 concentrations were measured in duplicate by a highly sensitive ELISA (Quantakine HS; R & D systems, Minneapolis, MN). This assay kit is validated for measuring IL-6 concentrations in the range 0.094–10 ng/L. In our laboratory the calibration curve loses linearity at 40 ng/L. The intraassay and interassay CVs for this assay are 3.8% and 7.1%, respectively. The study was repeated after 7 weeks of oral estrogen replacement therapy with 0.625 mg of conjugated estrogen (Premarin) daily. Serum cortisol concentrations were measured in duplicate by RIA (Coat-A-Count, Diagnostic Products Corp.) with an intraassay CV of 10.5%. The reported limit of detection of the assay was 2 µg/L (5.5 nmol/L).
Inspection of the first 25-h time series (Fig. 1a
) revealed considerable variation in IL-6 concentrations. IL-6
concentrations remained constant at 2.8–4.7 ng/L for 3 h after
catheter insertion, but this was followed by a steep increase to 35
ng/L, almost 10 times the baseline value, after 20 h of sample
collection. The IL-6 concentration at 0900 on the second day was much
higher than at 0900 on the first day.
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In a repeat study, after 7 weeks of estrogen replacement therapy, a
sample was drawn from the contralateral arm by direct venipuncture
11 h after the first sample (Fig. 1b
). The IL-6 in this sample
measured 2.6 ng/L, while a simultaneously drawn sample from the
indwelling catheter measured 24.2 ng/L. After 12 h of sampling, a
new venous catheter was placed in contralateral arm. The concentrations
of IL-6 from this new site were initially low but again showed an
abrupt increase at 3 h. After 22 h, a new catheter was again
placed in an arm vein proximal to the site of the first catheter. This
time, the IL-6 concentrations remained high (8–27 ng/L).
The cortisol concentrations measured in the same serum samples as IL-6
demonstrated a normal diurnal variation, with the peak values at 0530
after a nadir at 0200. No increase in cortisol was seen at the time of
the rapid increase in IL-6. After 24 h the cortisol concentrations
had returned to baseline value (Fig. 1c
).
The use of indwelling venous catheters for studying time series of hormones is the accepted experimental model. In most instances, hormones are secreted from glandular tissue and the measurements represent the concentrations in the systemic circulation. This case study indicates that the onset of increasing IL-6 concentrations 3 h after catheter insertion most probably represents local production of IL-6 and not the circulating concentrations—which indicates that this sampling method may have limitations when time series of immune mediators (e.g., IL-6) are being assessed. The local production of IL-6 is supported by several lines of evidence. First, the values for IL-6 were markedly lower when drawn by direct venipuncture or through a newly placed catheter from the opposite arm. Secondly, the blood concentrations of IL-6 were higher than values reported in other studies of postmenopausal women that used the same assay system (11)(12). Finally, the morning concentrations of IL-6 in samples drawn upon initial catheter insertion were much lower than those at the same time of day but drawn after the catheter had been in the vein for 24 h.
The increased concentrations of IL-6 found when the third catheter was placed in a vein proximal to the previous site of insertion supports the contention that the high IL-6 values derive from local tissue production rather than from the catheter system itself. Importantly, this local tissue production of IL-6 did not appear to increase the systemic concentrations of the cytokine for at least 12 h and did not seem to stimulate the HPA axis, as evidenced by measured cortisol concentrations.
One can speculate that interruption of the vascular endothelial layer by a venipuncture mediates a cytokine cascade sequence that results in increased IL-6 concentrations. Endothelin, which is produced by endothelial cells, has recently been shown to be a potent stimulator of IL-6 production (13). Other cells at the site of injury, such as vascular smooth muscle cells (14), have also been shown to secrete IL-6. One previous study suggested that the increased concentrations of IL-6 in bolus samples collected via indwelling venous catheters every 3 h for 9 h reflect local production of IL-6 rather than circulating concentrations (15). The continuous sampling, more-frequent measurements, and use of a sensitive IL-6 assay in our study confirm this finding and provide a picture of the timing of this local effect, which in this subject occurred at ~3 h.
Because of the local tissue production of IL-6, our study did not allow an assessment of the diurnal variation of IL-6. Although there appears to be a faster increase in IL-6 concentrations during estrogen replacement in this one subject, its exact magnitude is likely to vary, both from time to time and from one vein to another. The main reason for this is variation in the rate of blood flow, which ultimately will dilute the locally secreted IL-6 to various extents.
Our results suggest that circulating concentrations of IL-6 may be more reliably assessed by repeated direct venipunctures at a progressively more-distal venous site each time. However, discomfort and inconvenience associated with this method would obviate frequent sampling in human subjects. Researchers and clinicians measuring IL-6 for diagnostic or monitoring purposes should take into account the sample collection method used, particularly if high concentrations are encountered. This local production of IL-6 occurred as early as 3 h after insertion of a catheter at a fresh site and almost immediately from a catheter inserted downstream (proximal) to a previous catheter insertion site. Whether production of other cytokines is similarly triggered after insertion of a catheter is a matter for future investigation. Several previous reports of increased IL-6 in clinical settings may need reevaluation in view of our findings (16)(17).
Acknowledgments
This work was supported by a Merck/American Federation for Aging Research Fellowship in Geriatric Clinical Pharmacology to A.G.; NIH grants RR03186, RR29-DK40759, R01 AG-1071, and K07 AG-0451; the University of Wisconsin Medical School Research Fund; and the Department of Veterans Affairs.
References
The following articles in journals at HighWire Press have cited this article:
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  I. Liuba, H. Ahlmroth, L. Jonasson, A. Englund, A. Jonsson, K. Safstrom, and H. Walfridsson Source of inflammatory markers in patients with atrial fibrillation Europace, July 1, 2008; 10(7): 848 - 853. [Abstract] [Full Text] [PDF]
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 M. Mohlig, M. Freudenberg, T. Bobbert, M. Ristow, H. Rochlitz, M. O. Weickert, A. F. H. Pfeiffer, and J. Spranger Acetylsalicylic Acid Improves Lipid-Induced Insulin Resistance in Healthy Men J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 964 - 967. [Abstract] [Full Text] [PDF]
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 J. K. Kiecolt-Glaser, T. J. Loving, J. R. Stowell, W. B. Malarkey, S. Lemeshow, S. L. Dickinson, and R. Glaser Hostile Marital Interactions, Proinflammatory Cytokine Production, and Wound Healing Arch Gen Psychiatry, December 1, 2005; 62(12): 1377 - 1384. [Abstract] [Full Text] [PDF]
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 T. M. Buckley and A. F. Schatzberg Aging and the Role of the HPA Axis and Rhythm in Sleep and Memory-Consolidation Am J Geriatr Psychiatry, May 1, 2005; 13(5): 344 - 352. [Abstract] [Full Text] [PDF]
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J. H. Halpern, M. B. Sholar, J. Glowacki, N. K. Mello, J. H. Mendelson, and A. J. Siegel Diminished Interleukin-6 Response to Proinflammatory Challenge in Men and Women after Intravenous Cocaine Administration J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 1188 - 1193. [Abstract] [Full Text] [PDF]
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