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(Journal of Leukocyte Biology. 2003;73:564-573.)
© 2003 by Society for Leukocyte Biology

Monocyte and macrophage functions in M-CSF-deficient op/op mice during experimental leishmaniasis

Frank Schönlau*, Christian Schlesiger*, Jan Ehrehen*, Stephan Grabbe{dagger}, Clemens Sorg* and Cord Sunderkötter*,{dagger},{ddagger}

* Institute of Experimental Dermatology and
{dagger} Department of Dermatology, University of Münster, Germany; and
{ddagger} Department of Dermatology, University of Ulm, Germany

Correspondence: Cord Sunderkötter, M.D., Department of Dermatology, University of Ulm, Maienweg 12, 89081 Ulm, Germany. E-mail: cord.sunderkoetter{at}medizin.uni-ulm.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice with a naturally occurring Csfmop/Csfmop (op/op) gene mutation lack functional macrophage-colony stimulating factor (M-CSF) and are deficient of M-CSF-derived macrophages. They are severely monocytopenic, and their remaining M-CSF-independent macrophages were shown to differ in differentiation and distinct functions when compared with phenotypically normal mice of the same background. It is not known if osteopetrosis mice (op/op mice) are able to mount a specific immune response against intracellular pathogens, as this would require complex effector functions by macrophages. We therefore investigated the ability of op/op mice and their M-CSF-independent macrophages to combat infection with Leishmania major. op/op mice retained the ability to resist an infection with L. major by mounting a T helper cell type 1 cell response, eliminating parasites and resolving the lesions. Macrophages from op/op mice were able to sufficiently perform effector functions in vitro, such as phagocytosis, production of leishmanicidal nitric oxide (NO), killing of parasites, and release of interleukin (IL)-12. There were quantitative differences, as M-CSF-derived macrophages from hematopoietic organs of control mice showed significantly higher rates of phagocytosis and higher NO release after stimulation with lipopolysaccharides than corresponding macrophages from op/op mice. In contrast, when peritoneally elicited macrophages were used, those from op/op mice revealed a stronger response than those from control mice with regard to release of NO or IL-12. These differences suggest that M-CSF-independent maturation of op/op monocytes subsequent to their release from hematopoietic tissue exerts influence on their effector functions. However, M-CSF or M-CSF-derived macrophages are not necessary for an effective immune response against L. major.

Key Words: Leishmania major • osteopetrosis • IL-12


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The macrophage lineage comprises a system of cells of large diversity with respect to their morphological, functional, and metabolic properties. This distinguishes these cells from other myeloid cells, which are relatively homogeneous, such as neutrophils. The large diversity of the macrophage lineage is formed under the influence of growth factors, such as macrophage-colony stimulating factor (M-CSF, also termed CSF-1), granulocyte-M-CSF (GM-CSF), and interleukin-3 (IL-3) [1 , 2 ].

M-CSF presents the primary regulator of mononuclear phagocyte production in vivo and plays an essential role in the survival, proliferation, differentiation, and maturation of the macrophage myeloid lineage [1 2 3 4 ]. Its effects are mediated by CSF-1R, a high-affinity receptor tyrosinase kinase, encoded by the c-fms-proto-oncogene [5 ]. CSF-R1 is expressed by progenitor cells of mononuclear phagocytes, monoblasts, promonocytes, monocytes, tissue macrophages, and osteoclasts [4 ] and only few nonphagocytic cells. Cells of the mononuclear phagocyte lineage beyond the stage of primitive hematopoietic cells form colonies in response to M-CSF alone, and additional growth factors appear to be necessary to stimulate primitive multipotent hematopoietic cells via CSF-1R and other receptors [5 ]. As such, IL-3 and GM-CSF, which promote development of a great range of cell lineages, were demonstrated to stimulate proliferation and differentiation of primitive and early macrophage progenitors [6 ]. They bind to type I cytokine receptors composed of {alpha} and ß-common chain (ßC) subunits in which a region of the {alpha} cytosolic domain is decisive for receptor specificity [7 ].

During macrophage maturation, differences in macrophage populations arise as a result of a different responsiveness toward M-CSF or GM-CSF [8 , 9 ]. By raising bone marrow-derived progenitors with M-CSF or GM-CSF, it was possible to demonstrate that these macrophage populations present with several overlapping but also distinct functions. In this context, macrophages grown in M-CSF but in the absence of GM-CSF are referred to as M-CSF-derived or GM-CSF-independent macrophages, and macrophages grown in GM-CSF but in the absence of M-CSF were designated GM-CSF-derived or M-CSF-independent macrophages [8 , 9 ]. In these studies, M-CSF-derived macrophages were larger, constitutively ingested more latex, phagocytosed higher numbers of Listeria monocytogenes when stimulated with interferon-{gamma} (IFN-{gamma}) and lipopolysaccharides (LPS), and by far, generated more nitric oxide (NO) when activated with LPS [8 , 9 ]. Conversely, GM-CSF-derived macrophages displayed higher cytotoxic activity against tumor necrosis factor {alpha}-resistant tumor targets [8 ].

