Controls

Although most investigators prefer PBS or saline liposomes for control experiments and referees are usually asking for such controls if omitted, you should be aware that administration of PBS or saline liposomes does not represent the right control experiment in most cases. Compared to an experiment in macrophage depleted animals, the control animals should have normal healthy, non-blocked, non-suppressed and non-activated macrophages. As you may know, liposomes, as most other particulate compounds may block phagocytosis by saturation for certain periods of time. Moreover, it is not known exactly which other macrophage functions will be suppressed or reversely activated and for what duration. As a result the effects of macrophage depletion in your experiments may be somewhat less than expected, if these are compared with those in controls treated with PBS liposomes, since macrophage functions in the latter animals may also be affected to some extent. To mimic depletion treatments without affecting healthy macrophages, sham operations and injections of e.g. NaCl are sufficient. However if there is a chance that the observed effects are not due to macrophage depletion, both PBS or saline liposomes and free clodronate injections should be incorporated as control groups. See also J. Immunol.Meth. 174: 83-93 , 1994.

Please keep in mind that depletion of macrophages has to be checked in each new situation, e.g. when animal species, tissues or administration routes have not been studied before. This is rather easy in mice and rats where macrophage markers (e.g. monoclonal antibodies) are available, but could be a serious problem in other species. Also, Macrophages have quite a large effect (mainly via cytokines and chemokines) on non-phagocytic cells such as various subsets of lymphocytes (e.g. their homing and migration). As a consequence, especially at later time intervals after injection of clodronate liposomes, some of these cell types may temporally disappear from the spleen. If repeated injections with clodronate liposomes keep the macrophages away for longer periods, this may have a dramatic effect leading to a much smaller spleen.