Clodronate liposomes to study and manipulate macrophage and monocyte function
Macrophages form a first line of host defense against bacterial, viral and other forms of microbiological contamination penetrating into the bodies of vertebrates. They can be found in nearly all tissues and organs at strategic locations such as in the alveoli of the lung, at sites where the blood flow is entering spleen and liver and at a site where the afferent lymph vessels enter the lymph nodes.
From an evolutionary point of view, macrophages are older than the cells belonging to the acquired immune system of the higher vertebrates. As a consequence, during evolution, they did adapt many functions apart from ingestion and digestion of non-self particles and macromolecular complexes. These additional functions were mainly performed by production and secretion of cytokines and chemokines.
Via this system of “remote control”, several non-phagocytic cells could be influenced with respect to differentiation, migration or functional aspects.
Because of their multifunctional nature, specific depletion of macrophages was attempted in various ways in order to establish their role in numerous biomedical phenomena. However, just because macrophages were used to meet dangerous situations, most of these attempts did also influence non-phagocytic cells. Specificity was not achieved for that reason. Moreover, those macrophages that did not meet the agent in a sufficient high concentration to achieve their elimination, were often activated.
To solve these problems a highly specific liposome mediated macrophage “suicide” approach has been developed aimed at the specific depletion of macrophages, from organs or tissues. This method is now widely used for studies on functional aspects of macrophages as well as for manipulation of these functions. Important developments to which application of the approach did contribute will be reviewed.
Specific advantages of the liposome mediated macrophage “suicide” approach
* Applicable to macrophages in all vertebrate species (from fish to men). This is because specificity is based on functional activity of macrophages (phagocytosis) in stead of on antibody-mediated targeting of drugs.
*No counter-effects such as activation of non-depleted macrophages.
*Specific depletion of tissue- or organ-specific macrophage populations can be achieved by choosing the appropriate administration routes. In spleen, functional differences between macrophage subpopulations can be established based on differences in their repopulation kinetics.
*Clodronate is a frequently applied human medicine in its free form. It is inactivated after action on macrophages; clodronate released from killed macrophages will rapidly leave the circulation via the kidneys.
*Liposomes are composed of biologically inert compounds (phosphatidylcholine & cholesterol). They are already introduced in the clinic.
*Dependent on the administration schedule of clodronate liposomes, also bone marrow-derived monocyte precursors in the circulation can be depleted.
*The efficacy of drug targeting and gene transfer to non-phagocytic cells may be improved by prior transient depletion of macrophages in the target organ.
*Fine tuning of dosing and selection of administration routes may lead to therapeutic application in cases where drug targeting or gene therapy are hindered by excessive phagocytosis of non-self vehicles.
Disadvantages of macrophage depletion in general
*Since macrophages are influencing many other (non-phagocytic) cells by remote control (e.g. via cytokines and/or chemokines), long-term macrophage depletion may finally lead to cancelling of activities of non-phagocytic cells.
*Upon killing of macrophages, their remnants (both soluble and particular) may circulate for some time and influence non-phagocytic cells.
In the past three decennia. liposomes that encapsulate clodronate, a strongly hydrophilic bisphosphonate, have been developed to a research tool, widely applied for specific depletion of macrophages and monocytes from tissues, in order to study their role in numerous biomedical phenomena. After phagocytosis of the liposome, its phospholipid bilayers are disrupted by action of lysosomal phospholipases, followed by intracellular release of entrapped clodronate molecules. Subsequently, the clodronate molecules that are neither able to cross liposomal membranes nor cell membranes, accumulate in the cell. At a certain intracellular concentration of this calcium binding chelator molecule, the cell starts its apoptosis programme leading to its depletion . So, these liposomes are acting as “Trojan horses” enthusiastic welcomed by the citizens of the besieged city Troy. But the encapsulated clodronate molecules do behave as the soldiers, hidden in it, and unexpectedly coming out, once the walls around Troy are passed (Arctinus of Miletus, published but later lost).
