Developmental stages of myeloid dendritic cells in mouse bone marrow

The lineage relationship of dendritic cells (DC) with other hematopoietic cell types has been studied extensively, resulting in the identification of different bone marrow (BM) progenitors that give rise to distinct DC types. However, the identity of the different maturation stages of DC precursors in the BM remains unclear. In this study we define the in vivo developmental steps of the myeloid DC lineage in mouse BM. To this end, BM cells were separated according to their expression of CD31 (ER-MP12), Ly-6C (ER-MP20) and ER-MP58 antigens, and stimulated to develop into myeloid DC, using granulocyte macrophage colony stimulating factor as a specific growth factor. DC developed from three BM subpopulations: ER-MP12(hi)/20(-) (early blast cells), ER-MP12(+)/20(+) (myeloid blasts) and ER-MP12(-)/20(hi) (monocytes). The kinetic and phenotypic features of DC developing in vitro indicate that the three populations represent successive maturation stages of myeloid DC precursors. Within the earliest ER-MP12(hi)/20(-) population, DC precursors exclusively occurred in the myeloid-restricted ER-MP58(hi) subset. By using switch cultures, we show that these BM precursor subpopulations, when stimulated to develop into macrophages using macrophage colony stimulating factor, retain the ability to develop into myeloid DC until advanced stages of maturation. Together, these findings support a common ER-MP12/20-defined differentiation pathway for both macrophages and myeloid DC throughout their BM development.     

Milky spots in the mouse omentum may play an important role in the origin of peritoneal macrophages.

In the milky spots, precursors of cells belonging to the mononuclear phagocyte system (MPS), such as monoblasts, can be found on the basis of ultrastructural endogenous peroxidase cytochemistry. Therefore, in the present study, we investigated the milky spots using a panel of monoclonal antibodies, especially antibodies (ER-MP) that recognize macrophage precursor antigens. Early macrophage precursor antigens ER-MP12 and ER-MP58 were detected only on cells localized inside the milky spots. On the other hand, an antigen which disappears late in the course of macrophage differentiation, ER-MP20, was detected in high amounts on cells both inside and around the milky spots. This clearly indicates that macrophage precursors are centrally localized inside the milky spots, while more differentiated cells are found in peripheral areas. Moreover, long-term culture of milky spot tissue resulted in the forming of a monolayer of stromal cells which supported macrophage proliferation in vitro. In conclusion, both in situ and in vitro studies demonstrated that mouse milky spots have a microenvironment in which precursor cells of the MPS can home and proliferate, illustrating that milky spots play a role as a source of local macrophage generation, e.g. that of the free peritoneal macrophages.     

Murine macrophage precursor characterization. I. Production, phenotype and differentiation of macrophage precursor hybrids

This study reports on the earliest stages of mononuclear phagocyte differentiation. A crucial question in this developmental process is whether mature macrophage (M phi) heterogeneity is already appointed at the precursor cell level. For this purpose, we produced clonal populations of mononuclear phagocytes from bone marrow culture by somatic cell hybridization with two hypoxanthine, aminopterin, thymidine-sensitive myeloid cell lines. A panel of 22 stable hybrids was obtained from these fusions. Differentiation stage analysis of the hybrids indicated that all cell lines had immature mononuclear phagocyte characteristics. The hybrids exhibited typical myeloid morphology and mainly nonadherent growth. Mature M phi features, such as expression of the cell surface antigens Mac-1, Mac-2 and F4/80, phagocytosis of latex beads, and expression of nonspecific esterase and acid phosphatase activity, were virtually absent. The immature M phi markers Thy-1, MIV25 and MIV52, on the other hand, were readily expressed, although heterogeneity was observed among different hybrid cell lines. We then analyzed the differentiation potential of seven hybrids by culture of the cells in the presence of post-lipopolysaccharide serum supplemented with interferon-gamma and found that the expression of mature M phi characteristics was induced. However, the various hybrids showed divergent patterns of mature M phi marker induction. R0C2 cells, for instance, showed extensive morphological and phenotypical differentiation without concomitant induction of phagocytosis. In contrast, W1C4 cells showed significant induction of phagocytosis without simultaneous increase of phosphatase and esterase activity. R1C1 cells were unique in the strong induction of Ia antigen expression. Together, our data indicate that (a) early M phi differentiation stages can be rescued by somatic cell hybridization, and that (b) the obtained cell lines are able to mature according to divergent differentiation programs.     

