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The Journal of Immunology, 1999, 162: 3961-3969.
Copyright © 1999 by The American Association of Immunologists

Modulation of Monocytic Cell Activity and Virus Susceptibility During Differentiation into Macrophages1

Sameh Basta*, Sonja M. Knoetig*, Martha Spagnuolo-Weaver{dagger}, Gordon Allan{dagger} and Kenneth C. McCullough2,*

* Institute of Virology and Immunoprophylaxis, Mittelhausern, Switzerland; and {dagger} Department of Agriculture for Northern Ireland, Veterinary Sciences Division, Belfast, U.K.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A major component of innate immune responses relies on monocytes and macrophages, virus infection of which will pose a particular problem for immunological defense. Consequently, the monocytic cell differentiation pathway was analyzed in terms of cellular modulations therein and their relation to monocytotropic virus infection. Differentiation was characterized by down-regulation of CD14, MHC Ags, the monocytic SWC1 marker, and p53; concomitant up-regulation of the SWC9 macrophage marker, a putative porcine CD80 (detected with anti-human CD80 Ab), and acid phosphatase secretion were also characteristic. Elevated phagocytic and endocytic activities as well as endosomal/lysosomal acidification were identified as being important to the macrophage. In contrast, monocytes possessed high accessory activity. This was multifactorial, concomitantly requiring 1) high MHC Ag expression; 2) enzyme activity of esterase, peroxidase, myeloperoxidase, and 5' nucleotidase in preference to glucosidase, galactosidase, and glucuronidase; and 3) elevated capacity for spontaneous IL-1 production. Only with all parameters was efficient stimulation of Ag-specific lymphocytes possible. These results point to a continuous process during differentiation, involving inter-related characteristics linking the more accessory monocyte to the scavenger macrophage, both in vitro and in vivo. Of particular interest was how these characteristics related to monocytotropic virus infection, and how a particular virus could show a clear preference for the differentiating macrophages. Such results not only further our understanding of porcine immunology, but also provide evidence and a potential model for the determination and characterization of monocytotropic virus-host cell interactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages (Mø)3 belong to the mononuclear leukocyte system, differentiating from the monocytes therein, which had arisen from hemopoietic precursor myeloid cells in the bone marrow (1). Monocytes undergo maturation into M{phi} upon migration from the microvasculature into tissues, where they are exposed to specific signals that determine their functional and phenotypic heterogeneity (2, 3, 4). In vitro, monocyte maturation can be induced by serum factors and followed by the expression of specific maturation-associated Ags (5, 6, 7). Limited analyses have attempted to relate these differentiation steps to in vivo events (6, 7). Furthermore, the occurrence of new Ags during differentiation as well as the loss of those typically expressed by monocytes have been exploited to evaluate M{phi} diversity at the membrane level (5, 7, 8, 9, 10, 11).

Increased interest in porcine immunology in relation to xenotransplantation and an animal model for human research (12, 13, 14) requires greater understanding of the ontogeny of the porcine immune system. Such knowledge would also promote increased appreciation of host-pathogen interactions with monocytotropic virus infections. As with human cells, phenotypic differences between porcine monocytes and M{phi} have been observed (7, 15, 16, 17). Of particular interest were the down-regulation of CD14 and the appearance of the M{phi}-specific SWC9 marker (7, 16, 17). Enzymes specific for particular leukocyte subpopulations have also been used to quantify macrophage function (18, 19, 20). Nonspecific esterases are common positive markers for monocytes (18), while alterations in enzymes such as galactosidase have been related to the M{phi} maturation state (19). Reports on the relationship of cellular activity to phenotypic characteristics of myeloid cells are less in evidence (20). Furthermore, many of the studies to date have employed myeloid cell lines.

Consequently, the present work set out to analyze the functional changes occurring as blood monocytes differentiate into M{phi}. For this purpose, enzymatic activity, Ag expression, phagocytic and endocytic capacities, accessory functions, and the intracellular acidic environment of differentiating cells were concomitantly analyzed and compared. These comparative analyses are considered particularly important for studies on monocytic cells, the cellular activity and function of which are compromised by monocytotropic virus infection. Comparing the susceptibility to virus infection with the functional activity of the target monocytic cell should enhance our understanding of such potentially disastrous immunocompromising infections.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and cells

Monocytes were isolated over Ficoll-Paque (Pharmacia, Piscataway, NJ) from venous blood collected into Alsever’s solution from Swiss White Landrace pigs held under specific pathogen-free conditions as described previously (7). The cells (PBMC) were resuspended in DMEM/10% (v/v) FCS at 2–4 x 106 cells/ml, and nonadherent cells were removed following culture for 2–3 h at 37°C. Adherent cells were further incubated at 37°C in DMEM/30% (v/v) heparinized porcine plasma, 2 mM L-glutamine, and 10 mM HEPES (Life Technologies, Grand Island, NY). Following such culture for the different periods of time shown in Results, the adherent cells were recovered with the aid of 4°C PBSA/5 mM EDTA treatment. Cells cultured for up to 2 days were referred to as intermediate differentiating cells, while those that had been cultured for >=3 days were referred to as monocyte-derived M{phi} (MDMs), due to the phenotypic and functional characteristics subsequently elucidated.

Lung lavage macrophages (Alv-M{phi}) were isolated by repeated lavage with 4°C PBSA-0.03% w/v EDTA of lungs freshly obtained from slaughtered pigs. The cells in the lavages were washed three times with PBSA-EDTA and frozen in liquid nitrogen. When needed, they were thawed and cultured in DMEM/10% (v/v) FCS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin for 24 h.

