Spontaneous Knockout of CSF-1 Gene in the Mouse as a Model
to Study the Organization of the Macrophage System
 
Wieslaw Wiktor-Jedrzejczak, Andrzej Nowicki  
In: Zander AR et al. (eds) Gene Technolgy, Stem Cell and Leukemia Research, Nato ASI Series H: Cell Biology, Vol 94,
Springer-Verlag, Berlin Heidelberg New York London

Department of Immunology Central Clinical Hospital Military School of Medicine PLOO-909 Warsaw Poland

Physiological role of many molecules and cells can be investigated by evaluating animals possessing genetically determined alterations in the production of those molecules and/or cells. While animals with either knockouts of genes for various molecules or transgenic for these molecules could be at present experimentally created, there is still not fully explored potential of natural mutants, the so called "experiments of nature" (Good, 1991 ). In particular, there are several natural mutants with osteopetrosis: a disorder of osteoclasts i.e. cells related to macrophages, and they all may have alterations in other parts of the macrophage system (Wiktor-Jedrzejczak et al., 1981, Marks 1987). One of such models is the osteopetrotic op/op mouse (Marks and Lane, 1976) . This mutant was found to possess very severe deficiency of macrophages, secondary to deficiency of a growth factor (Wiktor-Jedrzejczak et al.,1982). In particular, it allowed the identification of the total absence of a major macrophage growth factor: colony stimulating factor 1 (CSF-1 or M-CSF; Wiktor-Jedrzejczak et al., 1990, Felix et al., 1990) as the cause of cellular deficiences. In turn, CSF-1 absence was found to be due to inactivating mutation of the gene for that factor (Yoshida et al., 1990). In consequence of these studies, the first mutant with severe congenital deficiency of macrophages became available for the investigation of the organization of the macrophage system, the function and diversity of cells belonging to that system and for verification of the evidence regarding this system obtained using other models (VanFurth et al., 1972, Nathan and Cohn, 1985, Gordon, 1986). Simultaneously, this model became molecularly defined.


Introduction to cells and regulatory molecules of the macrophage system

Macrophage lineage belongs to myelopoiesis but differs from other lines of myeloid differentiation in that: -cells leaving the bone marrow, i.e. monocytes are not yet fully functionally mature, and in some species including mice they may even proliferate; -there is a striking functional and phenotypic diversity among the end cells of the system, which include various forms of tissue macrophages as well as cells that are not phagocytes, such as dendritic cells and osteoclasts (Auger and Ross, 1992); -there is an additional very large potential to increase this diversity of mature cells by their activation, with dramatic enhancement of some functions already expressed by resting cells, and the appearance of many new functions (Adams and Hamilton, 1992) . Classically, macrophage differentiation in the bone marrow was considered to begin at the level of hematopoietic stem cell, and then to proceed through the stage of bipotent neutrophil-macrophage progenitor to the first identified cell of the lineage namely the monoblast, and subsequently to monocytes, and macrophages, as well as to the other end cell of the system (VanFurth, 1993) .However, in addition to these studies, also very primitive macrophage progenitor cells have been identified by Bradley and Hodgson ( 1979) suggesting that some macro phages may derive from progenitors unique to their line of differentiation and descending directly from the stem cells. Subsequent studies have suggested even greater heterogeneity of macrophage progenitors (Bertoncello et al., 1991, Suda et al., 1983). There are three identified growth factors, that when individually added in vitro to macrophage progenitors stimulate their growth and maturation to macrophages (Prystowsky et al., 1984, Koike et al., 1986, Falk and Vogel, 1988, Wiffeils et al., 1993). Such primary macrophage growth factors (Metcalf, 1991) include CSF-1, granulocyte-macrophage (GM)-CSF, and interleukin 3 (IL-3). CSF-1 is a large dimeric cytokine with homology to Steel Factor, and binding to a dimeric receptor of immunoglobulin superfamily with intrinsic tyrosine kinase activity: c-fms. Both GM-CSF and IL-3 belong to hematopoietin family and their receptors belong to the family of hematopoietin receptors. The gene for CSF-1 is located on chromosome 3 at the op locus (Gisselbrecht et al.,1989), is composed of 10 exons, and is producing five different mRNA by alternative splicing (reviewed by Stanley, 1994) .There are at least three different (N-terminus identical) protein forms of CSF-1 : -soluble proteoglycan with largest protein part of 522 aminoacids; -soluble glycoprotein of 406 aminoacids, and: -membrane-spanning glycoprotein of 224 aminoacids, which may be shed and also contribute to soluble CSF-1 . Genes for both GM-CSF and IL-3 are located on chromosome 11 and code for only one protein form of each factor (Gasson, 1991, I hie and Weinstein, 1986). Receptors for GM-CSF and IL-3 have common beta subunit (responsible for signal transduction to the cell inside) and unique alpha subunits (responsible for cytokine binding). In the mouse there is an additional beta subunit exclusive for IL-3 receptor (Miyajima et al.,1992). While IL-3 is only a paracrine factor acting at the vicinity of cells that produce it, GM-CSF is present in the circulation in small amounts (Cheers et al., 1988), suggesting that it may participate in steady-state regulation. However, the most of circulating macrophage colony stimulating activity is due to CSF-1, which is both endocrine (i.e. normally present in peripheral blood), paracrine, and cell contact molecule. Moreover, while both GM-CSF and IL-3 are considered to be mainly induced factors (Gasson, 1991, I hie and Weinstein, 1986), CSF-1 has considerable level of constitutive expression, and therefore, may playa major role in steady-state regulation as opposed to stress regulation, which may be a predominant role for GM-CSF and IL-3 as well as for other molecules. However, there is a considerable overlap between functions of CSF-1 , GM-CSF and IL-3, and it is extremely difficult to dissect the actual role of each molecule in the regulation of the system using normal mouse models.