ER-MP12 antigen, a new cell surface marker on mouse bone marrow cells with thymus-repopulating ability: I. Intrathymic repopulating ability of ER-MP12-positive bone marrow cells

We searched for new cell surface markers that allow a positiveidentification of thymusrepopulating cells in the bone marrow(BM) of the mouse. Recently we raised two rat monocional antibodies(ER-MP12 and ER-MP20) that recognize cell surface antigens expressedby mouse haematopoietic progenitor cells, among which are progenitorcells of the macrophage lineage. Here we show that the ER-MP12antigen, but not the ER-MP20 antigen, is also expressed by BMcells with thymus-repopulating ability. Using ER-MP12 and ER-MP20in two-colour immunofluorescence analysis six subpopulatlonsof BM cells can be identified. The thymusrepopulating abilityof each BM subpopulation was assessed after fluorescence-activatedcell sorting and subsequent intrathymic injection into sublethallyirradiated Thy-1 congenic recipient mice. Thymus-repopulatingactivity appeared to be exclusively confined to two subsetsof BM cells expressing either high or intermediate levels ofthe ER-MP12 antigen, but lacking ER-MP20 antigen expression.These BM subsets comprised 1–2% and 30% of total nucleatedBM cells respectively. The frequency of thymus-repopulatingcells was maximal in the minor BM subpopulation with the highestlevel of ER-MP12 antigen expression. We conclude that ER-MP12detects a hitherto unknown cell surface marker expressed byBM cells with thymus-repopulating ability.     

Distinct mouse bone marrow macrophage precursors identified by differential expression of ER-MP12 and ER-MP20 antigens

The characterization of early branch points in the differentiation of leukocytes requires identification of precursor cells in the bone marrow. Recently, we produced two monoclonal antibodies, ER-MP12 and ER-MP20, which in two-color flow-cytometric analysis divide the murine bone marrow into six defined subsets. Here we show, using fluorescence-activated cell sorting followed by macrophage colony-stimulating factor-stimulated culture in soft agar, that precursors of the mononuclear phagocyte system reside only within the ER-MP12^hi20^?, ER-MP12⁺20⁺ and ER-MP12^?20^hi bone marrow subsets. Together, these subsets comprise 15% of nucleated bone marrow cells. Furthermore, we provide evidence that the macrophage precursors present in these subsets represent successive stages in a maturation sequence where the most immature ER-MP12^hi20^? cells develop via the ER-MP12⁺20⁺ stage into ER-MP12^?20^hi monocytes.     

High-level expression of the ER-MP58 antigen on mouse bone marrow hematopoietic progenitor cells marks commitment to the myeloid lineage