Determination of surface Ag expression

These analyses employed the FACScan (Becton Dickinson, Mountain View, CA) and the following mAb: anti-SWC1 (76-6-7) found on porcine monocytes (21), anti-SWC3 (DH59) porcine pan-myeloid cell marker (22), anti-SWC9 (PM18-7, provided by Dr. Y. Kim, Finch University of Health Sciences, Chicago, IL) porcine macrophage marker (7, 16, 17), anti-swine MHC class I (MHCI; PT85A) (22), anti-swine MHC class II DQ (MHCII; TH16B) (22), anti-human CD14 (My4) (23), and anti-human CD80 (BB1) (24). DH59, PT85A, and TH16B were obtained from VRMD (Pullman, WA), My4 was purchased from Coulter-Clone (IG, Zurich, Switzerland), and BB1 was obtained from PharMingen (San Diego, CA). The other mAbs were prepared from the respective hybridomas.

For labeling, cells first received the mAb for 20 min at 4°C, then the appropriate isotype-specific conjugates (FITC-, phycoerythrin-, or biotin-conjugated goat F(ab')2 anti-mouse Igs; Southern Biotechnology Associates, Birmingham, AL) for 15 min at 4°C, followed by streptavidin-spectral red for 15 min at 4°C. Acquisition of the data employed gated monocytic cells; either a myeloid region was created in the forward/side scatter by gating SWC3+ cells, or data were acquired on CD14+ cells labeled in FL-3, yielding >98% myeloid cells.

Detection of extracellular enzymic activity

Tartrate-resistant acid phosphatase was measured using the Sigma Diagnostics kit (Poole, U.K.) on monocytes cultured for 0, 1, 2, or 3 days (day 0 corresponds to freshly isolated monocytes after adherence). Medium was replaced every 24 h, and the cells were cultured for an additional 24 h to detect de novo enzyme synthesis. Samples (1 ml) of culture media were added to the kit reagents, and absorbance at 405 nm was measured (Biochrom Ultrospec spectrophotometer, LKB, Rockville, MD). Absorbance values were converted into acid phosphatase activity (units per liter), and medium alone values were subtracted.

Phagocytosis and endocytosis assays

BODIPY-fluorescein-Zymosan (Molecular Probes, Leiden, Holland; 50 particles/cell; 30 min at 37°C) was used to measure phagocytosis by flow cytometry. Trypan blue (0.04%, v/v) was added to quench the signal from zymosan adsorbed on the cell surface but not yet internalized. Endocytic activity was analyzed with 20 µg/ml low density lipoprotein (BODIPY-fluorescein LDL complex, L-3483 Molecular Probes) for 30 min at 37°C as previously described (25).

pH analysis

Cl-NERF-conjugated dextran (D-3324, Molecular Probes) was employed for analysis of the pH within the relatively acidic endosomal/lysosomal compartments. Monocytic cells were harvested and incubated with the probe (0.25 mg/ml) for 5 min at 37°C, followed by washing. The data were acquired in three stages: stage I, before washing; stage II, immediately after washing; and stage III after washing plus further mAb labeling (anti-SWC9 and CD14) for a total of 40 min at 4°C.

Detection of intracellular enzyme activity and particular intracellular Ags

The following substrates (Molecular Probes) were employed to detect enzyme activity by flow cytometry: 5-(and-6)-carboxy 2,7-dichlorofluoresceinacetate (3 µM) for esterase, fluorescein di-ß-galactopyranoside (1 mM) for galactosidase, fluorescein-di-ß-glucopyranoside for glucosidase (1 mM), fluorescein di-ß-glucuronide for glucuronidase (1 mM), dihydrorhodamine-6G for peroxidase (25 µM), and the 5' nucleotidase kit (BODIPY FL-AMP). Probes were added to cells at 4°C immediately before acquisition with FACS to serve as negative controls of incorporation. The reactions were conducted following the manufacturer’s instructions and were stopped with cold CellWash (Becton Dickinson), followed by two washing cycles.

The tumor suppressor gene product p53 and the myeloperoxidase protein were the intracellular Ags studied. The FITC-conjugated anti-human p53 protein mAb (26) DO-7 and the FITC-conjugated anti-human myeloperoxidase mAb (27) MPO-7 (Dako, Copenhagen, Denmark) were incubated for 20 min at 4°C with cells permeabilized using a commercial kit (Harlan Sera-Lab, Loughborough, U.K.), according to the manufacturer’s instructions.

Accessory function analysis

Accessory cell-dependent stimulation was analyzed in a foot and mouth disease virus (FMDV) Ag-specific lymphoproliferation assay. PBMCs from a vaccinated animal were prepared, and lymphocytes (the nonadherent cells) were removed after 2 h adherence. The adherent cells were extensively washed and cultured for 4 days to differentiate into MDMs. Fresh PBMCs were obtained from the same animal to prepare fresh monocytes and fresh lymphocytes, separated by five successive 1-h adherence regimens (>96% pure by FACS analysis). Monocytes or MDMs were seeded in 96-well round-bottom microtiter plates (Nunc, Naperville, IL) at 5 x 104/well, to which lymphocytes were added at 2 x 105/well. A control culture employed unseparated PBMCs, seeded at 2.5 x 105/well. Following 7-day culture at 37°C with either FMDV (1 x 105 TCID50/well) or mock Ag (uninfected cell lysate), the wells were pulsed with 0.5 µCi/well of [methyl-3H]thymidine for 18 h at 37°C. The cells were collected onto glass fiber filters (Inotech Cell Harvester, Wohlen, Switzerland), and radioactivity incorporation was measured with a Trace 96 counter (Inotech).

Detection of IL-1 production and release

The ability of porcine monocytic cells to generate IL-1 during differentiation was determined by analysis of mRNA and protein production. Fresh monocytes were cultured in DMEM/10% (v/v) FCS without mitogen (control) or supplemented with LPS (10 µg/ml) or PMA (10 ng/ml). Additional cultures were incubated in DMEM/30% (v/v) plasma for 1, 2, or 3 days to permit M{phi} differentiation before replacing the medium with DMEM/10% (v/v) FCS with or without LPS and PMA. At 24 h after treatment supernatants were analyzed for IL-1 protein, and the cells were pelleted, frozen, and subsequently tested by RT-PCR for IL-1 mRNA.