Given the high natural resistance of macrophages against all kinds of enemies that they have to conquer, it is not surprising that many earlier approaches to deplete macrophages were suffering of side effects on other, often more sensitive, non-phagocytic cells. Moreover, macrophages that were not adequately depleted were activated, with as an overall net result e.g. more cytokines produced by less macrophages .
Macrophages did appear during evolution of vertebrates long before the cells forming together the complex immune system. Not surprisingly for that reason, they did also acquire various roles in the development, differentiation, migration and functioning of several of the latter non-phagocytic cells in mammals.
It has already been shown that the approach will do in men as it does in all other vertebrates investigated up to now, since it is independent of targeting by monoclonal antibodies and acts directly on phagocytic function . Clodronate, once released in the circulation from dead macrophages, has a short half life and non-immunogenic, inert phospholipids can be chosen as constituents of the liposomes.
Moreover, both liposomal phospholipids and the encapsulated clodronate molecules that are used in this liposome mediated macrophage “suicide” approach, did - though separately - already find their way to the clinic.
Given the high phagocytic activity of macrophages, it may not surprise that they can fulfill an important role in the mechanism by which antibodies can be efficacious in treating of e.g. B cell malignancies . On the other hand, where macrophages play a role in autoimmune phenomena, e.g. by rapidly removing platelets or erythrocytes, their depletion might help to reduce the immediate impact of immune thrombocytopenic purpura  or autoimmune hemolytic anemia  as shown in mouse models.
It is also obvious that macrophages may thwart a proposed therapy, e.g. when specific drugs or biological active molecules have to be carried to certain tissues or cells with the help of non-self vehicles as in drug targeting and gene-therapy.
So, although clodronate liposomes were primarily developed as a research tool, some studies might also lead to therapeutic applications .
Given their important role in the first line of host defense, depletion of monocytes/macrophages will not form an obvious solution when these cells are forming a serious barrier to therapeutic efficacy.
Nevertheless, selective administration routes and fine tuning of dosing may help to reduce the negative effects by restriction of depletion to defined organs or tissues and limiting the time period of this inactivation. New macrophages will replace the depleted cells by recruitment of monocytes from the bone marrow.
Each imaginable method of macrophage depletion suffers from its own success, if long term depletion by repeated injections of the agents is considered. Though macrophages remain indirectly responsible, inactivation of non-phagocytic cells as a result of dropping off cytokine/chemokine secretion by macrophages may finally form the direct cause of observed effects in long term depletion experiments.
Liposomes, clodronate and macrophages
Liposomes as structures were discovered by Alec Bangham (UK, died on March 9, 2010). He found that amphipathic phospholipid molecules, when dispersed in water, became organized in vesicles consisting of concentric phospholipid bilayers, separated by aqueous compartments. The hydrophobic fatty acid chains of each bilayer were opposed to each other in the inner part of the bilayer, whereas the hydrophilic head groups of the fatty acids were exposed to the water compartments on the outer part of the bilayer .
Hydrophilic molecules solved in the aqueous solution used for preparation of the liposomes could be encapsulated in the water compartments between the phospholipid bilayers, whereas lipophilic (hydrophobic) molecules could be associated with the phospholipid bilayers themselves, and it was Gregory Gregoriadis (Greece / UK), who proposed and initiated the application of liposomes as carriers of drugs in biology and medicine i.e. the liposomal drug carrier concept [9,10].
In the past decennia, liposomes appeared to be the ideal vehicles to carry antigens and drugs to cells of the monocyte-macrophage lineage.
Clodronate (dichloromethylene bisphosphonate) is a member of the family of bisphosphonates developed for the treatment of osteolytic bone diseases . It shows high affinity for calcium and as a consequence adheres to bone when administered to vertebrates. Osteoclasts, play a role in the physiology of bone by breaking it down, opposed to osteoblasts who are involved in its reconstruction. It appeared that the activity of osteoclasts could be affected by bisphosphonate molecules sticked to the bone.