Studies on the early events in the differentiation of the nonspecific immune system require the identification and isolation of myeloid-committed progenitor cells. Using the monoclonal antibodies (mAb) ER-MP12 and ER-MP20, generated against immortalized macrophage precursors, we have shown previously that the earliest macrophage colony-stimulating factor (M-CSF)-responsive cells in the bone marrow have the ER-MP12^hi20⁻ phenotype. In addition, we found that the ER-MP12^hi20⁻ subset (comprising about 2 % of total nucleated marrow) contains progenitor cells of all hematopoietic lineages. Aiming at the identification and purification of the myeloid progenitor cells within the ER-MP12^hi20⁻ subset, we used ER-MP58, a marker expressed at high level by all M-CSF-responsive bone marrow progenitors. With this marker the ER-MP12^hi20⁻ cell population could be divided into three subfractions: one with absent or low level ER-MP58 expression, one with intermediate, and one with high ER-MP58 expression. These subfractions were isolated by fluorescence-activated cell sorting and tested in vitro and in vivo for their differentiation capacities. In addition, the expression of ER-MP58 on stem cell subsets was examined in the cobblestone area-forming cell (CAFC) assay. Our data indicate that in the ER-MP12^hi20⁻ subpopulation myeloid-committed progenitors are characterized by high-level expression of the ER-MP58 antigen, whereas cells with other or broader differentiation capacities have an ER-MP58 negative/low or intermediate phenotype. These myeloid-committed progenitors have no significant repopulating ability in vivo, in contrast to the ER-MP58 intermediate cells. Primitive CAFC-28/35, corresponding to cells providing long-term hematopoietic engraftment in vivo, also did not express the ER-MP58 Ag at a high level. Thus, cells committed to the myeloid lineage can be separated from progenitor cells with other differentiation capacities by means of multiparameter cell sorting using ER-MP58 in combination with ER-MP12 and ER-MP20.     

Repopulation of murine Kupffer cells after intravenous administration of liposome-encapsulated dichloromethylene diphosphonate.

Kupffer cells were selectively eliminated in mice by the intravenous administration of liposome-entrapped dichloromethylene diphosphonate. At 5 days, small peroxidase-negative and acid-phosphatase-weakly-positive macrophages appeared, increased in number, and differentiated into peroxidase- and acid-phosphatase-positive Kupffer cells. Repopulating small macrophages actively proliferated, and the number of Kupffer cells returned to the normal level by day 14. The numbers of macrophage precursors in the liver as detected by the monoclonal antibodies ER-MP20 and ER-MP58 increased after liposome-entrapped dichloromethylene diphosphonate injection. ER-MP58-positive cells proliferated and differentiated into ER-MP20-positive cells and eventually into BM8-positive Kupffer cells in the liver. Bone-marrow-derived ER-MP58-positive cells were also detectable in the liver and differentiated into ER-MP20-positive cells, but they did not become BM8-positive macrophages. Macrophage colony-stimulating factor mRNA expression was enhanced in the liver 1 day after injection. The administration of macrophage colony-stimulating factor did not shorten the period of Kupffer cell depletion but increased the number and the proliferative capacity of repopulating Kupffer cells. These findings implied that repopulating Kupffer cells are derived from a macrophage precursor pool in the liver rather than from bone-marrow-derived monocytes. Local production of macrophage colony-stimulating factor in the liver plays a crucial role in the differentiation, maturation, and proliferation of Kupffer cells.     

Liver-associated macrophage precursor cells proliferate under impairment of regular hemopoiesis

We reported previously that immature macrophage precursor cells can be isolated from spleen and liver of cyclophosphamide or pyran copolymer-pretreated mice. We now extended our investigations to livers of normal, untreated specific pathogen-free mice. Using the response to the macrophage growth factor colony-stimulating factor-1 (CSF-1) and the presence of the mouse macrophage-specific F4/80 antigen as criteria of definition, in the liver of normal mice we could demonstrate macrophage precursor (M phi P) cells by means of proliferation assays and flow cytometric analysis. The amount of M phi P present in the normal liver was significantly increased after administration of pyran copolymer. Also an enhanced proliferative response to CSF-1 as well as augmented natural killer activity and cytostasis of Candida albicans was noted in liver nonparenychymal cells (LNPC) after treatment of bone marrow (BM)-irradiated, splenectomized mice with pyran copolymer. Since the irradiated BM was actually proven to be silent by assessment of BM number and proliferative capacity and by scoring white blood cells, our findings suggest a response of endogenous liver M phi P under the applied conditions. Further evidence for the presence of endogenous liver hemopoietic cells was obtained from transplantation experiments in which LNPC brought about the survival of lethally irradiated mice. The data point towards a significance of the liver in disposing hemopoietic cells to the organism under impairment of regular hemopoiesis.     