Bioactive IL-1 protein was assayed using the IL-1-sensitive A375 melanoma cell line (American Type Culture Collection, Manassas, VA) (28). Briefly, 5 x 103 cells/well of a 96-well microtiter plate (Costar, Cambridge, MA) were cultured for 24 h, when test samples or standards of recombinant human IL-1ß (Boehringer Mannheim, Mannheim, Germany) were added. After a further 72-h incubation the cells were fixed and stained with 0.05% (w/v) crystal violet, and the color was dissolved with acid ethanol and read at 590 nm.

RNA was pelleted from 106 cells using a commercial kit (Trizol, Life Technologies, Paisley, Scotland), resuspended in 10 µl of diethylpyrocarbonate-treated water, and stored at -80°C. A single-tube RT-PCR reaction using a commercial kit (Titan RT-PCR system, Boehringer Mannheim) was performed in duplicate with 4 µl of each RNA template in a 25-µl reaction. The reaction mixture contained 0.4 µM sense and antisense primers from GenBank porcine sequences for IL-1{alpha} (5'-ACA GAA GTG AAG ATG GCC AAA GTC-3' and 5'-TCA TGT TGC TCT GGA AGC TGT ATG-3') and actin (5'-ACA TCA AGG AGA AGC TCT GCT ACG-3' and 5'-GAG GGG CGA TGA TCT TGA TCT TCA-3'). To the reaction mix, the following were added; 200 µM dNTP mix, 5 mM DTT solution, 1.5 mM MgCl2, and 1 µl of an Expand TH High Fidelity enzyme blend (avian myeloblastosis virus reverse transcriptase, Taq DNA polymerase, and Pwo DNA polymerase). All reactions were effected in a Cyclogene Thermocycler (Techne, Cambridge, U.K.). RT was performed at 50°C for 30 min. The resulting cDNA was amplified using the following cycles: initial denaturation at 94°C for 45 s, 35 cycles of 30 s at 94°C for denaturation, 30 s at 55°C for annealing, and 2 min at 68°C for elongation. PCR products (10 µl) were electrophoresed at 80 V for 45 min on 1% (w/v) agarose gel in Tris-acetate/EDTA buffer, pH 8.7, containing 10 µg/ml ethidium bromide. A 123-bp DNA standard (Sigma, St. Louis, MO) was included in all gels, and the amplified fragments of the predicted size (385 bp for IL-1 and 366 bp for actin) were photographed.

Detection of susceptibility to monocytotropic virus infection

The KWH isolate of the porcine monocytotropic African swine fever (ASF) virus (provided by Dr. P. Wilkinson, BBSRC Institute for Animal Health, Pirbright, U.K.) was used. This virus had been passaged once in pigs and twice in cultured swine MDMs. Swine monocytes, day 2 monocytic cells, MDMs, or Alv-M{phi} cultures were infected with ASF virus at an m.o.i. of 10 infectious focus-forming units/cell. After adsorption for 2 h at 37°C, the inoculum was removed, and the cells were washed five to seven times with PBS containing 1% (v/v) FCS. The cultures were fed with DMEM/30% (v/v) plasma.

Virus Ag in infected cells was detected using the immunofluorescence assay or flow cytometry. Infected cells were taken at 20–24 h after infection, washed twice with PBS, fixed/permeabilized with ethanol for 10 min at -20°C, washed three times with PBS, and stained with mAbs against virus VP73 (provided by Dr. Fernando Alonso, CISA-INIA, Valdeolmos-Madrid, Spain) plus FITC-isotype-specific conjugate (Southern Biotechnology).

For determination of the number of cells productively infected, virus was adsorbed to the monocytes or MDMs for 2 h at 4°C, washed seven times with prewarmed PBS/1% (v/v) FCS, and then incubated for 2 h at 37°C to allow penetration of the cells by the adsorbed virus. The cells were then counted and titrated (0.01–1000 cells/well) on preformed 3- to 5-day-old MDMs (indicator cells) in 96-well microtiter plates. At 5 days postinfection, the final end point titer was determined by immunofluorescence assay for the presence of virus VP73 in the indicator cells. From such infectious center assays it was possible to calculate the percentage of cells in the monocyte or MDM culture that had been infected.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Surface Ag expressions identifying monocyte differentiation

These determinations employed freshly isolated blood monocytes as well as differentiating monocytic cells, intermediate (up to 2 days in culture) and MDMs (at least 3 days old), cultured in medium containing porcine plasma. Freshly isolated monocytes were typically high for SWC1, CD14, MHCI, and MHCII (DQ) expression, but were negative for SWC9 (Fig. 1Go, y1-axis). Monocytes were also negative when stained with the anti-human CD80 Ab. For those markers expressed on monocytes, all cells were seen to be positive (Fig. 1Go, y2-axis). In terms of surface Ag expression, only a single positive homogeneous monocytic population was detected, except for the MHCII (data not shown).



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  		FIGURE 1. FACS analysis of cell surface markers on differentiating monocytes. Adherent cells from PBMC were harvested after 0, 2, and 4 days of culture in DMEM containing 30% (v/v) plasma and were labeled with the mAb against SWC1 (a), SWC9 (b), MHCI (c), MHCII (d), CD14 (e), and CD80 (f). The relative intensity of CD marker expression on cultured monocytic cells (y1-axis; open histograms) is shown as the mean ± SEM from triplicate experiments and compared with that of Alv-M{phi} (y1-axis; filled histogram). Values for a relative intensity between 1–10 on the y1-axis correspond to the negative Ab control reaction. The percentage of positive cells (means only: SEM were <5%) for a particular marker are displayed on the y2-axis, as open and filled circles for monocytic cells and Alv-M{phi}, respectively.