The discovery of mice [10 ], which present a spontaneous point mutation of the M-CSF gene [Csfmop/Csfmop (op/op)] and therefore produce only nonfunctional M-CSF [11 ], has facilitated investigations about the function of this individual growth factor in vivo [1 ]. Studies have shown that the mutation results in a profound macrophage deficiency, which is generalized, but shows quantitative differences in various organs [12 , 13 ]. The deficiencies are similar, as observed in the recently generated CSF-1R-deficient mouse, but are less severe, as CSF-1R also appears to be required to mediate effects of other growth factors [5 ]. The residual M-CSF-independent macrophages in osteopetrosis mice (op/op mice) are small and round and show ultrastructural immaturity [12 , 14 ]. GM-CSF or IL-3 maintained their functional activity in vivo [14 15 16 ]. In vitro, macrophages from op/op mice were able to respond to M-CSF but better resembled their in vivo phenotype when raised under addition of GM-CSF. They are therefore referred to as M-CSF-independent, GM-CSF-derived macrophages [12 ].

Studies using the op/op mouse have provided the possibility to investigate distinct functions of the M-CSF-independent subtype of macrophages in vivo. They have revealed or confirmed that M-CSF-independent macrophages present defects with regard to phagocytosis, bactericidal activity, monokine release, ability to combat Escherichia coli-induced peritonitis, or recruitment of leukocytes to sites of infection. In contrast, the specific humoral immune response and parts of a delayed-type hypersensitivity (DTH) immune response appeared to be normal [13 , 15 , 17 18 19 ]. It has been hypothesized that the major role for M-CSF-independent, GM-CSF-derived macrophages in vivo is to interact with lymphocytes for mounting an immune response, and M-CSF-derived macrophages are major effector cells. [18 ].

Studies with the obligatorily intracellular parasite Leishmania major, which primarily invades macrophages, bear many advantages for studying the functions of macrophage subtypes [20 , 21 ]. It has been asserted that resistance to an infection with L. major is dependent on macrophages developed under the influence of GM-CSF, as the number of GM-CSF-secreting cells increased in the draining lymph nodes (LN) in resistant C57Bl/6 mice [22 ]. Also bone marrow macrophages derived under GM-CSF favored a T helper cell type 1 (Th1) response and were more responsive to IFN-{gamma} for leishmanicidal activity, whereas their impairment aggravated disease [22 ]. Yet, unexpectedly, mice unresponsive to GM-CSF (mice with a null mutation in the gene for the ßC of the receptors for GM-CSF, IL-3, and IL-5) were discovered to be resistant to infection with L. major [23 ].

We therefore wondered whether in contrast, the absence of M-CSF-dependent cells would impair resistance in leishmaniasis. The immune response of M-CSF-deficient op/op mice against infection with L. major provides an opportunity to elucidate the immunological functions of this monocyte and macrophage subtype at several levels. The events leading to resistance or susceptibility toward this infection are known to be associated with the expansion of Th1 cells or Th2 cells, respectively [21 , 24 ]. Infected macrophages, together with dendritic cells (DC), need to secrete IL-12 to enable and to sustain the development of Th1 cells [25 ]. The latter then secrete IFN-{gamma}, which is necessary to activate macrophages so that they produce leishmanicidal NO [26 , 27 ]. To initiate and to execute this complete immune response, M-CSF-independent macrophages in op/op mice therefore would have to fulfill or participate in all of the following critical steps: recruitment of sufficient numbers of monocytes and macrophages to the lesion; phagocytosis of the parasite; production of leishmanicidal NO upon activation; as well as secretion of IL-12 for supporting a L. major-specific Th1 response. Previous studies have indicated that op/op mice show deficits in some of these steps, and although T cells from op/op mice have been shown to proliferate in response to ovalbumin [17 ], the capability of op/op mice to generate a full Th1 or Th2 response is not known.

We therefore investigated the ability of op/op mice and their M-CSF-independent macrophages to combat infection with L. major. Our experiments reveal that mice deficient of M-CSF-dependent macrophages retain the ability to resist an infection with L. major. Infected op/op mice mount a Th1 response, eliminate parasites, and resolve the lesion. Macrophages from op/op mice were able to sufficiently perform effector functions in vitro, such as phagocytosis, production of leishmanicidal NO, and release of IL-12 upon activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
B6C3Fe-a/a-Cfsmop were obtained from the Jackson Laboratory (Bar Harbor, ME) and were bred at the Bomholtgard Breeding and Research Centre A/S (Denmark) using heterozygotic breeders as described before [10 ]. Offspring are genotypically op/op, +/+, or +/op mice, but +/+ mice as well as +/op mice are phenotypically normal and therefore commonly referred to as +/? mice without further genotyping [12 ]. Mutant op/op mice were phenotypically distinguished from normal siblings at 10–15 days of age by the absence of incisor eruption. Pups were weaned when they were 3–4 weeks old and were kept in separate cages from there on. The diet was composed of Nestle Powdered Baby Diet (fruit taste), mixed at equal parts with powdered standard Altromin 1314 mouse diet (Lage, Germany), and drinking water was acidified (pH=5) and additionally provided as gellified water to facilitate uptake. Mice not homozygous for Cfsmop (+/? mice) were used as control mice. They received mouse chow and acidified drinking water ad libitum. Bedding was changed twice a week using autoclaved Hahnflock bedding (Hahn and Co., Bredenbeck, Germany).