Given that both osteoclasts and macrophages both belong to the mononuclear phagocyte system (MPS), we decided to try clodronate as one of the first effector molecules in our planned “liposome mediated macrophage suicide technique”. This approach was developed in the first half of the 1980's in order to deplete macrophages from the spleen [12, 13].
Five subsets of macrophages had been distinguished in the spleen and their respective roles in immune responses were subject of investigation .
Liposomes had already been introduced in this research project, since they allowed to compare immune responses against liposome associated (particulate) antigens, that were either entrapped in liposomes or exposed on their outer surfaces, with the same antigens in a free soluble form . Combination of liposome encapsulated clodronate for depletion of macrophages, followed by immunization with liposomes as antigen carriers did bring us to the conclusion that macrophages in the spleen were involved in immune reactions against particulate antigens, but not against the same antigen in a free soluble form .
Macrophages in other organs such as alveolar macrophages in the lung , Kupffer cells in the liver , macrophages in lymph nodes [19, 20] and peritoneal macrophages in the peritoneal cavity , could also be depleted by this approach, provided that appropriate administration routes were chosen in order to avoid barriers between liposomes administered and the target macrophages. On the other hand, choosing the appropriate administration routes could also help to limit macrophage depletion to particular tissues or organs only.
Although subsequently we found several other hydrophilic molecules, that were also suitable as effector molecules in the approach, clodronate may still be considered the best choice because it shows maximum efficacy and minimal toxicity [2, 18].
Moreover, both liposomes and clodronate have been introduced in the clinic and it may be anticipated that their possible combination in the transient suppression of macrophage activity for human application would be easier to achieve than for any of the other candidate effector molecules which are known up to now .
Macrophages as multifunctional cells
Macrophages are involved in 'homoiostasis' of the body by ingesting and digesting microorganisms or non-self particles and macromolecules. Digestion is mediated by their lysosomal enzymes.
So, the natural fate of liposomes, once injected in the body is phagocytosis, followed by digestion of their phospholipid bilayers by phospholipases, and consecutive intracellular release of liposome-encapsulated compounds.
Whereas most cells participating in the immune system of vertebrates seem to be highly specialized, macrophages were long considered as simple vacuum cleaners, removing and degrading non-self particles such as microbial cells and antigen-antibody complexes from our body by direct contact.
From an evolutionary point of view, macrophages are ancient cells. They form the core of the natural immune system and did appear long before the cells forming together the complex immune system of the higher vertebrates. But during evolution, they did acquire multiple roles both in natural immune reactions and in the regulation of functions of other (non-phagocytic) cells. These tasks appeared to be mediated by soluble molecules such as cytokines and chemokines.
In the last decennia further evidence has been produced of macrophages as highly sophisticated cells, controlling many processes in the body by remote control. They are sending their soluble mediators to cells elsewhere, in order to regulate their development, differentiation, migration and functioning. For instance, cells of the monocyte/macrophage lineage control the differentiation of natural killer cells to an effector state [23,24]
In several studies, e.g. on pulmonary tuberculosis  and on pathogenesis and control of Salmonella infections in mice , a dual role of macrophages has been demonstrated.
Because of their multifunctional character, one of the first questions to be answered in many biomedical studies will be focused on the macrophage. Is the macrophage involved in a particular phenomenon, pathology or immune reaction to be studied? Is it interfering with therapeutic approaches, or has the macrophages itself to be considered as a suitable target cell for such therapeutic approaches?
Multilamellar liposomes of more than a few hundred nanometers will not be internalized by non-phagocytic cells. As a consequence, e.g. lymphocytes, granulocytes, endothelial cells and epithelial cells will not be affected by clodronate encapsulated in such liposomes, explaining the high specificity for phagocytic cells .
The purpose of the present review was to summarize shortly some of the most important of the widespread applications of the liposome mediated macrophage “suicide” approach.
Descriptions of the preparation of clodronate- and control liposomes as well as determination of the efficacy of clodronate encapsulation, can be found elsewhere in detail [28,29].
