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response of those cells in vitro—most often by measuring mitogen-induced cell proliferation, a normal immune response. Almost without exception, this approach has failed to demonstrate any reduction in leukocyte function. The major problem with the approach is that after blood samples are drawn from the study subjects the leukocytes must be isolated from whole blood before they are tested. That is done by high-speed centrifugation followed by extensive washing of the cells, which removes the cannabinoid; perhaps for this reason no adverse effects have been demonstrated in peripheral blood leukocytes from marijuana smokers.75^,91^,123^,160

Leukocyte Responses to THC. Another approach is to isolate peripheral blood leukocytes from healthy control subjects who do not smoke marijuana and then to measure the effect of THC on the ability of these cells to proliferate in response to mitogenic stimulation in vitro. One important difference between leukocytes isolated from a marijuana smoker, as described above, and leukocyte cell cultures to which THC has been added directly is in the cannabinoid composition. Marijuana smoke contains many distinct cannabinoid compounds of which THC is just one. Moreover, the immunomodulatory activity of many of the other cannabinoid compounds has never been tested, and it is now known that at least one of those—cannabinol (CBN)—has greater activity on the immune system than on the central nervous system,64 so it is unclear whether the profile of activity observed with THC accurately represents the effects of marijuana smoke on immune competence. Likewise, the extent to which different cannabinoids in combination exhibit additive, synergistic, or antagonistic effects with respect to immunomodulatory activity is unclear. The issue is complicated by the fact that leukocytes express both types of cannabinoid receptors: CB₁ and CB₂.

An additional factor that might affect the immunomodulatory activity of cannabinoids in leukocytes is metabolism. Leukocytes have very low levels of the cytochrome P-450 drug-metabolizing enzymes,20 so the metabolism of cannabinoids is probably different between in vivo and in vitro exposure. That last point is pertinent primarily to investigations of chronic, not acute, cannabinoid exposure.

Human-Derived Cell Lines. A third approach for investigating the effects of cannabinoids on human leukocytes has been to study human-derived cell lines.* As described above, the cell lines are treated in vitro with cannabinoids to test their responses to different stimuli. Although cell lines

*Cell lines are created by removing cells from an organism and then treating them so they are ''immortalized,'' meaning they will continue to divide and multiply indefinitely in culture. Cellular processes can then be studied in isolation from their original source.