Parasites and experimental infection
L. major (MHOM/IL/81/FE/BNI) were cultivated as described elsewhere [20 ]. Soluble L. major antigen (LmAg) was prepared by repeated freeze and thaw cycles [28 ].

Reagents
The recombinant (r) mouse proteins IFN-{gamma}, IL-2, IL-4, and GM-CSF were purchased from R&D Systems (Wiesbaden, Germany). All other chemicals were of the highest grade available (Sigma, Taufkirchen, Germany).

Preparation of macrophages from hematopoietic organs (bone marrow and spleen)
So-called M-CSF- and GM-CSF-dependent macrophages were cultured in vitro from bone marrow and spleen progenitors in modification of a method described previously [9 ]. Briefly, mice were killed, and the femurs and spleen were removed, cleansed of tissue, and flushed with Hanks’ balanced salt solution (HBSS). Erythrocytes were depleted by osmotic shock, and cells were washed with HBSS, collected by centrifugation, and were cultured at 1 x 107 cells in 10 ml Dulbecco’s modified Eagle’s medium containing 2 mM glutamine, 0.1 mM nonessential amino acids, antibiotics, and 10% heat-inactivated fetal calf serum (FCS; Biochrom, Berlin, Germany). During the first 24 h, the cells were cultured in 25 ml flasks, and the nonadherent cells were then transferred to Teflon bags (Heraeus, Hanau, Germany) and were provided with an additional 10 ml medium supplemented with 10% L cell-conditioned medium containing M-CSF (M-CSF-derived macrophages) or supplemented with 250 U/ml rGM-CSF (R&D Systems; GM-CSF-derived macrophages). After 3 days, Teflon bags were put on ice to loosen adherent cells, which then were gently removed and collected by centrifugation. Cells were then counted and seeded into 24-well plates at 2 x 105 cells/well in 2 ml medium containing L cell-conditioned medium or GM-CSF-conditioned medium. After 2 additional days, cells had grown adherent and were subsequently used for stimulation experiments. For phagocytosis, assay cells were kept in Teflon bags for 4 days before incubation with L. major.

Preparation of macrophages from peritoneum
Three days after intraperitoneal injection of 1 ml thioglycollate (3% in aqua dest.; Gibco, Paisley, UK), cells were obtained by peritoneal lavage with cold phosphate-buffered saline (PBS) containing 5 mM EDTA, washed, and subjected to lysis of erythrocytes. Inflammatory monocytes or macrophages elicited from peritoneum are viable without addition of M-CSF or GM-CSF and thus reflect more closely the genuine cells within op/op or +/? mice. Only killing assays could not be performed with these cells, as their longer duration would have required addition of growth factors.

Staining of L. major and phagocytosis assay
L. major were labeled with the fluorescent dye 5- (and 6)-carboxyfluoresceindiacetat succinimidylester (Molecular Probes, Leiden, The Netherlands), as described previously [29 ]. Fluorescent-labeled L. major were added to cultured bone marrow-derived macrophages grown in the presence of M-CSF or GM-CSF as described above in a ratio of 4:1 for 2 h at 37°C. Fresh mouse serum was added to allow complement opsonization of parasites. Cell suspensions of six wells per group were prepared for fluorescein-activated cell sorter (FACS) by removing the majority of excess, unbound parasites with repeated washing at 200 g for 7 min. The fluorescence of macrophages was measured to quantify the percentage of infected cells. Dead cells, which stained with propidium iodide, were gated out at the time of analysis. Unbound L. major were excluded by size using the forward-scatter.

Killing rate of macrophages
As described before [29 ], macrophages (6 days of culture) were seeded into LabTek® culture chamber incubation slides (Miles Scientific, Naperville, IL) in a number of 5 x 104 per chamber. On the following day, the adherent macrophages were incubated with stationary-phase L. major (ratio 10:1) for 2 h at 37°C. Thereafter, cultures were washed to remove extracellular parasites. The initial infection rate was estimated by counting phagocytosed L. major. The antileishmanial activity was assessed by monitoring the percentage of viable macrophages bearing viable L. major 48 h after infection and activation with IFN-{gamma} (100 U/ml). Presented are the mean values for 100 macrophages from six to seven chambers of one representative of three experiments.

NO release
For determination of NO release, supernatants were collected from op/op and +/? macrophages coincubated with L. major and stimulated with IFN-{gamma} as well as from controls. NO release was measured as nitrite concentration in the medium by a microplate assay using a Griess reagent as described [27 , 30 ].