Depletion of macrophages in the CNS
Whereas injection of clodronate liposomes into the fourth ventricle of the central nervous system (CNS) of rats resulted in a complete depletion of perivascular and meningeal macrophages in the cerebellum, cerebrum and spinal cord of rats  and microglia were successfully depleted from cultured slices of brain tissue , the blood brain barrier (BB) does not normally allow the passage of clodronate liposomes.
However, incorporation of mannose into the phospholipid bilayers of clodronate liposomes markedly suppressed expression of the clinical signs of experimental allergic encephalomyelitis (EAE) in rats after intravenous injection whereas similar liposomes without mannose did not have a comparable effect . In consecutive studies in mice, it was shown that invasion of the intraparenchymal tissues of the central nervous system (CNS) by F4/80+, Mac-1+ and MOMA-1+ macrophages was almost completely blocked after treatment with mannosylated clodronate liposomes . Given its affinity for mannose, it could be suggested that the depletion was caused by the fact that liposome/cell contacts were facilitated as a result of temporary immobilization of both macrophage-precursors and mannosylated clodronate liposomes at the level of the BB. Similar mannosylated clodronate liposomes appeared to be able to promote partial hindlimb recovery and neuroanatomical repair after experimental spinal cord injury .
Depletion of bone marrow macrophages and its effect on hematopoietic stem cell (HSC) mobilization
After intravenous injection of clodronate liposomes, not only spleen and liver macrophages were depleted, but also macrophages in the bone marrow [35,36].
Specifically a macrophage population with the unusual F4/80+, Ly-6G+, CD11b+ phenotype was reduced by 95% within 24 hours . Both research groups showed the concomitant mobilization of hematopoietic stem cells (HSC’s) from their HSC nishes.
Recently it has been independently shown in two papers that macrophages play a role in promoting erythropoiesis under homeostasis and stress  as well as in models of induced anemia, polycythemia vera and beta-thalassemia . Both papers suggested that modulation of the macrophage compartment might contribute to a strategy for therapeutical treatment of erythropoietic disorders.
Rhythmic modulation of a stem cell niche has been shown to be mediated by neutrophil clearance. Unique among leukocytes, neutrophils follow daily cycles of release from and migration back into the bone marrow, where they are eliminated.
The migration of aged neutrophils towards and engulfment by resident macrophages, raised the possibility that modulation of the bone marrow niches took place through the activity of macrophages rather than by neutrophils acting directly on niche components .
In agreement with this hypothesis, the authors were able to show that transfer of aged neutrophils after depletion of bone marrow macrophages, failed to elicit the changes in the hematopoietic niche that were observed in non-macrophage depleted controls .
Depletion of monocytes and indirect depletion of macrophages
For a long time, the generally accepted theory was that monocytes, as precursors of mature macrophages and dendritic cells (DC) were produced in the bone marrow and released in the circulation. After a short period (2-3 days) in the circulation, they left the circulation by extravasation and entered the parenchyma of tissues (organs). Here they did finally maturate into highly phagocytic resident macrophages, inflammatory macrophages (in case of an inflammatory area) or antigen presenting dendritic cells.
As a consequence, the finding that a single intravenous injection with clodronate liposomes did only deplete macrophages in organs where liposomes had an unhindered access to resident macrophages as in liver and spleen (in case of intravenous injection) was not surprising. Neither two or three injections, repeated with an interval of one week or more, appeared to be sufficient to deplete macrophages from organs where vascular endothelia did form a natural barrier between the circulation and the parenchyma of such tissues (early unpublished results).
To our surprise however, some collaborating investigators, who insisted to try the depletion of macrophages in such organs by injections given repeatedly and after shorter intervals, appeared to be successful in macrophage depletion. This could be explained by the results of a crucial study on subpopulations of mouse blood monocytes in the circulation, their maturation and their kinetics after depletion by clodronate liposomes .