Basic biology of the op/op mouse model

The op/op mouse is deprived of all forms of CSF-1 (Wiktor-Jedrzejczak et al., 1990) as a consequence of the insertion of thymidine in position 262 of CSF-1 gene (Yoshida et al., 1990). This insertion shifts the reading frame and produces stop codon 21 bases downstream. Such defect should lead to the production of truncated protein, shorter than any known bioactive form of CSF-1 (Heard et al., 1987). Although theoretically possible, there is no evidence for the repair of this lesion in mutant mice, and the described partial resolution of osteopetrosis in old op/op mouse (Marks and Lane, 1976, Begg et al., 1993) is considered to be the effect of other compensatory mechanisms rather than being due to the leak in the defect (Wiktor-Jedrzejczak, 1993b). Morever, the utilization of the op/op mouse model is facilitated by the availability of human recombinant CSF-1 (active in murine system) , that can be used in reconstitution experiments (Ladner et al., 1987, Halenbeck et al., 1989). Consequently, the role of CSF-1 in the regulation of the macrophage system can be studied by a combination of the analysis of deficiences of cells of this system in mutant mice with analysis of mutant animals having reconstituted circulating level of CSF-1 by the administration of recombinant form of the factor .


Pattern of macrophage deficiencies in the op/op mouse

The macrophage deficiency in the op/op mouse is severe but not absolute. Functionally competent macrophages are present (although in reduced number) in mutant animals (Wiktor-Jedrzejczak et al., 1992b) .Moreover, very profoud differences exist in the degree of affection of various local macrophage populations. This heterogeneity concerns not only different organs but also different specific locations within the same organ e.g. spleen. There are tissues, where the number of resident macrophages is reduced to less than 5% of the normal and sometimes it is even negligible. They include peritoneal cavity (where the defect was originally identified: Wiktor-Jedrzejczak et al., 1982) pleural cavity, muscle, skin, periostium, synovium, uterus, kidney and peripheral blood monocytes (Naito et al., 1991, Wiktor-Jedrzejczaket al., 1992b, Witmer-Pack et al., 1993, Cecchini et al., 1994) as well as spleen metallophils (Witmer-Pack et al.,1993, Cecchini et al.,1994) and osteoclasts (Marks and Lane, 1976, Marks, 1982). Organs, where the deficiency is less pronounced, and the number of macrophages is between 10 and 80% of the normal include the liver, other than metallophils populations of spleen macrophages, lung, intestine, salivary glands, adrenals, bladder as well as brain microglial cells (Naito et al., 1991, WiktorJedrzejczak et al.,1992b, Witmer-Pack et al.,1993, Cecchini et al., 1994). On the other hand, epidermal Langerhans cells, dendritic cells of lymphoid organs, as well as macrophages present in these organs are quantitatively normal in the op/op mice (Takahashi et al., 1992, Witmer-Pack et al., 1993, Takahashi et al., 1993, Cecchini et al.,1994), what suggests that they are completely CSF-1 independent. Restoration of circulating CSF-1 by systemic administration of recombinant form of the factor corrected only some near- completely depleted macrophage-related populations including osteoclasts, monocytes, kidney macrophages, and spleen metallophils, as well as some partially depleted populations including bone marrow, spleen and liver macrophages (Wiktor-Jedrzejczak et al., 1991, Kodama et al., 1991, Cecchini et al., 1994). Such populations as peritoneal cavity macrophages could only be restored by local CSF-1 administration, and it was hypothetized that this is due to the existence of blood-tissue barrier for circulating CSF-1 and to the exclusively local control of macrophage populations in many tissues (WiktorJedrzejczak et al., 1991 ). This hypothetical barrier was later found to be operative for almost all other local macrophage populations (Cecchini et al., 1994) in addition to the peritoneal and pleural cavities. On the other hand, these observations may also be explained by the requirement for increasing CSF-1 gradient for monocyte migration to tissues. According to this concept, monocytes could only migrate from locations with lower CSF-1 concentration (blood) to higher CSF-1 concentration (tissues) . Whatever the answer, the reported combined data suggest that the local macrophage populations could be divided into: -completely CSF-1 dependent; -partially CSF-1 dependent, and: -CSF-1 independent. The first two subpopulations could be further subdivided into: -dependent on systemic CSF-1 , and; -dependent on local CSF-1. Moreover, there appears to be a certain logic in distinction between organs with considerable CSF-1 independent resident macrophage population, and organs without such population. The organs with considerable CSF-1 independent macrophage population are mainly those that possess large total macrophage populations such as the liver, spleen, lungs, intestine, and that are at high risk of exposure to microorganisms, and particulate materials such as cell debris and other. Almost all other organs appear to have only CSF-1 dependent resident macrophage population, and they are generally at low risk of exposure to stimuli that require macrophage action. However, also in these latter organs in the op/op mouse, it is easy to elicit macrophages at the absence of CSF-1 by for instance injection of endotoxin (Wiktor-Jedrzejczak et al., unpublished observations). Furthermore, organs with normally large resident macrophage population such as the liver and spleen have their CSF-1 dependent macrophages under control of circulating, and not only of locally produced CSF-1 .Therefore, it appears that the presence of resident macrophage population is these organs is assurred in multiple ways, that is by circulating CSF-1 , local CSF-1 , and by other primary macrophage growth factors. In contrast, in most other organs resident macrophages are almost exclusively dependent on local CSF-1 (Fig. 1) .However, there are also organs such as the kidneys or specific subpopulations such as spleen metallophils, which are almost exclusively CSF-1 dependent, and which are dependent on circulating CSF1 (Cecchini et al., 1994). Additionally, it has to be pointed out that deficient macrophage populations in the op/op liver, spleen and other organs are maintained at the near absence of monocytes. Although diminished, these populations still constitute about 50% of the normal macrophage populations in these organs and are, therefore, of considerable size. This may suggest, that either only CSF-1 dependent resident macrophage populations require constant supply of monocytes or CSF-1 independent macrophages are replenished by a tiny monocyte subpopulation with very high turnover .



Fig.1. Growth factor dependency of various local macrophage populations. Organs such as the liver and spleen possess both CSF-1 dependent and CSF-1 independent macrophage populations of considerable size. Some of their CSF-1 dependent macrophages are under control of circulating, and some are under the influence of local CSF-1. Organs such as kidneys possess almost exclusively macrophages dependent on circulating CSF-1. Locations such peritoneal cavity and vast majority of others have macrophages dependent almost exclusively on local CSF-1 with only a few CSF-1 independent macrophages.


The range of CSF-1 action in macrophage maturation: medullary versus extramedullary role of CSF-1

Hematopoietic organs of the op/op mice possess large numbers of macrophage progenitors, with their frequency being normal in the bone marrow and increased in the spleen (Wiktor-Jedrzejczak et al., 1992b). Only after calculation of the total mouse macrophage progenitor number it was possible to detect a deficiency in this parameter in the op/op mouse and this deficiency could be a secondary consequence of reduced marrow due to osteopetrosis rather than specifically related to CSF-1 absence. This suggests that CSF-1 is not necessary for the generation of macrophage progenitors from the hematopoietic stem cells. Macrophages could be generated in vivo in the op/op mice after administration of CSF-1 within only 48 hours (Wiktor-Jedrzejczak et al., in preparation for publication). This suggests that even quite late macrophage progenitors are formed at the absence of CSF-1 , and that only then they reach block preventing further maturation.



Fig. 2. Sites of CSF-1 action in macrophage formation and survival in vivo. In contrast to previous assumptions, most of the CSF-1 action seems to be exerted extramedullary in the tissues.