ER-MP12 antigen, a new cell surface marker on mouse bone marrow cells with thymus-repopulating ability: II. Thymus-homing ability and phenotypic characterization of ER-MP12-positive bone marrow cells

In the accompanying paper we showed that six distinct subsetsof bone marrow (BM) cells can be identified using the mAb ER-MP12and ER-MP20 in two-colour immunofluorescence analysis. Uponintrathymic transfer into sublethally irradiated mice thymus-repopulatingability was restricted to ER-MP20^– BM cells expressingeither high or intermediate levels of the ER-MP12 antigen (1–2%and –30% of BM nucleated cells respectively). The highestfrequency of thymus-repopulating cells was found in the minorsubset of ER-MP12⁺⁺20^– BM cells. In the present studywe demonstrate that upon intravenous transfer, thymus-homingand-repopulating BM cells are exclusively confined to the ER-MP12⁺⁺20^–and ER-MP12⁺20^– subpopulations, the highest frequencybeing detected among ER-MP12⁺⁺20^– BM cells. Analysis ofthe peripheral blood leucocytes of reconstituted mice showedthat not only prothymocytes but also progenitorcells of theB cell lineage as well as the myelold lineage were present withinboth subsets. Three-colour flow cytometric analysis revealedthat ER-MP12⁺⁺20^– BM cells in particular were phenotyplcallyheterogeneous with respect to the expression of the cell surfacemarkers Thy-1, Sca-1, CD44, B220 and c-kit. Taken together ourdata demonstrate that ER-MP12 positively identifies BM cellswith the ability to home to and repopulate the thymus. The phenotypicheterogeneity displayed by the ER-MP12⁺⁺20^– BM subset,containing the highest frequency of thymus-homing and-repopulatingcells, provides a basis for further separation of prothymocyteactivity from other haematopoietic activities in the BM of themouse.     

Characterization of the rat leukocyte integrin, CD11/CD18, by the use of LFA-1 subunit-specific monoclonal antibodies.

We have attempted to characterize the rat leukocyte integrin, CD11/CD18, by the use of newly generated monoclonal antibodies (mAb) WT.1 (anti-CD11a) and WT.3 (anti-CD18) in conjunction with an mAb, OX42, reactive with a rat integrin-like molecule, with respect to the biochemistry, cellular distribution and function. The conclusion that the mAb WT.1 and WT.3 specifically recognize the rat CD11a and CD18, respectively, was based on: (a) their ability to inhibit homotypic aggregation of splenic concanavalin A (Con A) blasts; (b) sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the antigens recognized; (c) their ability to inhibit binding of Con A blasts to the purified ligand, namely the ICAM-1 antigen and (d) their blocking abilities in mixed leukocyte reaction. In the rat, CD18 has an apparent molecular mass of 95-100 kDa and can associate with at least three distinct alpha subunits of 160-170 kDa (CD11a), 140-150 kDa and 120-130 kDa. The latter two are precipitated by OX42 from M phi but not from unstimulated lymphocytes. They presumably represent the rat CD11b and CD11c, respectively. Rat thymocytes, PBL, thoracic duct lymphocytes, monocytes and neutrophils expressed differential levels of CD11a and CD18. Peritoneal M phi showed virtually no CD11a expression, although CD18 was expressed at levels similar to those seen on blood monocytes, showing an interesting pattern of LFA-1 expression regulation in this cell lineage. Both WT.1 (anti-CD11a) and WT.3 (anti-CD18) apparently recognize a "low-affinity" as well as a "high-affinity" form of LFA-1 and do not discriminate between the two.     