These authors found that virtually all peripheral blood monocytes were depleted at 24 hrs. after intravenous injection of clodronate liposomes. The first cells reappearing at two days after injection of clodronate liposomes were BM-like Ly-6chigh monocytes, whereas Ly-6clow monocytes were detected in the circulation from 7 days after depletion.
Ly-6chigh monocytes that reappeared on day 2 after depletion were pulse-labeled in vivo by injecting DiI labeled fluorescent liposomes without clodronate. By 3 days after the DiI labeling, Ly-6chigh monocytes had developed into Ly-6clow monocytes, whereas the vast majority of unlabeled monocytes at that time were again of the Ly-6chigh BM type.
These studies clearly demonstrated the rapid kinetics of monocytes in the circulation. Moreover, under inflammatory conditions, a significant increase in immature Ly-6chigh monocytes was observed .
So, depletion of monocytes from the circulation by injections of clodronate liposomes, repeated at shorter time intervals (e.g. two days), are needed for an effective depletion of mature macrophages and dendritic cells in organs where liposomes have no direct access to mature macrophages. Interestingly, this treatment caused a delayed disappearance (7-21days post injection) of macrophages and dendritic cells from the endocrine pancreas at a time when monocytes, macrophages and dendritic cells had already repopulated the circulation and spleen. The depletion of macrophages and dendritic cells from the endocrine pancreas was accompanied by a total disappearance of lymphocytes from the pancreas. All depleted cells started to reappear in pancreatic inflammatory infiltrates from 28 days onwards . Obviously, mature macrophages and dendritic cells are dependent on monocytes for their reappearance and lymphocytes in turn depend on cells of the monocyte/macrophage lineage for their entrance in both pancreas  and brain .
A long held concept of van Furth and Cohn was that monocytes originating from bone marrow were continuously replenishing tissue-resident macrophages . Later van Furth and Diesselhoff-den Dulk calculated that under steady-state conditions, 55% of the population of spleen macrophages was supplied by monocyte influx and 45% by local production, implicating a dual origin of spleen macrophages .
Quite a number of recent studies clearly demonstrated that tissue-resident macrophages may be self-maintaining with minimal contribution from circulating monocytes . Local macrophage proliferation can be more important than recruitment from the circulation  and classical monocytes can enter steady-state non-lymphoid organs and recirculate to lymph nodes without differentiation .
Mature tissue macrophages are often prevented from contact by clodronate liposomes, since the latter are particles that will not easily cross vascular endothelia if not provided with fenestrations that allow their passage, such as those in spleen and liver. The finding that monocytes can also be depleted, before these precursors leave the circulation  did extend the applicabilities of clodronate liposomes as a research tool to the complete monocyte/macrophage line.
Given the complexity of biological, pathological or therapeutical processes and the fact that various cells that are participating, are often under control of monocytes, it may not surprise that in some processes monocytes are playing more than a single role.
Monocytes are not only beneficial for the host itself, but may also be of some help for the pathogens that are triggering their expansion. So, during systemic infection in mice, Ly-6chigh monocytes are used by Listeria monocytogenes for its transport into the brain .
Ly-6chigh inflammatory monocytes were shown to be microglial precursors recruited in a pathogenic manner in West Nile virus encephalitis .
It has been shown that Ly-6chigh monocytes facilitate the progression of pulmonary fibrosis, but are not obviously engrafted into the lungs thereafter . The same authors did suggest a resolution-promoting role during the reversible phase of pulmonary fibrosis.
In hepatic fibrosis a profibrotic role of macrophages has been widely recognized. However also here, macrophages orchestrate the regression of murine liver fibrosis .
Importantly, inflammatory monocytes recruited after skeletal muscle injury switch into anti-inflammatory macrophages to support myogenesis and fiber growth .
Depletion of infiltrating macrophages markedly impaired wound healing and increased remodeling and mortality after myocardial injury, identifying the macrophage as a key player in myocardial wound healing .
