Introduction

The complement (C') system, a phylogenically ancient component of the immune system, is very important in defence against bacteria.^{1, 2} Genetic defects resulting in deficiencies in specific C' proteins have been linked to increased susceptibility to infections caused by bacteria such as Neisseria meningitidis.^{3, 4, 5} Derivatives of C' proteins, C3 and C4, are important opsonins for phagocytosis by macrophages,⁶ and macrophage surface receptors that take up particles coated with these cleavage products include CR1, CR3 and CR4.^{7, 8, 9, 10} CR1 is a single transmembrane receptor, whereas CR3 and CR4 are integrin heterodimers with a common 2 chain.^{7, 9, 10} These receptors have different affinities for different C' products and they are thought to collaborate in mediating the uptake of C'-opsonized particles.^{11, 12}

C'-mediated phagocytosis has assumed increasing importance in the era of HIV/AIDS, as the AIDS-related opportunistic pathogens Mycobacterium tuberculosis and Mycobacterium avium complex are phagocytosed by macrophages predominantly via the C' pathway.^{13, 14} Our observations that phagocytosis of M. avium complex is impaired by macrophages infected with HIV-1 in vitro and in vivo¹⁵ suggest a defect in C'-mediated phagocytosis in macrophages as a result of HIV infection. C' can also facilitate phago-cytosis of other mycobacteria, Legionella pneumophilia, Salmonella typhi and Staphylo-coccus aureus.^{16, 17, 18, 19}

Studies into C'-mediated phagocytosis by macrophages have been hampered by a lack of easy procedures for opsonization of target particles and convenient methods to quantify phagocytosis that can distinguish attached and phagocytosed particles. Common assays for investigation of phagocytosis by macrophages via C' receptors usually involve tedious microscopic evaluation of internalized C'-coated targets.^{20, 21, 22} Furthermore, the conventional coating protocols and targets used cannot simultaneously enable easy opsonization and convenient discrimination between attached and internalized particles.

In the present study, we describe a simple procedure using 2% human serum for C'-opsonizing SRBC as targets for phagocytosis, which can then be quantified by a modified colourimetric assay that is specific for ingestion via C' receptors. We further show, by use of this assay, that HIV-infected monocyte-derived macrophages are defective in their ability to phagocytose complement-opsonized SRBC.

Materials and Methods

Isolation and culture of human monocytes

Human peripheral blood monocytes were isolated from buffy packs of HIV-1, hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukaemia virus (HTLV) and syphilis-seronegative blood donors (purchased from the Red Cross Blood Bank, Melbourne, Vic., Australia) using Ficoll-Paque density gradient centrifugation followed by plastic adherence as described elsewhere.²³ Immediately after isolation, cell viability was greater than 95% as assessed by Trypan blue exclusion. The purity of monocytes was greater than 85% as determined by immunofluorescent staining with anti-CD14 mAb conjugated directly to phycoerytherin (CD14-PE; Becton Dickinson, San Jose, CA, USA) and assessed by flow cytometry using a FACS Star Plus flow cytometer (Becton Dickinson). Monocytes were resuspended and cultured in Iscove's medium (GibcoBRL, Life Technologies, NY, USA) supplemented with 10% heat-inactivated human AB-positive serum, 2 mmol/L L-glutamine (GibcoBRL) and 24 g/mL gentamicin (Delta West, Bentley, WA, Australia) at a concentration of 0.5–1 10⁶ cells/mL in polytetrafluorethylene (Teflon) jars (Savillex, Minnetonka, MO, USA).

Preparation of C'-opsonized sheep red blood cells

Sheep red blood cells (2 10⁸) (ICN-Cappel, Aurora, OH, USA) were washed three times in cold PBS (1430 g, 7 min), centrifuged and opsonized for 30 min at room temperature immediately prior to the phagocytosis assay using prewarmed (37°C) 2% AB-negative untreated human whole serum (NHS) from HIV-negative donors (a source of human C' components). To terminate opsonization, SRBC were pelleted by centrifugation at 660 g for 10 min at 4°C. Supernatant was collected and the concentration of released haemoglobin (Hb) was measured at 540 nm to determine the lysis of the SRBC resulting from destruction by C' components. Sheep red blood cells were then washed three times in cold PBS and resuspended at a concentration of 1 10⁸/mL. Sheep red blood cells used as controls were prepared using the same procedures but using heat-inactivated serum (iNHS); iNHS was derived from serum prepared from the same donor, heat-inactivated at 56°C for 45 min (iNHS) and clarified by centrifugation (20 000 g, 10 min). Freshly prepared NHS and iNHS were aliquoted into microcentrifuge tubes, typically in a volume of 50–100 L, snap-frozen in liquid nitrogen and stored at –80°C. Serum stored in this manner could be used for up to 12 months after preparation.