Immature macrophages derived from mouse bone marrow produce large amounts of IL-12p40 after LPS stimulation

Production of IL-12 is an important indicator of the macrophage's ability to regulate immune responses. In this study, we investigated the IL-12 production by macrophages in different developmental stages. To this end, macrophages were generated in vitro from precursors stimulated with M-CSF, GM-CSF or IL-3. Density separation yielded populations enriched in different maturation stages. Invariably, only cells banding at the 40-50% Percoll interface produced large amounts of IL-12p40 when stimulated with LPS, whereas only low levels of IL-12p70 were produced. These cells represented immature macrophages, as indicated by the absence of precursor markers CD31/ER-MP12, Ly-6C/ER-MP20 and ER-MP58, and by the low level of expression of mature-cell markers like ER-HR3, scavenger receptor and CD11b/Mac-1. Upon further maturation, the macrophages' ability to produce IL-12p40 decreased, coinciding with increased nitric oxide production upon LPS stimulation. These results show that immature macrophages produce high levels of IL-12p40 and thus may either contribute to IL-12p70 production or regulate it.     

Expression of the ED3 antigen on rat macrophages in relation to experimental autoimmune diseases.

Susceptibility to experimental autoimmune diseases (EAD) is rat strain dependent. Susceptible animals are reported to have a defective glucocorticoid response. Although many EAD are regarded as preferentially T cell-mediated, macrophages (M phi) play an important role in several different stages of these diseases. In this study we have investigated the possible effect of the disturbed hypothalamic-pituitary (HPA) axis on M phi phenotype. Therefore we studied M phi differentiation in several different rat strains, especially with regard to the M phi specific differentiation antigen as recognized by monoclonal antibody (mAb) ED3. This mAb is, in normal healthy rats, reactive with very restricted M phi subpopulations present in the lymphoid organs only. However, in autoimmune diseased tissues many of the infiltrated M phi are also ED3-positive. It appeared that M phi, in vitro derived from monocytes out of susceptible rat strains, showed a high ED3 expression in contrast to monocyte-derived M phi out of resistant rat strains. This difference in ED3 expression appeared to be T cell-mediated. Our results are suggestive for the fact that the impaired HPA-axis in EAD susceptible rat strains affects M phi differentiation. The relevance of the observed differences with respect to disease induction, maintenance, or suppression is discussed and obviously needs further investigation.     

The monoclonal antibody ER-BMDM1 recognizes a macrophage and dendritic cell differentiation antigen with aminopeptidase activity.

Here we describe the reactivity of monoclonal antibody (mAb) ER-BMDM1, directed against a 160-kDa cell membrane-associated antigen (Ag) with amino-peptidase activity. The aminopeptidase recognized by ER-BMDM1 is present on various mouse macrophage (M phi) and dendritic cell (DC) subpopulations as well as on microvillous epithelia. Analysis of ER-BMDM1 Ag expression in in vitro models of M phi maturation revealed that the Ag is expressed at increasing levels upon maturation of M phi. In vivo, high level expression of the ER-BMDM1 Ag occurs after the monocytic stage of maturation, since bone marrow cells and peripheral blood monocytes are essentially ER-BMDM1 negative. Analysis of isolated-resident and elicited M phi populations showed that ER-BMDM1 recognizes a specific subpopulation of mature M phi: only some resident peritoneal and alveolar M phi are ER-BMDM1 positive, whereas virtually all thioglycollate-elicited peritoneal exudate M phi bind the mAb. In lymphoid organs, a subpopulation of M phi is recognized as well as interdigitating cells (IDC) located in T cell areas. Phenotypic analysis of isolated DC--the in vitro equivalents of IDC--from spleen and lymph nodes confirmed that the majority of this important antigen-presenting cell population expresses the ER-BMDM1 aminopeptidase. The molecular characteristics of the ER-BMDM1 Ag suggest that it may represent the mouse homolog of human CD13.     