Monocytes in atherosclerosis
Monocytes participate critically in atherosclerosis and it has been shown that various monocyte subsets did imply different chemokine receptors for their accumulation in atherosclerotic plaques . The results of subsequent studies did suggest that treatments that were reducing cholesterol content of plaques also reduced their macrophage content . This reduction in macrophage content did not involve migratory egress from plaques. Instead, a marked suppression of monocyte recruitment was coupled with a stable rate of apoptosis as a cause of the loss of plaque macrophages .
Later studies indicated that extramedullary sites supplemented the hematopoietic function of the bone marrow by producing circulating inflammatory monocytes that infiltrate atherosclerotic lesions .
Since a number of recent studies clearly demonstrated that tissue-resident macrophages may be self-maintaining with minimal contribution from circulating monocytes  and local macrophage proliferation can be more important than recruitment from the circulation , Robbins et al.  revisited the mechanism underlying macrophage accumulation in atherosclerosis. They found that in murine atherosclerotic lesions, macrophages did turnover rapidly. Replenishment of macrophages in the atherosclerotic plaques appeared to depend predominantly on local macrophage proliferation rather than on monocyte influx. Macrophage proliferation was revealed as a key event in atherosclerosis and macrophage self-renewal was recognized as a therapeutic target for cardiovascular disease .
Transient depletion of macrophages to facilitate survival of human cells in mice
In a number of studies human cells to be studied in an “in vivo” environment were administered to severe combined immunodeficient (SCID) mice. However, even in these mice in which the acquired immune system is practically absent, human cells did normally not survive for more than a few days. Fraser et al.  were the first authors to show that a large proportion of these human cells did survive for much longer periods of time if macrophage depletion had been previously performed by administration of clodronate liposomes. Engraftment of acute myeloid leukemia cells and
human umbilicord blood cells was also facilitated by previous administration of clodronate liposomes [58,59].
In studies on the “in vivo” effects of specific antibodies on the human malaria parasite Plasmodium falsiparum, survival of concomitantly required human monocytes was also facilitated by previous depletion of mouse macrophages by clodronate liposomes .
Drug targeting and gene therapy facilitated by transient depletion of macrophages
Given the efficacy of macrophages in the clearance of non-self particles and macromolecular complexes, it will not surprise that transient macrophage depletion shortly before administration of vehicles carrying the appropriate effector molecules may greatly improve their targeting efficacy.
Clodronate liposomes given before so-called long circulating liposomes significantly prolonged the circulation time of the latter liposomes. So, even polyethylene glycol (PEG) conjugated liposomes, proposed to reduce their recognition and clearance by macrophages, appeared finally to be cleared by macrophages [61,62,63].
Wolff et al.  were the first authors to study the effects of macrophage depletion in a model of adenovirus-mediated gene transfer. They observed that depletion of Kupffer cells by administration of clodronate liposomes before administration of an Adenovirus vector, did result in a higher input of recombinant Adenoviral DNA to the liver, an absolute increase in transgene expression and a delayed clearance of the vector DNA and transgene expression.
Since, the efficacy of clodronate liposomes to improve gene transfer and expression by transient macrophage depletion, has been used in more than thirty published studies [e.g. 65-68].
Fraser et al.  were also the first authors to show that long term depletion of macrophages could be achieved by repeated injections of clodronate liposomes. In their protocol, the injections were given each five days intraperitoneally. Also depletion of monocytes could be prolonged by repeated injections, but given the more rapid repopulation of the circulation by monocytes from the bone marrow, the injections should be repeated each two days (Sunderkotter et al. 2004). The approach developed by Sunderkotter et al. has been followed in a lot of studies in the last decade.
However, in spite of the possibilities for long term depletion of monocytes and mature macrophages by clodronate liposomes, it should be kept in mind that these cells in turn regulate also development, differentiation, migration and functional activities of a lot of non-phagocytic cells. As a consequence, the latter cells may also be reduced in numbers or loose functional activities.
Finally this may lead to less specificity of the action of clodronate liposomes if the duration of the experiments is prolonged.
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