Experimental leishmaniasis
Cutaneous leishmaniasis was initiated by subcutaneous (s.c.) application of 2 x 107 promastigotes (stationary phase) of L. major in 20 µl PBS into the left hind footpad of 35 op/op and +/? mice. Footpad thickness was measured with a metric caliper. Footpads of six mice each were harvested 24 h, 72 h, and 2 weeks after infection; LN and spleens of five to seven mice each were obtained 6 and 12 weeks after infection.

Limiting dilution assay
Parasite numbers in lesions, LN, spleen, and bone marrow were determined 6 and 12 weeks after infection by a limiting dilution assay using a slant blood agar as described [31 ].

Antibodies and immunohistochemical staining
The following monoclonal antibodies (mAb) against mouse antigens were purchased from several commercial sources: F4/80 (Biozol, Eching, Germany), BM8 (BMA, Augst, Switzerland) [32 ], CD4 and CD8 (Becton Dickinson, Heidelberg, Germany), GR-1 (PharMingen, Hamburg, Germany), and 8E7 [33 ]. Immunohistochemical stainings were performed and evaluated as described elsewhere [20 ]. Percentages of positive cells were obtained by relating the number of all-present cells in a defined area (recognized by stained nuclei) to the number of positively stained cells (percentages) [20 ].

DTH
For assessment of the DTH responses, six infected op/op and +/? mice (5 weeks post-infection) were challenged by s.c. injection of soluble LmAg equivalent to 2 x 107 L. major into the contralateral foot. Control groups were also injected with LmAg but were not previously infected with parasites. DTH was determined after 3 h, 24 h, and 48 h via the degree of footpad swelling of the injected site compared with control mice.

Lymphocyte culture
Five weeks post-infection, the inguinal and popliteal LN of six mice each were collected and prepared as described [30 , 34 ]. LN cells were cultured in RPMI 1640 + 2 mM glutamine, 50 µM mercaptoethanol, and 10% FCS (1x106 LN cells/ml). After 48 h, LN cells were restimulated with soluble LmAg equivalent to 2 x 106 L. major. For control purposes, LN cells were also stimulated with mitogens phorbol 12-myristate 13-acetate (PMA; 3 ng/ml) and ionomycine (300 ng/ml). Culture supernatants were collected after 48 h and assayed for IL-2 using a bioassay with murine cytotoxic T cell lines (detection limit, <0.1 U/ml) and for IL-4 (detection limit, 0.2 U/ml) using a bioassay with the CTh4S cell line [34 ] and IFN-{gamma} (detection limit, <2.5 pg/ml) by enzyme-linked immunosorbent assay (ELISA) using paired mAb (PharMingen). A [3H]thymidine incorporation assay was performed to determine proliferation of cells.

Secretion of IL-12 by macrophages
M-CSF- and GM-CSF-derived macrophages, which have been cultured in 24-well plates as described above, as well as peritoneal macrophages, were primed with 100 U/ml IFN-{gamma} for 24 h. Macrophages were subsequently stimulated with LPS as positive control and with L. major and IFN-{gamma}. In the case of macrophages from hematopoietic organs, experiments were conducted with and without addition of fresh mouse serum containing complement as uptake via complement, and CR3 increases phagocytic efficiency but also inhibits release of IL-12 [29 ]. In the case of peritoneally elicited macrophages, assays were only performed with serum, as presence of serum cannot be excluded. After 24 h, the IL-12 amount present in the supernatant was determined by ELISA using paired mAb recognizing the p75 and p40 subunit (Laboserv, Giessen, Germany).

Statistics
The mean, SEM (data from individual samples), and SD (data obtained from replicates) of all numerical data were calculated. Statistical significance between groups was judged by the Student’s t-test. Comparison of data sets yielding P > 0.05 was considered not to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To investigate the effector functions of op/op macrophages and macrophages from phenotypically normal +/? mice (++ or +/op control mice), we analyzed phagocytosis, killing of L. major, as well as NO production. To imitate the in vivo conditions, we primarily compared GM-CSF-derived macrophages from op/op mice with M-CSF-derived macrophages form control mice. In addition, we performed most experiments with M-CSF-derived macrophages from op/op mice and GM-CSF-derived macrophages from control mice. As we found that peritoneal macrophages from both strains could be kept viable in vitro for 2–3 days without addition of M-CSF or GM-CSF, we additionally conducted corresponding experiments with peritoneal macrophages to see if they would yield similar results as those comparing M-CSF- and GM-CSF-derived macrophages.

Phagocytosis of L. major by macrophages from op/op and +/? mice
Fluorescent-labeled L. major were added in a fourfold excess to cultured macrophages for 2 h, and the amount of infected phagocytes was assessed by FACS. In the presence of fresh mouse serum, this resulted in infection of 56.5% (±5%, SD) of the GM-CSF-derived macrophages from op/op and of 77.0% (±7%, SD) of the M-CSF-derived macrophages from +/? mice (Table 1 ). We also investigated phagocytic activity of macrophages from op/op mice when cultured in M-CSF-containing medium. Although macrophages of op/op mice had been shown to be able to respond to M-CSF, their phagocytic activity (58.2%±4%, SD) was not significantly increased by 6 days of culture with M-CSF. With regard to macrophages from control mice, GM-CSF-derived macrophages revealed a lower phagocytosis rate when compared with corresponding M-CSF-derived macrophages. Thus, GM-CSF- and M-CSF-derived macrophages from op/op mice are capable of phagocytosing L. major, albeit at reduced rates compared with M-CSF-derived macrophages from control mice, thus both resembling GM-CSF-derived macrophages from controls.