 
During the first 48 h of culture, the profile of the above markers notably changed. A rapid down-regulation of SWC1 (Fig. 1Goa) concomitant with the appearance of SWC9 (Fig. 1Gob) was clear. Down-regulation was also noted with MHCI, MHCII, and CD14, but was comparatively slower than that of SWC1 (Fig. 1Goe). SWC9 was not the only molecule to be up-regulated; reactivity of the anti-human CD80 Ab was also now detected (Fig. 1Gof). No m.w. analyses have been performed on the porcine molecule detected with this anti-CD80 Ab. Consequently, it can only be referred to as a putative porcine CD80. The up-regulation of this molecule was not as rapid as that of SWC9 in terms of both the intensity of expression and the percentage of cells identifiable as positive (Fig. 1Go, b and f).

Although MHCI was still expressed on all cells by day 4 (Fig. 1Goc), the down-regulation of other markers rendered a certain population of the cells apparently negative; 20% of the cells now appeared CD14- (Fig. 1Goe), 30% were MHCII-DQ- (Fig. 1God), and 65% were SWC1- (Fig. 1Goa). In contrast, >95% of the cells on day 4 expressed the SWC9 macrophage marker (Fig. 1Gob); 40% were now positive for the putative porcine CD80 (Fig. 1Gof).

The expression of the above surface Ags on Alv-M{phi} was, in general, most similar to that expressed on monocytic cells cultured for 4 days (Fig. 1Go, a-f, Alv-M{phi}). There were two exceptions; MHCII expression was high on Alv-M{phi}, higher than even that on blood monocytes (Fig. 1God), and the putative CD80 levels were more akin to those found on intermediate differentiating monocytic cells after 48 h in culture (Fig. 1Gof).

Tartrate-resistant acid phosphatase in monocytic cell cultures

The differentiation of porcine monocytes into M{phi}, characterized phenotypically as shown in Fig. 1Go, was also noted in terms of morphological changes in the cells. Increased forward and side scatter, determined through flow cytometry, concomitant with increased size and process formation, observed microscopically, were typical of MDMs (data not shown). Both the phenotypic and morphological changes were associated with a steady increase in the activity of the secreted M{phi} marker, tartrate-resistant acid phosphatase (Fig. 2Go). This was virtually absent when freshly isolated monocytes were employed, tested after 24 h of incubation (Fig. 2Go, time zero). Up-regulation of the activity was observed as the cells differentiated into M{phi} with time in culture. If the medium was unchanged during the 3-day culture period, the activity was nearly twice that of 3-day-cultured cells that had received a medium change 24 h earlier. This confirmed a continual secretion of the enzyme into the extracellular environment.



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  		FIGURE 2. Effect of time in culture on the activity of tartrate-resistant acid phosphatase in differentiating monocytes. Samples (1 ml) from adherent cell cultures were assayed for the secreted enzyme at 0, 1, 2, and 3 days after an additional 24 h of culture. The medium was replaced every 24 h, except on one occasion (asterisk) when it was left unchanged to show cumulative enzyme production. The data shown are minus the values for control medium and are expressed as the mean ± SEM from triplicate experiments.

 
Phagocytosis and endocytosis capacity of monocytes/macrophages

Considering these characteristic modulations as monocytes differentiated toward M{phi}, it was of interest to determine how cellular functional activities important to monocytes/M{phi} were being modified. The ability of the monocytes/macrophages to phagocytose unopsonized BODIPY- fluorescein-labeled zymosan was employed to measure the phagocytic capacity of differentiating cells (Fig. 3Goa). Almost 60% of fresh monocytes had a phagocytic capacity (Fig. 3Goa, line graph, y2-axis). Nevertheless, these cells could only phagocytose relatively few zymosan particles compared with the cells at later time points of culturing (Fig. 3Goa, histogram, y1-axis). In fact, as the differentiation advanced with time in culture, there was an increase in both the number of phagocytic cells and the quantity of zymosan engulfed. FACS analysis further illustrated that the phagocytosing population was homogeneous on day 0, but became heterogeneous, with respect to its phagocytic capacity, on days 2 and 4 as differentiation proceeded (data not shown). This gave the impression, as shown in Fig. 3Goa, that the capacity to phagocytose increased before the number of phagocytosing cells. Analysis of Alv-M{phi} tended to support this. The phagocytic capacity of Alv-M{phi} was akin to that of the MDMs, but only 50% of the cells possessed that capacity (Fig. 3aGo, Alv-M{phi}).



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  		FIGURE 3. Comparison of phagocytic and endocytic activities of differentiating adherent monocytic cells isolated from PBMC. a, Influence of in vitro culture-dependent differentiation on the capacity of monocytic cells to phagocytose BODIPY fluorescein-Zymosan (50 particles/cell). The values for fluorescence intensity of the phagocytosed zymosan are expressed as the mean ± SEM from triplicate experiments for monocytic cells and Alv-M{phi} (y1-axis; open and filled histograms, respectively); the values for the percentage of active phagocytes show means only, because the SEMs were <5% (y2-axis; open and filled circles for monocytic cells and Alv-M{phi}, respectively). b, Differentiation of monocytes in vitro is associated with increased expression of LDL receptors, measured using the BODIPY-fluorescence LDL complex. The data were expressed in a similar manner as in a.

