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. 2001 Feb;123(2):239–246. doi: 10.1046/j.1365-2249.2001.01465.x

The impact of CMV on the respiratory burst of macrophages in response to Pneumocystis carinii

A L Laursen* *, S C Mogensen †, H M K Andersen ‡, P L Andersen* *, S Ellermann-Eriksen †
PMCID: PMC1905976  PMID: 11207654

Abstract

Infection of human monocyte-derived macrophages with CMV decreased the respiratory burst when cells were stimulated with opsonized zymosan or Pneumocystis carinii (P. carinii). Such an effect, though smaller, was also seen with heat-inactivated CMV, but only when triggered by zymosan. The effect was most pronounced in cells obtained from CMV antibody-negative donors. Dexamethasone further reduced the respiratory burst, both in uninfected and CMV-infected cells. Interferon-γ increased the response in uninfected cells and, to a lesser extend, in cells treated with heat-inactivated CMV, whereas no effect was seen with infective CMV. No overt productive infection or cytopathology could be detected, however, the monocytes incubated with infective but also heat-inactivated CMV formed clusters, a phenomenon that was equally pronounced in cultures from CMV antibody positive and negative-donors. These results might help explain the worse prognosis of P. carinii pneumonia in patients coinfected with CMV and receiving dexamethasone.

Keywords: Macrophage, CMV, P. carini, dexamethasone, interferon-γ

INTRODUCTION

In immunosuppressed patients cytomegalovirus (CMV) and Pneumocystis carinii are well known causes of severe pneumonia. However the relative importance of the two agents depends on the underlying cause of the immunsuppressed state. Thus patients with HIV-induced immunodeficiency are more prone to infections with P. carinii than to pneumonia caused by CMV [1], whereas patients immunosuppressed due to treatment for haematological malignancies or undergoing transplantation are prone to both infections [2]. The reason for this difference is not clear.

In studies performed before the widespread use of corticosteroids in P. carinii pneumonia (PCP) conflicting results as to the impact of CMV on PCP were reported [1,3].

More recent studies have suggested that the use of steroids as adjunctional treatment in patients with PCP may carry with it a worse prognosis when the patient is coinfected with CMV [4,5]. Additionally steroids have been shown to accelerate CMV infection in patients with HIV infection [6] and in vitro to activate CMV [7].

To elucidate the influence of CMV on PCP, we have studied the effect of the virus on the ability of opsonized P. carinii organisms to elicit a respiratory burst in cultivated human mononuclear cells. Furthermore, the effect of dexamethasone and interferon-γ (IFN-γ) on activation of the respiratory burst in uninfected-and CMV-infected cells was investigated.

MATERIALS AND METHODS

Production and opsonization of Pneumocystis carinii

Wistar male rats were immunosuppressed with dexamethasone 1 mg/l added to the drinking water, which was furthermore supplemented with tetracyclin 1 g/l to prevent secondary infections. After two weeks of immunosuppression the animals were inoculated intratracheally with P. carinii (obtained from the American Type Culture Collection), as previously described by Boylan and Current [8]. When pneumonia ensued, usually after 6–8 weeks, animals were sacrificed, the lungs were removed aseptically, and imprints of lung sections were examined for bacterial and fungal infections other than PCP. Lung tissue was cut into small pieces, digested with collagenase and hyaluronidase, and P. carinii organisms were purified by percoll gradient centrifugation as previously described [9]. P. carinii cysts were enumerated by microscopy and stored at −70 °C until use.

P. carinii organisms were trypsinized at 37 °C for 30 min using 0·25% trypsin (Sigma type XI; St Louis, MO) to remove bound antibodies and proteins covering the parasites, followed by incubation at 37 °C for 30 min in 70% serum from a blood donor. The presence of antibodies and complement on the surface of P. carinii was examined by direct immunofluorescence (FITC-conjugated rabbit antihuman IgG and anti C3c, DAKO, Copenhagen, Denmark).

Opsonization of zymosan

Immediately before each experiment zymosan, purified from Saccharomyces cerevisiae (Sigma, St Louis, MO), was opsonized in human serum, obtained from a blood donor, by incubating the suspension for 30 min at 37 °C. After centrifugation at 400 × g the pellet was dissolved in Krebs-Ringer-phosphate buffer containing 10 mm glucose to a final concentration of 1·5 mg/ml.

Production and quantification of CMV

Monolayers of outgrown MRC-5 cultures grown in Eagle's Minimal Essential Medium (Eagle's) supplemented with 10% foetal calf serum, 200 IU/ml penicillin and 200 μg/ml streptomycin were inoculated with the Ad 169 strain (1 virus/cell). Ten to 12 days later, when the cultures showed 90–100% CPE, the supernatant was poured off, centrifuged at 3000 × g for 30 min to remove cell débris, aliquoted, and stored at −70 °C until use. To inactivate virus, samples were heated to 56 °C for 30 min, a procedure that destroyed all infectivity.

Quantification of the virus was performed in the same cells grown in the rapid shell vial centrifugation culture assay system. Undiluted or serial 10 fold diluted fluid material or 10% cell suspension in PBS was each inoculated onto 2 MRC−5 coverglass cultures in shell vials (25 μl material in 75 (l PBS). The shell vials were then centrifuged for 40 min and overlaid with cell culture medium (Eagle's). After 44 h at 37 °C the cultures were fixed in 80% acetone in PBS for 10 min and washed twice in PBS. The number of CMV-positive cells stained with IMAGEN™ (DAKO) immunofluorescence monoclonal antibodies antibody to CMV immediate-early antigen were counted after which the amount of virus/ml fluid could be calculated.