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  		Table 1. Phagocytosis of L. major

 
When peritoneal cells were used instead of growth factor-treated, bone marrow-derived macrophages, cells from op/op mice presented an infection rate of 46.2% (±6.7%, SD; n=6), and control mice had a significantly higher infection rate of 66.0 (±5.9%, SD; n=7). Thus, peritoneal macrophages from op/op mice also revealed a lower rate of phagocytosis than macrophages from control mice.

Killing activity by macrophages from op/op and +/? mice
To investigate the potential of op/op macrophages to eliminate intracellular parasites, we determined infection rates of macrophages cultured on Labtek® culture chambers directly after phagocytosis and 48 h after activation with IFN-{gamma}. As GM-CSF-derived macrophages from op/op mice phagocytosed slightly less L. major, we incubated all macrophages with subordinate numbers of L. major so that as a result of the limited number of parasites, both macrophage species showed low and comparable infection rates. The coculture of L. major with macrophages in ratios 1:10 led to low infection rates of 23.8% (±3.2%) in the case of control macrophages raised with M-CSF and of 16.6% (±2.4%) in the case of op/op macrophages raised with GM-CSF (mean±SD; n=6–7). At 48 h after incubation in the presence of IFN-{gamma} (100 U/ml), infected GM-CSF-dependent macrophages from op/op mice had eliminated 52.0% of internalized L. major, whereas M-CSF-derived control macrophages had killed 54% of ingested L. major (mean±SD; n=6–7; Table 2 ). This indicates that after activation with IFN-{gamma}, GM-CSF-dependent macrophages from op/op mice present a similar killing rate as M-CSF-dependent macrophages from +/? mice. The killing rate was not significantly changed when macrophages from op/op mice were raised with M-CSF instead of GM-CSF. It was lower in GM-CSF-derived control macrophages, but this reduced killing rate did not reach statistical significance in all three experiments.


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  		Table 2. Killing Activity of Infected Macrophages

 
Release of NO by macrophages from op/op and +/? mice
As destruction of L. major by macrophages requires sufficient production of NO [26 , 27 ] and as M-CSF-dependent macrophages were reported to release more NO constitutively [8 ], we compared secretion of NO between GM-CSF-derived macrophages from op/op mice and M-CSF-derived macrophages from +/? mice. As presented in Figure 1 , NO release is higher by +/? macrophages after stimulation with LPS, but NO production in op/op macrophages in response to IFN-{gamma} and L. major is only slightly lower than in +/? macrophages. The higher release of NO by macrophages from +/? mice at a ratio of 4:10 could be a result of a higher infection rate of these macrophages. Control experiments showed that at ratios of 1:10, infection rates were similar, which then was associated with comparable levels of NO release (Fig. 1) . In one separate set of experiments, we used macrophages from op/op mice cultured in M-CSF; they showed slightly higher NO release than GM-CSF-derived macrophages from op/op mice, but this difference was not statistically significant (P<0.05; data not shown).



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  		Figure 1. NO release by macrophages from hematopoietic organs (bone marrow, spleen) in response to LPS, to L. major in two different effector/target ratios, and to L. major in the presence of IFN-{gamma} (100 U/ml) after 24 h. Different effector/target ratios were used to compensate for potential differences induced by higher infection rates. The higher release of NO by macrophages from +/? mice at a ratio of 4:10 can be explained by a higher infection rate; infection rates were similar at ratios of 1:10. Macrophages from op/op mice were raised with GM-CSF, macrophages from control mice raised with M-CSF (six wells, mean±SD, one of four experiments with similar results).

 
As previous studies on NO release have yielded different results, probably depending on the culture conditions and on stimuli of M-CSF- or GM-CSF-derived macrophages [6 , 8 ], we decided to repeat our NO assay with inflammatory monocytes/macrophages from op/op and +/? mice, which were elicited from peritoneum. For determination of NO (24 h), they survive long enough in medium without having to be supplemented with M-CSF or GM-CSF. Here, NO release after stimulation with LPS or L. major was significantly higher by peritoneal macrophages from op/op mice than by macrophages from +/? mice (Fig. 2 ).



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  		Figure 2. NO release by elicited macrophages from peritoneal cavity of op/op and +/? mice in response to LPS, L. major in two different effector/target ratios, and L. major in the presence of IFN-{gamma} (100 U/ml). This experiment shows that elicited macrophages from op/op mice exert full NO production. They are able to release even significantly more NO than macrophages from +/? mice (designated by stars), independent of the stimulus LPS or L. major and of the effector/target ratio (five to six wells, mean±SD, one of four experiments with similar results; P<0.05 for comparisons between macrophages from op/op and +/? mice after stimulation with LPS or L. major).