Consequently, only macrophage formation and not macrophage progenitor formation appears to be considerably CSF-1 dependent (Fig. 2). The process of macrophage formation may be further divided into formation of monocytes from macrophage progenitors and formation of macrophages from monocytes. Absence of more than 95% of monocytes in the op/op mouse and their restoration after CSF-1 treatment (Kodama et al.,1991) suggests that this process is near completely CSF-1 dependent. This agrees to some extent with published models of the development of macrophage system, where CSF-1 and other macrophage factors have been suggested to act mainly in the bone marrow effecting the production of monocytes (VanFurth, 1993, Johnson, 1993}. However, the very presence of profound macrophage deficiencies in the op/op mice suggests that the role of CSF-1 in transition of monocytes to the tissues, and in their further maturation to macrophages resident there, is much more important. Clearly, this suggests that the essential CSF-1 role in macrophage maturation is extramedullary, and that CSF-1 is not necessary in the bone marrow, but for most of the tissues (listed earlier) it is necessary in those tissues to maintain supply of monocytes, formation of macrophages, and local survival of these latter cells. This is supported also by the observation, that the CSF-1 treated op/op mouse has plenty of monocytes and still possess profound local macrophage deficiencies (Wiktor-Jedrzejczak et al., 1991, Cecchini et al., 1994). The data from other models suggest that CSF-1 is not necessary for macrophage activation (Evans, 1991) .Our studies suggest that it is clearly not necessary for the in vivo activation of those CSF-1 independent macrophages that are still present in the op/op mouse (Wiktor-Jedrzejczak et al., unpublished observations). However, the dependency of activation of CSF-1 dependent macrophages on CSF1 has not been experimentally approached in the mutant mice. This is testable by restoring CSF-1 dependent macrophages in the op/op mouse with exogenous CSF1, withdrawing this factor and testing macrophage activation at CSF-1 absence. Therefore, the results of such experiment should provide more definite answer to this Question.


The role of other primary macrophage Growth factors in the regulation of the macrophage system in relation to CSF-1

In addition to the op/op mouse, animals with knockouts of a gene for the 2nd major primary macrophage factor: GM-CSF have been created recently (Dranoff et al., 1994, E. Stanley et al.,1994). Moreover, also a double knockout: GM-CSFknockout-op/op mice have been bred and partly characterized (lieschke et al., 1994). These models complement very well the advantages of the op/op mouse. Similarly to the op/op mouse, these mutants possess large numbers of macrophage progenitors (E. Stanley et al.,1994, lieschke et al., 1994), confirming that CSF-1 is not necessary for their formation, and suggesting that also GM-CSF is playing only a minor role in that process. In agreement with a notion that most of monocytes are produced under CSF-1 influence, GM-CSF knockout mouse possess normal number of these cells (Dranoff et al., 1994, E. Stanley et al., 1994). However, also double knockout: GM-CSF knockout-op/op mouse still possess some monocytes (lieschke et al., 1994), suggesting that some other factor at the absence of both GM-CSF and CSF-1 is capable of maintaining formation of a few monocytes, and that CSF-1 independent macrophages present in the op/op mouse are not exclusively GM-CSF dependent macrophages. One possible candidate molecule for the supporting the production of CSF-1 independent macrophages is IL-3. However, the fact that it is produced only by activated T cells (Ihie and Weinstein, 1986) may suggest that also some other molecules may playa role in that process. The discussion of contribution of other than CSF-1 factors to macrophage presence in the tissues has to begin from the appreciation that considerable numbers of resident macrophages are present in the op/op mice in such organs as liver and spleen suggesting that they can reach full functional maturity without CSF-1. This may suggest, that in agreement with the model proposed earlier (Wiktor-Jedrzejczak, 1993b) other than CSF-1 primary macrophage growth factors also playa role in terminal maturation of macrophages, and contribute to the formation and maintenance of resident (as opposed to induced) cells. Both published reports concerning GM-CSF knockout mice failed to identify numerical macrophage deficiencies and pointed out pulmonary proteinosis as the major abnormality present in these mice. Whether this proteinosis due to surfactant accumulation is secondary to the deficiency of a few alveolar macrophages dependent on GM-CSF is still an open question. Theoretically, it is possible that GM-CSF may control surfactant production also by a macrophage-independent mechanism. However, the analysis of macrophage deficiencies in these mice was not sufficiently detailed to exclude the presence of even quite significant local deficits. Until such data are available, it is difficult to answer such basic question as: whether CSF-1 dependent and GM-CSF dependent macrophage subpopulations in organs such as liver are regulated independently of each other or whether the total liver macrophage population is maintained by the combined activity of all macrophage growth factors? However, the regular character of macrophage deficiencies in the op/op mouse may suggest that they are created by subtraction of an inherently regulated CSF-1-only- dependent subpopulation. In fact, data are available from other model that CSF-1 level and CSF-1 dependent macrophage subpopulation that is consuming CSF-1 form an integral regulatory feedback system (Bartocci et al., 1987). Besides, the op/op mouse under stress conditions such as bacterial infection can produce large numbers of macrophages. For instance in the course of E. coli fecal peritonitis the mutant mice are capable of eliciting millions of macrophages in peritoneal cavity within 48 hours postinfection (Wiktor-Jedrzejczak et al., submitted for publication). This suggests that CSF-1 independent (i.e. dependent on other macrophage growth factors) formation of induced macrophages is efficient in the op/op mouse, in contrast to deficient formation of resident macrophages. Interestingly, the induced local expansion of macrophages may originate from local progenitors. This is supported by a recent observation that in the presence of inflammatory stimulus such as glucan, local (liver) macrophages can considerably expand, at the absence of CSF-1 , and at the near absence of monocytes (Takahashi et al., 1994).