A new epitope of the T200 molecule family defined by the 3A35 monoclonal antibody and expressed by macrophages and activated T lymphocytes

A monoclonal antibody (mAb), 3A35, produced against mouse macrophages (M phi) was found to react against certain activated T cells. This mAb, a rat IgM, resulted from a cell fusion between a mouse plasmacytoma and rat lymphocytes immunized against mouse M phi. It bound more avidly to activated than to resident M phi. It did not react against B cells and resting T lymphocytes but recognized certain dividing T cells like EL4 lymphoma, concanavalin A-activated and interleukin 2-expanded spleen cells, and helper T cell hybridomas. By contrast, other T lymphocyte-derived cell lines such as YAC-1 and CTLL2 were unreactive. No clear relationship was found between the binding of 3A35 to cells and the expression of L3T4 and Lyt-2 antigens. The specific stimulation of T cell clones with antigen rapidly induced a strong reactivity with 3A35 mAb which declined thereafter to a low (helper clones) or non-reactivity (cytotoxic clones) after 10 days of culture. Immunoprecipitation experiments, performed with M phi derived from bone marrow cell cultures, surface iodinated with 125I or metabolically labeled with [35S]methionine, showed that 3A35 bound to a 200-kDa molecule, shifting to 175 kDa under reducing conditions. In peritoneal M phi activated in vivo, in addition to the 175-kDa band, new bands migrating at 140, 120 and 85 kDa were identified by 3A35 and could be absorbed on a commercial anti-T200 mAb bound to Sepharose beads. After strengthening the cell binding of 3A35 to EL4 lymphoma cells by a cross-linking agent, only a 85-kDa molecule was immunoprecipitated. Thus, 3A35 identifies a new epitope of the T200 molecule family which is expressed on M phi and activated T cells.     

Myeloid dendritic cell precursors generated from bone marrow suppress T cell responses via cell contact and nitric oxide production in vitro

Tolerogenic activity of myeloid dendritic cells (DC) has so far been attributed mostly to immature or semi-mature differentiation stages but never to their precursor cells. Although myeloid suppressor cells (MSC) have been isolated ex vivo, their developmental relationship to DC and their precise phenotype remained elusive. Here, we describe the generation of MSC as myeloid DC precursors with potent suppressive activity on allogeneic and OVA-specific CD4+ and CD8+ T cell responses in vitro. These MSC appear transiently in DC cultures of bone marrow (BM) cells after 8-10 days under low GM-CSF conditions or after 3-4 days under high GM-CSF conditions. They represent CD11c- myeloid precursor cells with ring-shaped nuclei and are Gr-1low (i.e. Ly-6C+, Ly-6Glow), CD11b+, CD31+, ER-MP58+, asialoGM1+ and F4/80+. Sorted MSC develop into CD11c+ DC within 6 days. Their suppressor activity partially depends on IFN-gamma stimulation. Suppression is mediated through mechanisms requiring cell contact and nitric oxide but is independent of TNF, CD1d and TGF-beta. Together, our data describe the generation of MSC with distinct suppressor mechanisms in vitro preceding their development into immature DC.     

Microenvironmental organization and stromal cell associations of B lymphocyte precursor cells in mouse bone marrow