 
The expression of LDL receptors on monocytic cells differentiating toward M{phi} was employed as a measure of receptor-mediated endocytosis, using the pH-insensitive BODIPY-LDL probes. A positive correlation was noted between monocyte to M{phi} differentiation and LDL receptor expression, indicative of increased endocytic activity (Fig. 3Gob). More than 85% of intermediate cells in the differentiation (day 2) were already endocytosis positive compared with <30% of monocytes (Fig. 3Gob, line graph, y2-axis). Although this percentage of LDL-receptor positive cells increased only slightly between days 2 and 4 (Fig. 3Gob, y2-axis), it was the relative LDL binding to the cells that notably increased (Fig. 3Gob, histogram, y1-axis). Such results would be indicative of additional maturational changes as the monocytic cells approached more mature M{phi}. Compared with Alv-M{phi}, only around 50% of the cells were endocytosing, and their activity was closer to that of intermediate in vitro differentiating cells (Fig. 3Gob, Alv-M{phi}).

Intracellular pH environment of monocytic cells

The efficiency of the phagocytic and endocytic pathways is dependent on the activity of the vacular H+-ATPases, and their ability to acidify cytoplasmic vesicles. Macrophages should therefore have a more acidic intracellular vesicle environment than monocytes due to their increased capacity to endocytose or phagocytose material (see Fig. 3Go). Consequently, the loss of fluorescence by internalized pH-sensitive probes was compared with that of monocytes as they differentiated into M{phi} (Fig. 4Go). It was clear that these conjugated dextrans encountered a lower pH (more acidic vesicles) within differentiating cells and more mature M{phi} than in monocytes. With the monocytes, the signal acquired after stage I of the process (Fig. 4Goa, I) was higher than its counterpart with day 2 differentiating cells (Fig. 4Gob, I), which, in turn, was higher than that with the more mature macrophages obtained on day 4 (Fig. 4Goc, I). After the stage II measurement, subsequent to the washing step (Fig. 4Go, II), it was noted that the environment in which the probe was found had continued to acidify. The apparent rate of acidification during this time was similar for monocytes (Fig. 4Goa), intermediate cells (Fig. 4Gob), and more mature M{phi} (Fig. 4Goc). After stage III of the process, measured following the incubation with the mAb (Fig. 4Go, III), the signal was again further reduced. It was virtually quenched (similar to the negative control value) in the more mature cells (Fig. 4Goc), but less so in the differentiating cells. Such a quenching would indicate a pH environment 5 according to the manufacturer’s information. The intracellular pH environment of Alv-M{phi} (Fig. 4God) was similar to that of the in vitro intermediate differentiated cells, but was more homogeneous and relatively advanced in acidity. One other notable observation with the Alv-M{phi} was that the stage III signal was not as low as that obtained with the intermediate cells in vitro, suggesting that the final degree of vacuolar acidification by Alv-M{phi} was lower or attained more slowly.



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  		FIGURE 4. FACS analysis of dextran-FITC (pH indicator) internalization in differentiating monocytes and Alv-M{phi}. Monocytes (adherent cells) were isolated from PBMC and tested after 0 (a), 2 (b), or 4 (c) days of culture and compared with Alv-M{phi} (d). The cells were left for 40 min on ice before incubation with the probe (0.25 mg/ml) for 5 min at 37°C. Data were acquired at three stages: stage I, before washing; stage II, immediately after washing; and stage III, after washing plus subsequent labeling with anti-SWC9 and anti-CD14 mAbs (data not shown). The results are representative of a single experiment of three replicates.

 
It was interesting that the acidification process was already apparent at stage I, particularly with the more mature cells (Fig. 4Goc). Although all cells had a similar capacity to further acidify, the stage II and stage III peaks almost overlapped with those of more mature cells (Fig. 4Goc), suggesting that maximum acidification detectable with this probe was almost attained by stage II. The more mature M{phi} (Fig. 4Goc) were also the most homogeneous in terms of the pH-sensitive signal given by the probe. This would suggest a relatively greater homogeneity in the internalization and/or acidification of the probe compared with monocytes and intermediate differentiating cells.

Cell-associated enzyme activity and intracellular Ags during monocyte differentiation

For endocytosis/phagocytosis to reach completion, the internalized material must encounter cytoplasmic organelle enzymes of the monocytic cells. The enzymes relative activities should differ dependent on the characteristic of the cell in question. Freshly isolated monocytes were strongly positive for cell-associated nonspecific esterase activity (Fig. 5Goa, day 0). During differentiation toward M{phi} the activity was notably down-regulated (Fig. 5Goa, days 2 and 4). The predominant down-regulation occurred during the earlier phases of differentiation (Fig. 5Goa, day 2), although the majority of the more mature macrophages (SWC9+) remained positive for this marker (Fig. 5Goa, day 4). From the contour plots it was also evident that the cells became more heterogeneous in their cell-associated esterase activity during differentiation.



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  		FIGURE 5. Double immunofluorescence analysis of intracellular enzyme activity and intracellular Ag expression vs expression of the M{phi} marker SWC9. Monocytic cells harvested from PBMC by adherence were cultured for 0 (first row), 2 (second row), and 4 (third row) days. These cells were compared with Alv-M{phi} (fourth row) when labeled with the probes for esterase (a), ß-galactosidase (b), glucuronidase (c), 5' nucleotidase (d), peroxidase (e), and myeloperoxidase (f). The results shown are representative of one experiment of three replicates.

 
Monocytes were also positive for ß-galactosidase activity (Fig. 5Gob, day 0). This remained relatively unchanged as differentiation progressed, as monitored by SWC9 up-regulation (Fig. 5Gob, days 2 and 4). With cell-associated glucuronidase activity, monocytes were negative/dim (Fig. 5Goc, day 0). Upon differentiation, there was a marginal increase in the cell-associated activity of the enzyme, primarily with a subpopulation of SWC9+ cells (Fig. 5Goc, days 2 and 4); a small subpopulation of SWC9+ cells with higher ß-galactosidase activity was also noted (Fig. 5Gob, day 4). The measurements obtained for glucosidase activity were almost identical with those of glucuronidase in Fig. 5Goc (data not shown).