For inhibition studies of FcR-mediated phagocytosis, SRBC were opsonized with IgG by incubation with rabbit antiserum to SRBC (ICN Cappel) at a dilution of 1 : 300 for 30 min at room temperature.

Gel electrophoresis and western blotting

For analysis of C3 derivatives (major C' opsonins) on opsonized SRBC preparations, SRBC samples were lysed in cold 0.2% NaCl (Merck, Kilsyth, Vic., Australia) solution, and SRBC membranes pelleted by centrifuging at 20 800 g for 5 min at 4°C. The SRBC membrane fragments were washed once with cold 0.2% NaCl solution and the pellet was boiled in SDS sample buffer (10 mmol/L Tris pH 8.0, 2 mmol/L EDTA, 1% SDS, 5% -mercaptoethanol, 5% glycerol) at 95°C. Samples were analysed by SDS-PAGE using the discontinuous buffer system at 100 V. Proteins were transferred to nitrocellulose (Amersham International, Buckinghamshire, UK) at 70 V for 2 h. The nitrocellulose filters were blocked in 5% skim milk in Tris-buffered saline containing 0.3% Tween-20 (Astral, Gymea, NSW, Australia) for 2 h at 37°C and then probed with goat polyvalent antiserum against human C3 (ICN-Cappel) at 4°C for 16 h. Following five washes in 1 TBST, blots were probed with a rabbit-antigoat antibody conjugated with horseradish peroxidase (Dako, Carpinteria, CA, USA), followed by three washes in 1 TBST, and developed for enhanced chemiluminescence (ECL) according to the manufacturer's instructions (Amersham International).

C'-mediated phagocytosis assay

On days 5–14 of postisolation, monocyte-derived macrophages (MDM) were adhered in quadruplicate wells onto flat-bottomed 96-well plates at 5 10⁴ cells/well in 200 L of supplemented Iscove's medium for 24 h at 37°C in a 5% CO₂ humidified incubator. The volume of the medium was adjusted to 85 L before the addition of 5 L of phorbol-12-myristate-13-acetate (PMA; Sigma) in 10% dimethylsulfoxide (Merck) (final concentration of PMA of 200 nmol/L per well) for 10 min prior to the assay. Phagocytic targets (SRBC coated with NHS or iNHS) were added to MDM at the ratio of 20 SRBC to 1 MDM and centrifuged at 57 g for 5 min at 4°C onto the adherent MDM. The plate was placed at 37°C in 5% CO₂ for phagocytosis to proceed. Phagocytosis was terminated after 60 min by placing the plate on ice and washing non-phagocytosed SRBC away with cold (4°C) PBS. The amount of internalized SRBC was measured using a colourimetric assay.²⁴ Briefly, unbound SRBC were washed off the wells with cold PBS, whereas adhered but non-phagocytosed SRBC were lysed with 0.2% NaCl solution for 3 min and removed by washing three times with prewarmed (37°C) Iscove's medium. Monocyte-derived macrophages were lysed with 6 mol/L urea solution containing 0.2 mol/L Tris-HCl buffer (pH 7.4). The number of phagocytosed SRBC was determined by measuring the amount of fluorene blue that had been converted from 2,7-diaminofluorene (Sigma) by the pseudoperoxidase activity of Hb.

Absorbance was determined at 620 nm in an ELISA plate reader (Labsystems, Helsinki, Finland) and corrected against a blank, which contained only 2,7-diaminofluorene solution and urea–Tris buffer. The adjusted absorbance was compared to a standard curve, which was generated for each assay plate using known numbers of SRBC (twofold dilutions in triplicate ranging from 8 10³ to 1 10⁶ per well) and fitted by four-parameter analysis using a microplate analysis program (Deltasoft 3, BioMetallics Inc., Princeton, NJ, USA). The number of SRBC determined from this standard curve was divided by the number of MDM in the well (5 10⁴) and multiplied by 100 to obtain the phagocytic index (the number of SRBC phagocytosed per 100 MDM). The data from quadruplicate wells were averaged and expressed as mean SD.