 
Thus, macrophages from op/op mice showed full capability to release leishmanicidal NO and even reveal higher activity in the case of freshly elicited peritoneal cells.

Course of experimental leishmaniasis in op/op and control mice
Infection was accomplished by injection of 2 x 107 stationary L. major into the hind footpad of op/op and +/? control mice as well as of susceptible BALB/c mice for comparison with susceptible mice. In op/op as well as +/? mice, an initial increase in footpad swelling was observed during the first 3 weeks post-infection (Fig. 3 ). Thereafter, the swelling decreased continuously in both groups of mice during the observed time span of 85 days. The lesions of all mice healed, and the mice generally appeared in good health. In contrast, BALB/c mice showed the known course of infection, which encompasses continuous swelling of the lesion and ulceration after 5 weeks.



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  		Figure 3. Footpad thickness of infected op/op, wild-type (WT), and BALB/c mice (mean±SEM, n=5).

 
Evaluation of the number of L. major in popliteal LN 6 and 12 weeks post-infection did not reveal significant differences between +/?- and op/op mice (Table 3 ). Both showed continuous elimination of the parasite. In spleens after 6 weeks, we detected L. major only in one out of six infected control mice and in none of five op/op mice; after 12 weeks, we did not detect L. major in any of the spleens. Bone marrow was free of parasites after 6 as well as after 12 weeks. In contrast, BALB/c mice, as expected, revealed increasing numbers of parasites in LN, spleen, and finally, bone marrow (data not shown).


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  		Table 3. L. major Content in Popliteal LN of +/? and op/op Mice

 
Cellular composition of the infiltrate
As op/op mice have marked monocytopenia and as L. major are obligatorily intracellular parasites, we looked at the development of the infiltrate. Serial sections of infected footpads were immunohistochemically stained for the antigens F4/80 (mature macrophages), GR-1 (granulocytes), and CD4 and CD8. The composition of the infiltrate in op/op mice 24 h post-infection differed from that in +/? mice with regard to macrophages (Table 4 ). While granulocytes prevailed in lesions of op/op and op/? mice, there were some F4/80-positive macrophages in +/? mice but almost none in op/op mice. After 72 h and 3 weeks of infection, however, the percentage of macrophages in the infiltrate of op/op mice largely matched with that of +/? mice.


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  		Table 4. Composition of Infiltrates of op/op and WT Mice, 24 h and 72 h Post-Infection

 
Lymphokine profile of op/op mice infected with L. major
To find out whether a Th1 or Th2 cell response prevails in infected op/op mice, popliteal and inguinal LN were collected 5 weeks post-infection. LN cells were restimulated with soluble LmAg, and the supernatant was analyzed for IL-2, IL-4, and IFN-{gamma}. Mitogen stimulation using PMA and ionomycine was performed to prove general responsiveness of LN cells. op/op as well as +/? mice showed cytokine patterns typical for a Th1 response (Fig. 4 ). In op/op and +/? mice, the amount of IL-4 was below the detection limit of 0.2 U/ml, whereas they both produced considerable amounts of IFN-{gamma}. These results suggest that op/op and +/? mice mount a Th1 response. For control purposes, BALB/c mice were investigated simultaneously. They showed the typical pattern for a Th2 response in terms of low IFN-{gamma} but high IL-4 production. We also found that LN cells from BALB/c mice produced considerably more IL-2, which was in line with the proliferation of Th2 cells. The proliferation of LN cells was examined by a tritium incorporation assay following restimulation with LmAg. Proliferation of LN cells from op/op and +/? mice was not significantly different (125,419.7±23,579 degradations per minute (DPM) and 127,144±23,579 DPM, respectively).



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  		Figure 4. Cytokine pattern of inguinal and popliteal LN 6 weeks post-infection. LN cells were restimulated with LmAg (hatched bars) or mitogen (open bars) for 48 h, and cytokine content in supernatant was determined (mean±SD, quadriplets, n=4).

 
DTH response to L. major
We investigated the DTH reactivity of op/op and +/? mice 5 weeks after infection by injecting LmAg s.c. into the contralateral foot. The increase of footpad thickness as compared with a group of nonimmunized mice injected with antigen is presented in Table 5 . Control (+/?) mice showed a vigorous reaction already 3 h after antigen administration, reaching its peak after 24 h and declining at 48 h. The DTH reaction in op/op mice developed more slowly, continuously rising over the observed period of 48 h.