With cell-associated 5' nucleotidase activity, two populations were distinguished by their enzymatic activity in monocytes. This enzyme underwent a gradual down-regulation in both cell populations during differentiation into M{phi} (Fig. 5God). The relative difference in activity between the populations remained throughout the differentiation, although the more active cells tended to dominate the relatively SWC9high population. Likewise, there was a clear and rapid reduction in cell-associated peroxidase activity (Fig. 5Goe). As with esterase, the reduction was most marked during the earlier phases of differentiation, and the majority of cells, albeit low to dim, were still positive by day 4. The majority of monocytes were also positive for the intracellular myeloperoxidase Ag (Fig. 5Gof, day 0), the detectability of which was rapidly lost during differentiation into M{phi} (Fig. 5Gof, days 2 and 4). A similar profile of down-regulation was obtained with the intracellular protein p53 (data not shown).

Analysis of the enzyme activity and the intracellular Ag expression associated with Alv-M{phi} (Fig. 5Go) revealed a state of activity more similar to that of intermediate differentiating cells (day 2) than to that of the more mature MDMs (day 4). This was most clear with the 5' nucleotidase and peroxidase activities. Interestingly, however, the Alv-M{phi} did not present a double population for the 5' nucleotidase, but, rather, presented a skewed population. The dominant area of activity related to the population of MDMs on day 4, which possessed the higher enzyme activity, while the skew related more to the nucleotidasehigh populations of intermediate cells and monocytes.

Accessory functions of monocytes/macrophages

One difference between monocytes and M{phi} that might be consequential to different enzyme patterns as well as the relative endocytic/phagocytic activities and associated vacuolar acidification concerns accessory cell function. Monocytes exhibited a relatively high accessory activity, which was lost upon differentiation into M{phi} (Fig. 6Go). Culturing PBMCs from FMDV-vaccinated animals in the presence of virus Ag (Fig. 6Go, PBMC, filled histograms) resulted in a specific proliferation compared with that produced by the control Ag (Fig. 6Go, PBMC, open histograms). PBMCs from nonvaccinated control animals gave low proliferative responses to both the FMDV and mock control Ag (data not shown). When the majority of adherent cells were removed from PBMCs, the remaining lymphocytes could not be induced into FMDV-specific proliferation (Fig. 6Go, Ly), as expected due to the lack of adequate accessory cell activity. If such nonadherent lymphocyte preparations were added back to autologous adherent monocytes, the FMDV-specific lymphoproliferative response was restored (Fig. 6Go, mo+Ly). In contrast, if the autologous adherent monocytes were allowed to differentiate into M{phi} before addition of the lymphocyte preparations from which the majority of monocytes had been removed, no sign of FMDV-specific proliferation was detected (Fig. 6Go, MDM+Ly). The latter results would even imply a loss in proliferative capacity.



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  		FIGURE 6. Accessory activity of monocytes (mo) and MDMs for mock (open histograms) or FMDV (filled histograms) Ag presentation, measured by [3H]thymidine incorporation into proliferating lymphocytes. The monocytes were obtained by adherence of PBMC and removal of nonadherent cells after 2 h of culture at 37°C. MDMs were derived from these monocytes by culture in DMEM containing 30% (v/v) porcine plasma for 3 days at 37°C. Nonadherent PBMC from which the majority of monocytic cells had been removed by multiple adherence cycles were designated Ly. The data were expressed as the mean ± SEM from triplicate wells and are shown for one representative experiment of four replicates.

 
One reason for the poor accessory activity of M{phi} compared with monocytes might lie with the lower MHCII expression (Fig. 1God). Yet, another important parameter is cytokine production, particularly that of cytokines such as IL-1, which are essential for interleukocyte communication. Indeed, when the supernatants from the monocyte cultures depicted in Fig. 6Go (mo+Ly, filled histograms) were transferred to the M{phi} cultures (MDM+Ly, filled histograms), the lymphoproliferative response was partially restored (data not shown). However, lymphocyte cultures (Ly, filled histograms) that received the same supernatants also exhibited increased proliferation (data not shown), indicating that the presence of soluble factors is important for lymphocyte proliferation even when monocyte concentrations are limited.

IL-1 production during monocyte differentiation

A more detailed analysis of cytokine secretion as monocytes differentiated into M{phi} focused on IL-1 because of its importance in lymphocyte proliferation. Freshly isolated monocytes secreted high IL-1 levels over a 24-h period even without external stimuli (Fig. 7Goa, day 1). Between 24 and 48 h of culture the protein secreted by the unstimulated cells declined sharply, to reach almost background levels, at which it remained for the rest of the differentiation process. When the cells were pulse treated with PMA, IL-1 production by 24-h-old cells (measured after a further 24 h (=24 h post-treatment)) was enhanced to the levels in monocytes; LPS stimulation was also effective, but less efficient than PMA. The efficiency of PMA-induced IL-1 production declined with continued cell differentiation, but remained higher than that obtained with LPS or nonstimulated cells (day 3). Endogenous productivity and LPS inducibility of IL-1 were lost in the more mature cells and were reduced to a minimal level for PMA induction (day 4 cells). Alv-M{phi} (Fig. 7Goa, Alv-M{phi}) were analogous to the day 4 MDMs in having low IL-1 production and poor inducibility.



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  		FIGURE 7. Kinetics of IL-1 secretion and mRNA transcription in monocytes and MDMs prepared as described in Materials and Methods. a, Supernatants from adherent monocytic cells in culture for 0–3 days were collected at 24 h after treatment of the cells with medium alone (circles), 10 µg/ml LPS (stars), or 10 ng/ml PMA (squares) and were assayed for the presence of IL-1. The background values of IL-1-like activity in the medium (triangles) was also tested. The production of IL-1 in Alv-M{phi} is represented as filled markers. For the purpose of standardization and comparison, all tests employed a recombinant human IL-1 standard, against which the results were compared and converted into relative picograms per milliliter. b, IL-1 mRNA levels were analyzed by RT-PCR followed by agarose gel electrophoresis. Equal amounts of extracted RNA were employed in each lane, as indicated by the actin PCR (data not shown). M, DNA markers; C, control cells; L, LPS-treated cells; P, PMA-treated cells; N, negative control.