Measurement of C'-mediated phagocytosis in HIV-infected monocyte-derived macrophages

Monocyte-derived macrophages prepared from individual donors' monocytes were subdivided into two and either infected with HIV-1_Ba-L or mock infected, as described elswhere¹⁵ on day 5 of postisolation. Infected cells were cultured for a further 6 days and then plated for 24 h onto 96-well plates for the measurement of C'- and Fc-mediated phagocytosis. Infection was confirmed by the measurement of reverse transcriptase activity in culture supernatants on day 7 of postinfection. Experiments were carried out under strict PC3 containment until the virus was inactivated by the urea lysis step, after which the plates were transferred to the PC2 laboratory for colourimetric determination of released Hb.

Statistical analysis

The significance of the data from various phagocytosis experiments was assessed using a paired Student's t-test; significance depended upon a P-value of 0.05, two-tailed.

Results

Sensitivity of the colourimetric assay for sheep red blood cells

In order to quantify the number of SRBC phagocytosed by macrophages, Hb released following lysis of cells was determined by reaction with 2,7-diaminofluorene and the fluorene blue product was measured spectrophotometrically.²⁴ To determine the relationship between the fluorene blue generated and the number of SRBC, a standard curve was constructed with SRBC ranging from 8 10³ to 1 10⁶ per well. Under the conditions used, colour development was not saturated up to 4.5 10⁵ SRBC per well (data not shown). The range of values that could be read from the standard curve (from the first data point significantly above the background to the point of saturation) equated to a phagocytic index of between 15 and 900 in experiments using 5 10⁴ MDM plated per well.

Effect of variables on phagocytosis of sheep red blood cell targets

Conditions for opsonization of targets The FcR-mediated phagocytosis assay described by Gebran and coworkers²⁴ was modified to measure C'-mediated phagocytosis without the requirement of target opsonization by purified C' components. Untreated whole serum (NHS) was evaluated as a source of C' components. Various serum concentrations were tested to achieve adequate opsonization with insignificant lysis of the SRBC targets. When MDM were incubated with SRBC for 60 min, a similar extent of phagocytosis of target particles was achieved using targets opsonized at room temperature at final serum concentrations of 2% (phagocytic index SD = 389 33.0) and 5% (phagocytic index SD = 433 53.1); however, the latter concentration resulted in significantly more lysis of the target SRBC (62% vs 14%, respectively). Incubation periods of 30 min, 1 h and 2 h were evaluated with incubation at 4°C and/or room temperature (20–25°C); the optimal incubation conditions were found to be 30 min at room temperature (data not shown). On the basis of these results, the target opsonization parameters were set at 2% serum at room temperature for 30 min. This concentration was subsequently used successfully with AB serum prepared from several individuals, but should be optimized for each serum batch.

Deposition of C' on SRBC opsonized under the optimal conditions was subsequently demonstrated by the detection of C3 products on SRBC ghosts using SDS-PAGE followed by immunoblotting with polyclonal goat-antiserum against human C3 components. Multiple bands between 40 kDa and 200 kDa (C3 products) were detected on SRBC opsonized with NHS but not iNHS (Figure 1).

Figure 1.

Deposition of C' opsonins on SRBC targets. Sheep red blood cell ghosts were obtained by lysing SRBC with 0.2% NaCl solution. C3 opsonins were detected by immunoblotting with a polyclonal goat-antiserum against human C3 following enhanced chemiluminescence detection, and were present in SRBC opsonized with untreated serum (US) but not in SRBC samples treated with heat-inactivated serum (HIS).

Full figure and legend (37K)

Conditions for phagocytosis assay incubation time for phagocytosis To determine the optimal incubation time for phagocytosis, the kinetics of C'-mediated phagocytosis (0–120 min) by MDM were assessed using a target to MDM ratio of 20 : 1. The number of phagocytosed SRBC, as assessed by the content of internalized Hb, increased as a function of time, reached a maximum by 90 min and plateaued thereafter (Figure 2). Adherence time of MDM prior to phagocytosis assay As macrophage function, including phagocytosis, might be augmented as a result of activation induced by adherence,²⁵ the effect of altering the adherence period prior to conducting a phagocytic assay was examined. Monocyte-derived macrophages from three donors were adhered to plastic 96-well plates for 24 h prior to phagocytosis assay and compared to control MDM from the same donors adhered for only 2 h (as described for FcR-mediated phagocytosis²⁴). Adherence for 24 h resulted in higher levels of phagocytosis than 2 h of adherence (86% increase; Figure 3). Increasing the adherence time to 48 h and 72 h did not result in higher levels of phagocytosis when compared to 24 h (D Doischer, pers. comm., 2000).

Figure 2.