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  		Table 5. DTH Reactivity of op/op and +/? Mice as Established by Increase of Footpad Thickness after s.c. Injection of LmAg (n=5, SEM)

 
IL-12 production by macrophages from op/op and +/? mice
As IL-12 is one of the decisive cytokines in priming T cells toward a Th1 response, we investigated the ability of macrophages from op/op mice to produce IL-12 in response to infection with L. major. For primary comparison, we used M-CSF-independent (GM-CSF-derived) macrophages from hematopoietic organs of op/op mice and macrophages from +/? mice cultured with M-CSF. Macrophages were primed with 100 U/ml rIFN-{gamma} before the addition of L. major, as this is known to be necessary for substantial production of IL-12 by macrophages [36 ]. IL-12 release revealed no significant difference between M-CSF-derived macrophages from +/? mice and GM-CSF-raised macrophages from op/op mice in response to stimulation with L. major (Fig. 5 ). This applied for release after uptake of nonopsonized L. major and inhibited release after uptake of opsonized parasites via CR3, which is known to down-regulate IL-12 production [29 ]. Thus, both regulatory pathways function well in M-CSF-independent (GM-CSF-derived) macrophages from hematopoietic organs of op/op mice. Incubation of macrophages from op/op mice in M-CSF instead of GM-CSF neither resulted in significantly increased release of IL-12 in comparison with GM-CSF-derived macrophages from op/op mice nor with M-CSF-derived macrophages from +/? mice (data not shown).



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  		Figure 5. IL-12 secretion by macrophages from hematopoietic organs (bone marrow, spleen) primed with 100 U/ml IFN-{gamma} for 24 h and then challenged for another 24 h in the presence IFN-{gamma} with LPS (20 ng/ml) and L. major (L.m.; ratio 1:5), LPS, L. major only, or IFN-{gamma} only (control). The ratio of 1:5 yielded similar infection rates of macrophages. Macrophages from op/op mice were raised with GM-CSF; macrophages from +/? mice were raised with M-CSF (quadriplets; mean±SD, one of three experiments with similar results). Comparisons between macrophages from op/op mice and +/? mice did not yield statistically significant differences (P<0.05). NMS, normal mouse serum.

 
When inflammatory monocytes/macrophages from peritoneum were compared, those from op/op mice showed a moderately higher production of IL-12 in response to LPS (Fig. 6 ), which was more accentuated after addition of L. major. Thus, compared with +/? mice, macrophages from op/op mice show unimpaired release of IL-12, independent from their culture conditions; similarly as observed with NO release, they even show higher production when freshly elicited from peritoneum.



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  		Figure 6. IL-12 secretion by elicited macrophages from peritoneum from op/op and +/? mice, primed with 100 U/ml IFN-{gamma} for 24 h and then challenged for another 24 h in the presence of: IFN-{gamma} with LPS (20 ng/ml) and L. major (ratio 1:5), LPS, L. major only, or IFN-{gamma} only (control). These experiments were all done in the presence of mouse serum. After uptake of parasites, activated cells from op/op mice released significantly more IL-12 (signified by star; quadriplets; mean±SD, one of three experiments with similar results; P<0.05 for comparisons of macrophages between op/op and +/? mice).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study shows that op/op mice are able to fulfill all the requirements to mount a protective immune response toward infection with L. major despite their lack of M-CSF and of M-CSF-derived macrophages. As such, in vitro, their M-CSF-independent macrophages phagocytosed parasites and were capable of eliminating them, and they secreted sufficient NO and IL-12 after uptake of L. major. In vivo, they revealed recruitment of sufficient numbers of macrophages to the lesion within 3 days, and their M-CSF-independent phagocytes were able to mount a L. major-specific Th1 response. Thus, M-CSF and M-CSF-dependent phagocytes are not necessary to generate a Th1 response and to generate efficient effector cells in experimental leishmaniasis.

The diversity of the monocytic lineage has stipulated attempts to assign discrete functions to defined subgroups of these cells. These subgroups can partially be defined by use of surface markers, but these markers have not yet succeeded to separate all subpopulations [32 , 37 ]. The fact that monocytes/macrophages originate from bone marrow progenitors under the influence of primary macrophage growth factors such as M-CSF, GM-CSF, and IL-3 has led to alternative approaches. A number of comparative in vitro studies with bone marrow-derived macrophages grown under the influence of M-CSF or GM-CSF have revealed some distinct functions among these subtypes [8 , 9 , 38 ]. The discovery of op/op mice with a spontaneous mutation leading to the complete absence of functional M-CSF has made possible the comparative investigation of M-CSF-derived and -independent cells in vivo [14 , 15 ]. To imitate the in vivo conditions in vitro, the most appropriate comparison would be between op/op macrophages raised in the absence of M-CSF but in the presence of GM-CSF and macrophages from control (+/?) mice raised in the presence of M-CSF [2 , 18 , 19 ]. Previous comparisons between M-CSF-dependent macrophages and GM-CSF-dependent macrophages from hematopoietic tissue (bone marrow or spleen progenitors) have yielded different results for some functions, which probably were partially a result of variations in culture conditions or in activating stimuli for macrophages [2 ]. Therefore, we included experiments in which macrophages from hematopoietic organs of op/op and control mice were treated with M-CSF and GM-CSF, respectively. In addition, we investigated phagocytosis, as well as NO and IL-12 release of peritoneally elicited macrophages from op/op and +/? mice, as these inflammatory monocytes or macrophages need neither M-CSF nor GM-CSF for the duration of the assay, and they resemble more closely the type of monocyte or macrophage encountered in inflamed tissue.

