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Published online before print August 7, 2006
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Klinik für Anaesthesiologie und Operative Intensivmedizin, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany
1 Correspondence: Klinik für Anaesthesiologie und Operative Intensivmedizin, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, Berlin D-12200, Germany. E-mail: alexander.brack{at}charite.de
Key Words: inflammation • pain • PMN
We thank Cunha and Verri [1 ] for their constructive comments about our manuscript "Selective local PMN recruitment by CXCL1 or CXCL2/3 injection does not cause inflammatory pain" [2 ]. They raise several important issues concerning the role of polymorphonuclear cells (PMN), of chemokines, and of IL-1ß in the induction of inflammatory hyperalgesia.
Initially, the authors mention that their group has previously shown that certain chemokines and cytokines can induce hyperalgesia [3 , 4 ], whereas in our study, chemokine (CXCL1, CXCL2/3) injection did not cause hyperalgesia. We agree with their comment that this discrepancy is most likely a result of different behavioral methods used. This issue is discussed in detail in our manuscript [2 ]. Cunha and Verri then show data that local PMN infiltration [measured indirectly by myeloperoxidase (MPO) activity] and mechanical hyperalgesia (measured by the von Frey test) are low in early inflammation [i.e., 2 h post-complete Freund’s adjuvant (CFA)] and increase in parallel within the first 6 h of CFA injection into the rat paw. Based on these correlative data they postulate that i) early time-points of inflammation are not ideal, and ii) PMN depletion at later time-points would be required to analyze the role of PMN in inflammatory hyperalgesia.
In response, we would first like to point out that in line with our previous study [5 ], we indeed detected a significant increase in the number of PMN at 2 h after CFA injection (1.9x105 PMN/paw) compared with noninflamed paws (0.3x105 PMN/paw; Fig. 4 in ref. [2 ]). In contrast, Cunha and Verri detected a nonsignificant increase in MPO activity at 2 h after CFA (calculated equivalent of 0.5x104 PMN/mg paw tissue) compared with noninflamed paws (0.25x104 PMN/mg tissue). However, there are important differences between the two methods of PMN quantification and data reporting. We quantified infiltrating PMN by enzymatic tissue digestion, preparation of single cell suspensions, and flow cytometry using a PMN-specific antibody, and Cunha and Verri measured MPO activity in whole paw tissue and indirectly calculated the number of PMN. PMN quantification of tissue extracts by spectrophotometric MPO measurement is affected by tissue myoglobin and hemoglobin, and gel filtration chromatography is recommended to obtain a linear relationship between MPO activity and PMN numbers [6 ]. Thus, in addition to MPO activity from infiltrating PMN, Cunha and Verri might have measured MPO activity from other sources, which may have resulted in an artifically high MPO activity in noninflamed tissue, and thus, they were unable to detect significant differences between noninflamed tissue and 2 h of inflammation. In our experiments, in line with previous immunohistochemical studies [7 , 8 ], we found very few infiltrating immune cells in noninflamed tissue and a significant rise in PMN at 2 h of inflammation (Fig. 4 in ref. [2 ]).
Furthermore, Cunha and Verri found a significant increase in infiltrating PMN and the intensity of hyperalgesia at 6 h after CFA injection and suggested that PMN depletion studies should be performed at this time-point. To address this issue, we performed additional experiments and determined PMN infiltration in the paw and mechanical nociceptive thresholds in the presence and absence of prior systemic PMN depletion. In these experiments, injection of CFA, PMN depletion, leukocyte quantification by flow cytometry, and measurement of nociceptive thresholds were performed as described in our manuscript [2 ] with one modification: Experiments were performed at 6 h post-CFA injection. We found that PMN (Fig. 1A ) and monocytes/macrophages in the paw were reduced significantly by prior systemic PMN depletion (CFA: 1.2±0.4 vs. CFA+PMN depletion: 0.3±0.1x105 cells/paw, P<0.05; t-test). In both cases, mechanical nociceptive thresholds were decreased significantly in comparison with noninflamed paws, and no significant differences were found as a consequence of prior PMN depletion (Fig. 1B , P<0.05, P>0.05, respectively; t-test). Thus, in line with our results at 2 h of inflammation [2 ], systemic PMN depletion effectively reduced the number of PMN at the site of inflammation but did not alter hyperalgesia at 6 h post-CFA. Thus, even at later time-points when PMN infiltration is higher, depletion does not affect inflammatory pain.
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Taken together, we disagree with Cunha et al. [1 ] that 2 h of inflammation is not a suitable time-point for analyzing the role of PMN on hyperalgesia for three reasons: i) PMN depletion does not alter nociceptive thresholds at either time-point; ii) PMN depletion is only selective for PMN at 2 h but not at 6 h post-CFA, as monocytes/macrophages are also decreased at the later time-point; and iii) significant hyperalgesia to mechanical and thermal stimuli was detectable at 2 h post-CFA, in line with the findings of Cunha et al. In summary, we still conclude that PMN seem to be less important in inflammatory hyperalgesia than previously thought.
Received May 19, 2006; accepted May 19, 2006.
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