Kinetics of C'-mediated phagocytosis by monocyte-derived macrophages (MDM) measured using the colourimetric assay. Monocyte-derived macrophages were prestimulated with PMA (,) or DMSO control (,) for 10 min at 37°C. Sheep red blood cells (opsonized with untreated serum (NHS) (,), or heat-inactivated serum (iNHS) (,)) were added at a MDM to target ratio of 1 : 20, and phagocytosis was allowed to proceed for the times indicated. Phagocytosis was then quantified as described in Materials and Methods. The figure is a representative of three independent experiments. Each data point was performed in quadruplicate and error bars indicate standard deviation. Phagocytic indices from 30 min onwards that were below the lowest point on the standard curve are arbitrarily defined as 15 (the phagocytic index equivalent to the lowest limit of the standard curve).

Full figure and legend (8K)

Figure 3.

Phagocytic index is increased in monocyte-derived macrophages (MDM) that have adhered for 24 h in comparison to those that have adhered for only 2 h prior to phagocytosis assay. Phagocytosis assays were performed on MDM cultured for 12 days in Teflon pots after their initial isolation and then adhered to plastic for 2 h or 24 h prior to assay. The figure is representative of three independent experiments using cells from three different donors (mean SD).

Full figure and legend (12K)

Specificity of C'-mediated phagocytosis assay

Sheep red blood cells treated with NHS as described earlier were verified to be phagocytosed via a C'-mediated pathway. First, phagocytosis was enhanced by PMA, a known activator of C' receptors (Figure 4a, see also Figure 4b,c). There was no phagocytosis of SRBC targets when the SRBC were treated with iNHS (Figure 4a–c). In addition, phagocytosis measured by this assay was inhibited by a specific PKC inhibitor, RO 31-8220 (Figure 4b), but not the tyrosine kinase (PTK)-specific inhibitor, genistein (Figure 4c). The PKC inhibitor did not inhibit the FcR-mediated phagocytosis of Ig-opsonized SRBC (Figure 4d).

Figure 4.

Specificity of C'-mediated phagocytosis assay. (a) Monocyte-derived macrophages (MDM) were pre-incubated with the indicated concentrations of PMA in 0.5% DMSO (, and ) 10 min prior to the addition of SRBC coated with untreated serum (NHS; ) or heat-inactivated serum (iNHS; ). Data represent mean SD from a single experiment performed in triplicate. For all subsequent experiments, a PMA concentration of 200 nmol/L was used. (b,c) Adhered MDM were treated with varying concentrations of the indicated kinase inhibitors for 10 min. After a further 10 min of incubation with PMA (,) or the DMSO vehicle control (), opsonized SRBC (,) or iNHS-treated SRBC () were added to the MDM for phagocytosis at 37°C for 1 h. Quantification of ingested SRBC was performed as described in Materials and Methods. Shown here are results (mean SD) from one experiment. Each data point was performed in triplicate and error bars indicate standard deviations. (d) Monocyte-derived macrophages were plated for 2 h prior to assay. Immunoglobulin-opsonized SRBC () were then added to MDM for phagocytosis at 37°C for 30 min. Quantification of ingested SRBC was performed as described in Materials and Methods. Shown here are representative results (mean SD) from three independent experiments.

Full figure and legend (34K)

Quantification of phagocytosis of C'-opsonized sheep red blood cell targets by monocyte-derived macrophages

C'-mediated phagocytosis by MDM was assessed under the conditions established using cells from eight different donors cultured for a mean of 12 days (range 6–15 days) after isolation. As shown in Table 1, the mean phagocytic index of MDM was 199 59 (mean SEM; range, 47–490). For all experiments, iNHS-coated SRBC controls were below or very close to the lower limit of detection of the assay (data not shown).

Table 1 - C'-mediated phagocytosis by monocyte-derived macrophages.

Full table

Inhibition of C'- and Fc-mediated phagocytosis by HIV-1 infection of monocyte-derived macrophages

Monocyte-derived macrophages from three separate donors were infected with HIV-1_Ba-L, which is an M-tropic laboratory- adapted strain of HIV-1. Infected and mock-infected cells were then assayed at the same time for their ability to phagocytose either C'-opsonized or IgG-opsonized SRBC. The data show that C'-mediated phagocytosis was strongly inhibited by HIV-1 infection, as was Fc-mediated phagocytosis (Table 2).

Table 2 - Inhibition of C'- and Fc-mediated phagocytosis by HIV-1 infection of monocyte-derived macrophages.