In summary, we found that macrophages from op/op mice were able to sufficiently perform effector functions in vitro, such as phagocytosis and production of leishmanicidal NO, and that production of NO by their macrophages from hematopoietic organs was consistent with their killing rate of parasites. In addition, their release of IL-12 upon activation was similar to that of control macrophages and therefore associated with induction of a specific Th1 response. This applied for IL-12 release after uptake of nonopsonized L. major and inhibited release after uptake of opsonized parasites via CR3, which is known to down-regulate IL-12 production [29 ]. Thus, both regulatory pathways function well in M-CSF-independent (GM-CSF-derived) macrophages from hematopoietic organs of op/op mice. There were quantitative differences in the degree of some responses. M-CSF-derived macrophages from control mice showed significantly higher rates of phagocytosis than the other subpopulations and higher NO release after stimulation with LPS. This may indicate that these functions are to some extent dependent on M-CSF. The fact that NO release did not differ markedly in response to L. major suggests that its production is additionally dependent on the number of phagocytosed parasites, as we had kept the phagocytosis rate similar in our experiments for better comparison.

It is interesting that comparisons between peritoneal macrophages of both strains revealed a stronger response by activated op/op macrophages than by activated +/? macrophages with regard to release of NO and IL-12. These differences between hematopoietic macrophages and inflammatory macrophages infiltrating the peritoneum indicate that after monocytes are released from hematopoietic organs, their differentiation is influenced by the growth factor or cytokine milieu of tissue or blood; this may be different in op/op mice as a result of the absence of M-CSF.

Although op/op mice were shown to generate a DTH immune response [13 , 17 ], this is the first study to directly demonstrate that they are capable of elaborating a L. major-specific Th1 cell response. Besides the ability of op/op macrophages to release IL-12, such an immune response largely depends on the action of DC. Previous studies found normal development of Langerhans cells in op/op mice except for minor deviations in their ultrastructure [39 , 40 ]. Although functional in vitro studies on DC from op/op mice remain to be done, our work and other studies [13 , 17 ] indicate that DC have normal functions in op/op mice. Although GM-CSF is a decisive growth factor to induce differentiation of cells from the macrophage-phagocyte system into DC, the GM-CSF-dependent cells analyzed in this study were macrophages according to culture conditions, adherence, and morphology.

As resistance was not disturbed in op/op mice, it is remarkable that mice rendered unresponsive to GM-CSF also appear to remain resistant to L. major [23 ]. These mice have a null mutation for the ßC of the receptors for GM-CSF, IL-3, and IL-5 [23 ]. Peritoneal macrophages from these mice and from WT mice showed no significant difference in phagocytosis within 6 h, but similar to our data, there was lower NO release by WT cells when compared with cells from mice with dysfunctional GM-CSF. This may indicate that in the absence of one of the growth factors, macrophages display a more activated phenotype. In contrast to our study, they did not report or investigate IL-12 release form macrophages nor the type of T cell response generated after infection. The authors were speculating that a lack of local lesion development observed in mice unresponsive to GM-CSF may be a result of deficits in granuloma formation [23 ] so that they would be independent from the presence of a Th1 or Th2 response.

In our study, we investigated lesion development in op/op mice in light of the marked monocytopenia [41 , 42 ] and the known reduction of monocyte-derived macrophage populations in various organs and tissues [12 ]. The number of resident macrophages in skin of op/op mice was reported to be normal [41 ]. However, at an early stage, 24 h post-infection with L. major, the percentage of macrophages in the infiltrate was lower, and lesion size or swelling of infected footpads did not differ markedly compared with +/? mice. Yet, 72 h after infection, the percentage of macrophages in lesions of op/op and +/? was not significantly different any more. Similarly, the initially retarded but then rapid formation of the DTH response in L. major-infected mice may also be related to a sudden rise of monocytic cells in the infiltrate.

In conclusion, op/op mice are able to compensate a lack of M-CSF when mounting an effective and complete immune response against L. major. Therefore M-CSF and M-CSF-dependent monocytes or macrophages are not necessary for combatting infection with L. major. Functions of M-CSF- and GM-CSF-derived macrophages in experimental leishmaniasis and probably other infections or inflammations apparently overlap widely enough so that they can mutually cover against an invading microorganism for diverse tasks in the immune defense. To find out that M-CSF may indeed be optional, despite the major role that macrophages play in infection with L. major, also bears relevance for trials that investigate addition of GM-CSF and other growth factors to vaccines against L. major.


   ACKNOWLEDGEMENTS
 
This work was supported by grants to C. Sunderkötter from the German Research Association (SFB293/A8) and the Interdisciplinary Center of Clinical Research in conjunction with the German Ministry of Research (IZKF-C2, D15). F. S. and C. Schlesiger contributed equally to this study. We thank Claus Christensen (Bomholtgard Breeding and Research Centre A/S) for breeding the op/op mice. We also thank Mrs. S. Merfeld, Mrs. E. Nattkemper, and Mrs. M. Steinert for their skillful technical assistance.

Received December 5, 2001; revised December 1, 2002; accepted January 21, 2003.


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