 
The assay employed in Fig. 7Go had to use a recombinant human IL-1 standard due to problems encountered with commercial recombinant porcine IL-1. Consequently, the concentrations shown are only relative to the human IL-1 standard. Confirmation of these results therefore made use of the RT-PCR to detect porcine IL-1-specific mRNA (Fig. 7Gob). In this way the observations on IL-1 production shown in Fig. 7Goa were confirmed to be occurring at the transcriptional level and were applicable for both IL-1{alpha} and IL-1ß (Fig. 7Gob shows RT-PCR for IL-1{alpha}; results were similar for IL-1ß).

Modified susceptibility to virus infection as monocytes differentiate into M{phi}

A number of viruses are known to be monocytotropic, but many are capable of infecting other cells, and monocytic cells are a major target. In contrast, ASF virus has a particularly high predilection for cells of the reticuloendothelial system. For this reason it was chosen to analyze the influence of monocyte differentiation to M{phi} on the susceptibility to infection. An m.o.i. of 10 infectious units/cell was chosen so as to increase the likelihood that each living cell in the culture had a >99% chance of encountering at least one infectious virion, according to Poissonian distribution. Fig. 8Goa shows that MDMs had a high susceptibility to infection by ASF virus, detected by both the infectious center assay and virion VP73 production. In contrast, with freshly isolated monocytes <5% of the cells were identified as being infected in the infectious center assay. It was possible that this was reflecting an incomplete virus replicative cycle in the apparently uninfected cells. This did not seem to be the case, because again <5% of the cells were seen to have produced VP73 at 24 h after infection. Attempts to detect virus associated with the monocytes by PCR also gave a weak signal, compared with the strong signal obtained with the infections of MDMs (data not shown).



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  		FIGURE 8. Analysis of the capacity of ASF virus to infect (m.o.i., 10 infectious focus-forming units/cell) porcine monocytic cells (a, freshly isolated monocytes or MDMs; b, Alv-M{phi} and intermediate differentiating monocytic cells). The infected cells were incubated at 37°C for 20–24 h before titration on preformed 3-day-cultured MDMs. These cocultures were incubated at 37°C for 5 days, then analyzed by immunofluorescence for the presence of virus Ag. From this infectious center assay titration, it was possible to determine the percentage of cells (monocytes or MDMs) in the original infected cell preparation in which a productive infection had occurred (y1-axis, dark gray histograms). In replicate experiments, the infected cells were stained with anti-VP73 mAb for the presence of de novo synthesized virus Ag (this method did not detect input virus during the initial infection). The percentage of cells positive for the VP73 is shown on the y2-axis (a) or the y1-axis (b) with the light gray histogram.

 
Alv-M{phi} displayed a susceptibility to ASF virus infection that showed 10–25% of cells VP73 positive at 24 h postinfection (Fig. 8Gob). These results demonstrate that recently isolated lung lavage M{phi} exhibit a sensitivity to ASF virus related to that of intermediate differentiating monocytic cells (Fig. 8Gob). Alv-M{phi} were clearly susceptible to infection, unlike monocytes, but were not as sensitive as MDMs.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The study of human M{phi} differentiation has employed in vitro models of monocyte culture (5, 6), although little information is available concerning porcine cells (7). Knowledge of the latter is becoming increasingly important due to the application of porcine systems as animal models for immunological analyses (12, 13, 14) as well as in the transplantation area (12). Consequently, the present work aimed to determine details of the temporal sequence and mechanisms, especially their interrelationships, in monocyte to M{phi} differentiation. While being important for porcine immunology per se, for which such elements have not been analyzed in depth, these studies also provide more general immunological information concerning cell function interdependency during the differentiation. The analyzed characteristics also permitted more detailed analysis of cell susceptibility to infection by monocytotropic viruses capable of perturbing immune defenses.

Monocytic cells are a heterogeneous population, seen by their variety of functions (3), probably due to differences in their maturation and/or activation state. The activity of particular cytokines in vivo and interactions with cells such as those of the vascular endothelium are plausible explanations. Such activities could certainly explain the heterogeneity of monocyte MHCII expression noted in this study and previously (7). An inverse relationship between the SWC1 Ag expressed on porcine monocytes (21) and the SWC9 found on porcine M{phi} (7, 16, 17) as well as the putative porcine CD80 related to the differentiation stages of monocytic cells. SWC1 down-regulation, concomitant with SWC9 and CD80 up-regulation, was linked to an increase in activity of the M{phi} enzyme acid phosphatase. Although particular to porcine cells, the results relate to observations on human monocytic cells (29, 30, 31). In contrast, CD14 was down-regulated during porcine M{phi} differentiation (7), whereas human cells may down-regulate or up-regulate CD14 (5, 32), possibly relating to granulocyte-macrophage CSF activity (33).

As the phenotypic modulations progressed with increasing differentiation of the monocytes toward M{phi}, the phagocytic and endocytic activities also became more prominent, probably relating to mannose and ß-glucan receptor activity (34) as well as LDL receptor expression (35). The efficiency and rate of intracytoplasmic vesicle acidification increased concomitantly with endocytic activity as the monocytes matured into M{phi}, relating to the acidification characteristics of the endocytic pathway (36). With these changes, modulations of endosomal and lysosomal enzyme activities were also noted. Porcine monocytes clearly down-regulated endosomal enzyme activity during differentiation to M{phi}; such a relationship was variable in analyses of human monocytic cell differentiation (18, 19, 37, 38), but could be related to advanced differentiation or activation (37, 39, 40). In contrast, the activities of the lysosomal enzymes ß-galactosidase, glucuronidase, and glucosidase correlated in a positive manner to porcine M{phi} differentiation. A similar relationship had been reported with the maturation of human mononuclear phagocytes (19, 41). Interestingly, the highest activity was associated with porcine cells expressing the higher levels of the SWC9 macrophage marker.