B lymphocyte precursor cells expressing B220 glycoprotein have been examined in mouse bone marrow (BM) by the in vivo binding of monoclonal antibody (mAb) 14.8 visualized by light and electron microscope radio autography. Young mice were injected intravenously with 125I-labeled mAb 14.8 and then perfused to remove unbound antibody. Quantitative analysis of radioauto graphic sections of femoral BM revealed many labeled mAb 14.8-binding cells which were situated both singly and in groups throughout the extravascular BM parenchyma. Groups of large 14.8+ cells were located in patchy areas in the peripheral regions of the BM near the endosteum. These cells were shown to include proliferating precursor B cells by using mice given vincristine sulfate to stop cells in metaphase and mice treated from birth with anti-IgM antibodies to delete mature B lymphocytes. Electron microscopy revealed clusters of 14.8+ cells intimately associated with the processes of stromal reticular cells. Other 14.8+ cells were in close contact with macrophages; in some instances the intervening cell membranes were indistinct and the macrophages contained 14.8+ material in their cytoplasm. In addition, 14.8+ small lymphocytes were highly concentrated within the lumen of some sinusoids. The present method of detecting B lineage precursor cells in situ has led to a working model of the microenvironmental organization of primary B cell genesis in vivo. The model proposes (a) a centrally directed sequence of differentiation initiated by early precursor cells situated peripherally near the surrounding bone; (b) close associations between precursor B cells and stromal reticular cells; (c) deletion of ineffective B cells by macrophages, and (d) an intravascular maturation phase before B lymphocytes are finally delivered into the blood stream.     

Regulation of Forssman antigen expression during maturation of mouse stromal macrophages in haematopoietic foci

During a trial to develop a monoclonal antibody (mAb) specific to stromal macrophages (M phi) in haematopoietic foci, we have created a mAb, designated F10, that stains the stromal M phi more selectively than any other mAb reported. As F10 was found to react with Forssman glycosphingolipid (GSL) specifically and to give clearer immunostaining than anti-Forssman GSL IgG, we have studied Forssman antigen expression during maturation of the stromal M phi in splenic haematopoietic foci using F10 and a system of allogenic bone marrow transplantation which allows us to know the turnover of the stromal M phi in vivo. C3H/He mice (H-2k) were lethally irradiated and intravenously infused with the bone marrow cells of BALB/c nu-nu mice (H-2d). Splenic frozen-sections and cytocentrifuge preparations of splenic haematopoietic clusters from the recipient mice were stained with F10 and with mAb against major histocompatibility class I antigens. H-2d-type stromal M phi began to appear in the haematopoietic clusters at Week 5 and they gradually replaced H-2k-type stromal M phi. The percentage of Forssman+ stromal M phi gradually decreased and reached a nadir at Week 6, when most stromal M phi were already of the donor type. At Week 8, however, Forssman+ stromal M phi levels returned to normal. The delayed expression of Forssman antigen on the stromal M phi in haematopoietic foci following genotypic conversion suggests that Forssman antigen is regularly expressed on the subpopulation of stromal M phi, which mature well under specific microenvironmental factors in vivo.     

Monoclonal antibodies specific for porcine monocytes/macrophages: macrophage heterogeneity in the pig evidenced by the expression of surface antigens

Macrophages are widely distributed in most tissues of the body, where they play important roles in host defense and repair of tissue damage. In this report we describe the production and characterization of a panel of six monoclonal: antibodies (mAb) against porcine macrophages and their use for phenotyping tissue macrophages. All mAbs were produced by immunizing mice with porcine alveolar macrophages. Three of them (2A10/11, 3B11/11 and3F7/ll) react mainly with macrophages and, at a lower extent, blood monocytes, whereas the others (1E12/11, 2C12/10 and 4E9/11) also recognize granulocytes. Antigens recognized by these antibodies could be characterized by Western blot and/or immunoprecipitation, with the exception of that one recognized by 2C12/10. By their behavior in SDS-PAGE under reducing and nonreducing conditions, all seem to be single polypeptides, whose apparent molecular weight under reducing conditions are: 1E12/11 and 3B11/11 larger than 204 kDa; 2A10/11, 150 kDa; 4E9/I1, 125–170 kDa; and 3F7/11, 135 kDa. Immunohistochemical analyses of both lymphoid and non-lymphoid organs using these mAbs reveal important antigenic heterogeneity among tissue macrophages. These mAbs are, therefore, useful tools for the study of porcine macrophage maturation and differentiation and for determining their heterogeneity both in normal and pathological conditions.     