Full table

Discussion

Phagocytosis assays for monocytes often use non-adherent cells cultured in suspension and flow cytometric assessment of internalization of fluorescently labelled targets.^{26, 27} Adherent cells may better reflect the in vivo physiological environment of macrophages as effector cells. Conventional assays using adherent cells usually involve microscopic analysis of internalized particles,^{20, 21, 22, 28} and investigator bias can diminish their usefulness. The C'-mediated phagocytosis assay reported in the present study employs an adaptation of a colourimetric method described previously by Gebran and coworkers²⁴ for FcR-mediated phagocytosis, eliminating the need for manual microscopic counting, and is highly reproducible and independent of investigator bias. The protocol also enables the measurement of only ingested targets and not attached particles. In addition, a simple and inexpensive method to produce C'-opsonized SRBC as phagocytic targets is provided.

Commonly used targets for measuring C'-mediated phagocytosis are C'-coated zymosan, latex beads and erythrocytes.^{21, 22, 26} Zymosan is resistant to lysis by C' components and can be opsonized adequately using NHS.^{29, 30} Latex beads are also resistant to lysis but cannot be fixed directly with C' and they must be coated by BSA and anti-BSA Ig prior to incubation with serum.²⁶ Despite the relative ease of opsonization of these lysis-resistant particles, their use in assays is not ideal because attached and engulfed particles cannot be differentiated easily. The extent of attachment must be additionally measured (e.g. by using cytochalasin D to inhibit microtubules required for phagocytosis but not the attachment of particles²⁶). Another commonly used procedure is to use fluorescently labelled targets followed by quenching of the attached fluorescent targets using a non-cell permeable quenching agent.²⁹ However, the reliability of quenching is debatable and, in our experience, these methods may be associated with a high background.²⁷

In contrast, attached erythrocytes can be hypotonically lysed under conditions that do not lyse macrophages, thus allowing precise quantification of internalized particles.^{20, 21, 31} As SRBC are susceptible to C' lysis, the published protocols for opsonizing SRBC use purified C' components (e.g. C3, factor I, factor H), or sera deficient in one of the later C' components that constitute the membrane attack complex that is responsible for lysis^{20, 31} instead of whole serum. The present study reports for the first time conditions that allow opsonization of SRBC by untreated whole serum without generating substantial lysis, a rapid and inexpensive protocol for the production of C'-opsonized targets.

Because C' is heat labile,⁶ iNHS was used to coat SRBC as a negative control. The negligible phagocytosis of iNHS-treated SRBC, as well as the PKC dependency of NHS-opsonized SRBC phagocytosis observed in the present study, confirms the specificity of the assay and demonstrates that opsonization of SRBC by xenoantibodies present in human serum³² does not result in significant phagocytosis via Fc-mediated pathways under the conditions used here for opsonization and phagocytosis. The dependence of phagocytosis on PMA stimulation (and thus PKC activity) and inhibition with the PKC inhibitor RO 31-8220, but not the tyrosine kinase inhibitor genistein, are in agreement with earlier studies by other investigators showing the dependence of C'-mediated phagocytosis on PKC activation.^{22, 33, 34} This assay is particularly useful for such pharmacological investigations because the protocol employs 96-well plates, allowing a comparison of C'-mediated phagocytosis of MDM from the same donor with numerous different treatments.

C'-mediated phagocytosis requires C' receptors to be activated. Despite pre-incubation of MDM with PMA to activate C' receptors, the phagocytic index remained variable and low. Because it has been reported that lengthy adherence of MDM to either plastic or glass can activate C' receptors for phagocytosis,²¹ the protocol has been modified to adhere cells for 24 h instead of 2 h prior to assay.

We have shown previously that HIV-infection of human MDM impairs their ability to phagocytose M. avium complex,¹⁵ implying that complement-mediated phagocytosis is impaired in MDM because M. avium complex is principally ingested via a complement-dependent path-way.^{13, 14} In the present study, we show directly, using specifically opsonized target particles, that complement-mediated phagocytosis is impaired when MDM are infected with HIV-1_Ba-L and that the degree of inhibition is at least as strong as that of Fc-mediated phagocytosis. We are currently using this assay in the laboratory to investigate the signalling pathways involved in C'-mediated phagocytosis by macrophages and the effect of HIV-1 infection on this function. In addition, this assay provides a useful tool for the examination of the effects of therapeutic agents on C'-mediated phagocytosis and, thus, may assist in assessing new adjunctive therapies for restoration of activity against pathogens entering cells via this pathway (e.g. M. avium complex) in immunodeficient patients.

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Acknowledgements

The authors wish to thank Prof. J Mills for his critical review of the manuscript and Mr D Doischer for his technical assistance. This work was supported by a grant to SMC from the Australian National Council on AIDS and Related Diseases through the Australian National Centres in HIV Virology Research and the Macfarlane Burnet Centre Research Fund. KK is a recipient of an NHMRC PhD scholarship.