Macrophage growth factor dependency and functional diversity of macrophages

We have suggested earlier, that CSF-1 dependent macrophages (absent in the op/op mouse) play mainly regulatory role through release of cytokines, while CSF-1 independent macrophages (present in the op/op mouse) are responsible primarily for the immune functions of macrophages (Wiktor-Jedrzejczak et al. , 1992a) .It has to be understood that in order to be called macrophages, cells have to possess common set of functions including phagocytosis. Other studies comparing CSF-1 induced and GM-CSF induced macrophages in vitro have shown that GM-CSF induced macrophages expressed higher levels of class II MHC (Doherty et al., 1993) what is in agreement with suggestion on primarily immune function of these cells. However, the same authors have also suggested that GM-CSF induced macrophages are better producers of cytokines including IL-1 , IL-6 and TNF-alfa than CSF-1 induced macrophages. This is in contrast to the postulated major role of these latter cells in that process. On the other hand, not only efficiency of production on the per cell basis counts in vivo, but also availability of cells. From that point of view, in most locations the vast majority of resident macrophages are dependent on CSF-1 and they have to provide the respective cytokines during early phases of tissues response to trauma or infection (Fig.3). This notion is supported by the presence of TNF-alfa, and IL-1 alfa deficiencies in the op/op mouse (WiktorJedrzejczak et al., 1992a, Szperl et al., 1995).



Fig. 3. Growth factor dependency of macrophage diversity. The diversity in vivo is a function of availability of cells, with much greater availability of CSF-1 dependent macrophages, and functional diversity. Most of the functions are shared by both subpopulations, which is indicated by shaded area, while some functions are unique to each subpopulation.

At present, there appears to be no data supporting the existence of separate CSF-1 dependent and CSF-1 independent macrophage progenitors. All macrophage progenitors seem to be able to respond to both CSF-1 and GM-CSF (Metcalf and Nicola, 1992). There is no synergy between these factors, what may suggest that they act on the same cell population. However, after macrophages are formed, in addition to functions shared by macrophages formed under the influence of any factor, there appear to be functions that are exclusively dependent on CSF-1 or GM-CSF. Only CSF-1 dependent resident peritoneal macrophages are capable of recruiting lymphocytes to peritoneal cavity (Kalinski et al.,1993). On the other hand, eicosanoids production has been recently shown to be an exclusive function of GM-CSF and IL-3 produced macrophages, and not of CSF-1 induced macrophages (Shibata et al.,1994).


Concluding remarks

The studies employing the op/op mouse challenge several established views concerning the organization of the macrophage system and provide novel insights into regulation of macrophages at the tissue level. The model is far from being explored and awaites application to the studies reappraising various roles classically assigned to macrophages. Moreover, its possibilities may be increased by breeding mice combining CSF-1 deficiency with deficiencies of other earlier or later acting macrophage growth factors and macrophage activators such as interferon-gamma. An example of this approach: breeding the op/op mouse possessing knockout of GMCSF gene is already available (Lieschke et al., 1994) .It is a strong belief of the present author that these models would allow final dissection of the organization and function of the macrophage system.


Acknowledgements

This study was supported in part by grants 4131791 01 and 44381 91 02 from
the Polish National Committee for Scientific Research to W .W-J .


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