Confirmation that the in vitro observations relate to in vivo processes came from studies on Alv-M{phi}. Interestingly, Alv-M{phi} resembled intermediate differentiating cells in terms of endocytosis and expression of the putative porcine CD80 as well as endosomal enzyme activities. Yet their vesicle acidification related more to the MDMs, and therefore to the role of vacuolar H+-ATPase in vesicle acidification (42, 43). This demonstrates that not all characteristics of in vivo differentiating M{phi} are modulated with the same kinetics as those in vitro. It would appear that in in vivo derived M{phi} differentiation would occur earlier, but they would have experienced a degree of activation or priming compared with in vitro MDMs.

Combining the monocytic cell functional activity and enzyme profiles with MHC expression suggests that porcine monocytes should possess greater accessory cell potential than M{phi}. This was indeed the case. However, the early phases of human M{phi} differentiation have shown enhanced accessory capacity over monocytes (44). Although a CD80 up-regulation might relate to this, monocytic costimulation signals of T cells are not solely B7 mediated (31). Furthermore, advanced differentiation to a cell resembling the pulmonary M{phi} resulted in a loss of accessory capacity (45). It is not certain whether the human analyses, which employed mitogenic stimulation to assess monocyte accessory function, relate to Ag-specific responses. Autoreactive human T cells respond less efficiently than Ag-specific lymphocytes to monocytic accessory cells (46). Moreover, despite reports of human macrophage accessory capacity (44, 45, 46, 47), it is the nonphagocytic monocyte-derived dendritic cell that is the more efficient (48, 49). With porcine monocytes, accessory activity is clearly lost upon differentiation into M{phi}. In the absence of a defined dendritic cell population in the pig, it is unclear whether other cells types can differentiate from the monocytes to give the equivalent of the human dendritic cell.

The relative accessory capacity of porcine monocytic cells related to their ability to produce IL-1, both spontaneous and induced. Higher IL-1 production has also been reported for human monocytes compared with M{phi}, but this could not relate to accessory function (46, 50, 51). Through the current analyses, the accessory cell capacity associated with monocytes was found to require concomitantly high MHCII expression, endosomal enzyme activity, and IL-1 productivity. Differentiation in vitro to more mature M{phi} reduced these characteristics concomitant with loss of accessory function. Although porcine Alv-M{phi} can retain high MHCII expression, their IL-1 productivity is poor, and their endosomal enzyme activity is less than that of monocytes. Such characteristics would explain why Alv-M{phi} are reported to be poor accessory cells (45).

As differentiating M{phi} lose their accessory capacity, their phagocytosis and endocytosis increase. Thus, the modulations associated with changing from accessory monocyte to scavenger macrophage are multifactorial. Efficient phagocytosis, endocytosis, and cytoplasmic vesicle acidification plus a shift from endosomal to lysosomal enzyme activity are required for scavenger M{phi}. These modulations might be reflecting alterations in the change from a processing to a more destructive endocytic pathway (49, 52).

Such multifactorial characteristics have particular consequences for the susceptibility of the cells to infection by monocytotropic viruses. ASF virus actually targets the phagocytic and lysosome-active scavenger M{phi}, not the more accessory monocyte. Alv-M{phi} again displayed an intermediate differentiating cell characteristic in terms of susceptibility to ASF virus. In fact, when Alv-M{phi} were cultured in vitro over 72 h, both their activities and susceptibility to ASFV became were more akin to those of MDMs (data not shown). This confirmed that the lung lavage M{phi} are indeed not fully mature; further differentiation, similar to that seen with MDMs, is possible. It is conceivable that the virus may not adsorb to the monocytes, but this would not explain the low percentage of monocytes that can be infected. Alternatively, the cellular activity may not be favorable to initiate virus replication. This would imply a greater requirement for high phagocytosis/endocytosis in virus entry as well as a rapid vesicle acidification and lysosomal enzyme activity for virus uncoating. Certainly, ASF virus has an obligate requirement for efficient cytoplasmic vesicle acidification and a potent vacuolar H+-ATPase activity (43), properties that would be more prominent with M{phi} than monocytes. Delineation of these events should now prove beneficial in the study of the effect of viral infection on different monocytic cell populations, and the functional parameters that aid viruses to escape potential degradation by the innate immune cells.


   Acknowledgments
 
We thank H. Gerber, E. Blanco, and R. Tschudin for technical assistance, D. Brechbühl for animal bleedings, and Drs. A. Summerfield and C. Griot for critical discussions of the results.


   Footnotes
 
1 This work was supported by the Swiss National Science Foundation (Grant 31-47239.96; to S.B.) and the Swiss Federal Veterinary Office (to S.K.). Back

2 Address correspondence and reprint requests to Dr. Kenneth McCullough, Institute of Virology and Immunoprophylaxis, Postfach, CH-3147 Mittelhausern, Switzerland. E-mail address: Back

3 Abbreviations used in this paper: M{phi}, macrophages; PBSA, calcium/magnesium-free PBS; SWC, swine workshop cluster designation; MDM, monocyte-derived macrophage; Alv-M{phi}, lung lavage macrophages; MHCI, Class I; MHCII, MHCII class II; LDL, low density lipoproteins; FMDV, foot and mouth disease virus; ASF, African swine fever; m.o.i., multiplicity of infection. Back

Received for publication September 28, 1998. Accepted for publication January 11, 1999.


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