Changes in macrophage populations: phenotypic differences between normal and tumor-bearing host macrophages

As a tumor grows, changes occur in the function of macrophages (M phi). This is concomitant with changes in their phenotype. Flow cytometric analysis of monoclonal antibody (mAb)-labeled thioglycollate-elicited peritoneal, and resident splenic, M phi showed a tumor-induced shift of Mac-1, -2, -3, and Ia antigen expression. During tumor growth, the percentage of peritoneal Mac-2+, -3+, and Ia+ M phi decreased significantly (22%, 14%, and 58%, respectively), while Mac-1+ M phi remained unchanged. By analyzing the data on two-dimensional histograms and comparing the sizes of M phi to cell-surface antigen expression, we identified distinct subpopulations of peritoneal M phi. Three distinct size versus antigen expression M phi subpopulations were detected by flow cytometry and consisted of 10-16, 17-22, and 23-27 microns for the small-, medium-, and large-sized populations, respectively. Large-sized Mac-1+ and -2+ M phi decreased (37% and 38%), while large-sized Mac-3+ M phi did not decrease during tumor growth. Medium-sized Mac-3+ M phi decreased 33% during tumor growth, while no differences could be seen in medium-sized Mac-1+ or -2+ M phi. Concomitant with the decrease in large-sized Mac-1+ M phi was an increase in small-sized Mac-1+ M phi. Peritoneal Ia+ M phi were mostly small-sized (4-7-fold increase over the medium-sized and none in the large-sized population). M phi Ia antigen expression was nearly absent in the 21-day tumor-bearing host, with less than 4% of the cells labeling positive (a 73% drop from normal host M phi). In splenic M phi, the percentage of Mac-1+ M phi significantly increased (90%) during tumor growth, while Mac-2+ and -3+ M phi showed a smaller, but still significant, increase (48% and 40%, respectively). Additionally, splenic Ia+ M phi significantly decreased (29%) during tumor growth. More important than the decreased cell numbers was the significant decrease in Ia antigen expression per cell. Unlike the peritoneal M phi, the splenic M phi did not show distinct size versus antigen expression subpopulations, although there was an overall difference in M phi size between normal and TBH. These data suggested that M phi from different anatomical sites are phenotypically different and tumor growth mediates phenotypic alterations in peritoneal and splenic M phi populations. This may be the source of tumor-induced dysfunction of M phi-mediated immune activity.     

Effects of granulocyte/macrophage colony-stimulating factor on the development and differentiation of CD5-positive macrophages and their potential derivation from a CD5-positive B-cell lineage in mice.

In co-cultures of either the murine pre-B cell line J13, fetal liver cells, or adult peritoneal or bone marrow cells with ST2 mouse bone marrow stromal cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), the development of CD5+ macrophages was demonstrated by immunohistochemical staining and flow cytometry. Although CD5+ macrophages were not present in the peritoneal cavities of normal mice, approximately 30% of the peritoneal macrophages in viable motheaten (mev/mev) mice, deficient in SHP-1 protein tyrosine phosphatase, expressed cell surface CD5 and B220, markers for B cells. In the mev/mev mice, GM-CSF level in peritoneal fluid was increased significantly. At 5 days after daily intravenous injection with GM-CSF, many CD5+ macrophages appeared in the peritoneal cavity and in omental milky spots of normal mice but fewer in osteopetrosis (op) mutant mice, deficient in macrophage (M)-CSF. These results indicate that GM-CSF, in combination with M-CSF, induces the development and differentiation of CD5+ macrophages in the peritoneal cavity, particularly in the omental milky spots of mice. In the peritoneal cavity of GM-CSF-treated mice, the percentages of hematopoietic progenitor cells doubly positive for CD5 and CD34 or c-kit and of macrophage precursor cells doubly positive for CD5 and ER-MP58 or ER-MP20 were increased significantly during the development of CD5+ macrophages and CD5 B cells, suggesting that CD5+ macrophages and B cells may share a bipotential progenitor in vivo.