ABSTRACT
The interactions between different cell signaling pathways governing ciliary beat frequency (CBF) are as complex as the interpretation of the resultant CBF profiles, leading to many arguments about the best analytical methods. Here we are not concerned with those arguments but will concentrate on the alternative modes of presentation of the resultant data. For example, when CBF is studied in ciliated cells brush-biopsied from the human inferior turbinate using our previously described protocol (1), they do not all have the same setpoint of CBF. Thus, even if we accept that the in vitro CBF acts as a surrogate marker for the integrated output from such signaling pathways, then the wide range of intrabiopsy CBF values found in freshly isolated cells incubated in medium 199 (Fig. 1) suggests intercell differences in the regulation of the CBF response. For example, note how individual cells differ so substantially in the set point of their intrinsic CBF. Is this difference a true reflection of cell biology or a methodological artefact due to cell injury? The individual patient data sets in Figure 1 represent the averaged CBF responses from at least two cell borders (typically three cell borders) per patient recorded co-temperaneously from the microscopic field of view in an aliquot of each normal subject’s nasal brushing. The average of the curves shown, 91
as recommended by conventional statistical advice, provides measurements that are truly independent of one another, and typically, our publications (1-3) have reported such averaged data from approximately 10 different patients (over 30 cells per study). However, if the hypothesis that the spectrum of observed CBF is a true manifestation of different cell biology has any credence, then the averaged response calculated from Figure 1 may be misleading. Figure 2 shows two different groups of underlying raw data: first from those apparently normal subjects whose cilia had an unstable CBF in one or more cells (upper panel) or, second, from the more typical subject whose CBF showed both internal consistency and stability between different cell borders (lower panel). Which is the “ true” response? Methodological differences cannot explain this dichotomy because postharvest, the CBF was recorded identically for the two groups from cell borders which were immediately placed in medium 199 on ice, transferred to the perfu-
sion chamber, and the chamber temperature restored to the normal nasal temperature of 32°C, all within 60 minutes. Despite such standardization, the spectrum of CBF is wide. We have previously not studied cells beating at around 6 Hz (at 32°C) assuming that they are in some way damaged, thus explaining a decay in their CBF that is so extreme that ciliary arrest occurs in some cases. However, this application of such a notion must then also include a cell beating at approximately 12 Hz (having been stable enough for inclusion at the beginning of the study), which nevertheless also shows the same decay to ciliary arrest phenomenon. Are such cells dead? Figure 3 shows that this conclusion is also premature because reperfusion with medium transiently restores CBF in some cells, whereas for others, CBF reverts to predecay values (or higher; not shown). The simplest conclusion is that such cells are “ resting” since reperfusion does not merely refresh the chamber with new energy from the medium (or wash out toxins) because throughout the phase of ciliary arrest adjacent cells within the same field of view continue to beat normally (see Fig. 2). Could ciliary arrest reflect an adaptive response to the high energy requirements induced by ciliary beat? We
do not know the answers to such questions at present but obtained a clue from another set of studies. We were investigating the effects of pM concentrations of the phorbol ester PMA. Figure 4 shows a composite pattern of results. Note that in the left panel, which shows raw frequencies against time, CBF does not decay to zero in any of the cells. Is this a chance (false-negative) observation, or does pM phorbol ester promote the stability of CBF? The right panel shows comparative box plots of the difference in CBF (ACBF) between the beginning and end of the study for each cell. Note that (1) a few cells increase their CBF, but only when PMA is present, (2) the median decline (solid line intra-box) is less than with medium alone, and (3) the mean CBF (dotted line) lies on different sides of the median for the two groups, suggesting a different distribution of CBF. Conventionally, pM PMA might be thought to be too low for a biological effect, and our preliminary data suggest that this notion may have to be revised for intact cells.In the above examples, the study temperature was chosen to reflect the normal nasal value of 32°C. The temperature used for CBF studies varies between investigators-indeed, it has been our experience over the last decade that many referees have asked why we report our results at the “ nonphysiological’ ’ 20°C
(1-3). We have argued that the nasal temperature in the antarctic deep winter will be substantially different from that in the tropical rain forest. Thus, the nasal epithelium will need to adapt to climate-induced temperature change, thus making the study of CBF relevant whatever the temperature. We (and others) have noted that CBF is more stable at room temperature compared to 32°C (see Fig. 5), which probably explains why so many studies are undertaken at the ‘non’ physiological temperature. Our recent work (3) suggests that at 20°C the stability of CBF is partially dependent on the immediate history of the cell-specifically the flow-stress imparted on that cell by fluid impinging on the cell surface. When freshly biopsied human nasal epithelial cells were subjected to a flow rate of 0.5 mL/min, we found that there was a precipitate decline in CBF of —15% within 30 seconds of the applied flow followed by an attempt at recovery over the next 30 minutes to preflow baseline. Figure 5 shows that this decline in ciliary response is also present at 32°C and may therefore play an important regulatory role. Our data at 20°C (3) suggest that PKC activation prior to the flow stress eliminates this flow-induced decline in CBF. We do not know what happens at other temperatures.The observations reported here lead to many questions. First, does the control of cell calcium differ in cells that show a decay to arrest phenomenon in
respect of their CBF? Second, do phorbol esters control the temporal profile of CBF? Third, if they do, then do they act via cell calcium or not (4)? However, when we provide our peers with answers to such questions, we will have to present our data in a transparent manner. Only then will we take peer review to a new dimension. In 1690, John Locke at the age of 57, having published virtually nothing in his academic life, wrote in the third book of his classic essay on human understanding, that total ignorance was a “ want of ideas” and partial ignorance was a want of “ connexions between ideas.” Three hundred and ten years later we remain partially ignorant about the subtleties of CBF control (5,6). Perhaps there should be a MCC website (CBF.org) where researchers can place the underlying raw data from their publications for all to analyze remotely. Only then will Locke rest easy knowing that we are all getting connected. ACKNOWLEDGMENTS
XM and RM are supported by the Wellcome Trust and the Anonymous Trust. XM holds an International Research Training Fellowship. REFERENCES
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