Isolation and cultivation of mononuclear cells

Human peripheral blood mononuclear cells (PBMC) from blood donors were isolated from heparin-stabilized blood by flotation on Isopaque-Ficoll (Nyegaard, Oslo, Norway), washed twice in PBS, and resuspended in RPMI medium (Gibco, Paisley, UK) supplemented with 10% fetal calf serum (FCF), penicillin 100 U/ml, netilmycine 100 mg/ml and glutamine 2 mmol/l to yield a final concentration of 2 × 106 cells/ml. Aliquots of 1 ml were transferred to chemiluminescence vials (Sarstedt, Nümbrecht, Germany) and incubated at 37 °C in 5% CO2. The mean fraction of CD14 positive cells, estimated by immunofluorescence using anti CD14 antibodies (FITC-conjugated rabbit anti-CD14, DAKO) was 35% (SD 10%). Plasma samples from the cell donors were analysed for IgG antibodies against CMV by a semiautomated ELISA system (BioMérieux, Marcy-l'Etoile, France) as recommended by the manufacturer.

Experimental procedure

In all experiments 2 × 106 blood-derived mononuclear cells were grown in chemiluminescence vials for 8 days, allowing monocytes to differentiate into macrophages. On day one after establishment of the cultures, the cells were infected with CMV at a multiplicity of infection (m.o.i.) of 0·5 viral p.f.u. per cell, treated with an equivalent amount of heat-inactivated virus or left untreated for the remaining 7 days. The virus infection was performed as follows. The culture medium was substituted with 0·5 ml of a virus suspension containing 1 × 106 pfu/ml, centrifuged at 500 × g for 90 min and left for 30 min at 37 °C. The viral suspension was then removed and substituted with RPMI medium. Control cells and cells receiving heat-inactivated virus were treated in the same manner.

In some experiments dexamethasone (Merck Sharp and Dohme, NJ) or human IFN-γ (PeproTech, Rocky Hill, NJ) was added at various timepoints in doses indicated in individual experiments.

In order to study the ability of cultured mononuclear cells to sustain a productive infection, half of the supernatants was removed from each of triplicate cultures at various time points after infection. These supernatants and the remainder of the cultures, were frozen at −70 °C for later determination of the amounts of extracellular and intracellular virus, respectively, by titration in MRC-5 cells as described above. To calculate the amounts of intracellular virus, the estimated amounts of extracellular virus was subtracted from the total yield of virus from the frozen culture

To study the ability of CMV to induce cytopathic changes mononuclear cells were grown in flat-bottomed culture vials and treated as above with infective and heat-inactivated CMV. The appearance of the cultures was recorded and photographed in an upside-down microscope at various time points

Measurement of respiratory burst by chemiluminescence

The respiratory burst was measured by chemiluminescence (CL) in a luminometer (model 1250; LB-Wallac, Turku, Finland) with a water-jacket cuvette holder ensuring a steady temperature of 37 °C. Cuvettes containing 2 × 106 mononuclear cells and 80 μM lucigenin (Sigma) were temperature-equilibrated for 5 min and the response without P. carinii was recorded. The response after addition of 10 × 106 opsonized P. carinii cysts was then measured every 2 min for 60 min and the maximal CL response was recorded and expressed as mV.

Statistics

Student's t-test was used to analyse differences in the chemiluminiscence response between different groups. The analysis was performed using the Microsoft Excel® programme.

RESULTS

Chemiluminescence response with opsonized zymosan

In preliminary experiments the basal conditions for activation of the respiratory burst in mononuclear cells were established regarding number of cells, length of cultivation, and the dose of CMV. Serum-opsonized zymosan was used as the stimulant in these experiments. Infection of 1–5 × 106 mononuclear cells for 7 days with CMV at a m.o.i. of 0·5–2·0 CMV pfu/cell gave a consistent reduction of superoxide production as compared with uninfected cells (data not shown).

In the following experiments we chose to infect 2 × 106 cells for 7 days at a m.o.i. of 0·5. Using these conditions we analysed mononuclear cell cultures from a total of 7 blood donors. As seen from Fig. 1(a), infection with CMV reduced the CL response by approximately 50% (P < 0·001), whereas treatment of the cells with heat-inactivated virus resulted in a much less pronounced, yet statistically significant, reduction (P = 0·02). The difference between the effect of infectious and noninfectious CMV was also statistically significant (P = 0·009).

Fig. 1.

Fig. 1

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Effect of CMV on Zymosan-induced respiratory burst of mononuclear cells. The cells were seeded and after 24 h infected with CMV at a multiplicity of infection of 0·5 or treated with an equivalent amount of inactivated virus or with pure medium. After 7 days of culture, the cells were assayed for zymosan-induced chemiluminescence. (a) represents the results from all examined donors (n = 12); (b) the stratified results from CMV seronegative (n = 7) and (c) the stratified results from CMV seropositive (n = 5) donors. All examinations were performed with triplicate cultures. Columns represent the mean results from the indicated experiments, and bars indicate the S.E.M. of the means from each experiment.

To examine whether the observed effect of CMV was related to the CMV seropositivity of the cell donors, we analysed the results in relation to the CMV antibody status of the donors (Fig. 1b,c). The depression of CL was observed in both groups of donors, but even though the effect of both infectious and heat-inactivated virus seemed to be highest in seropositive donors, this difference was not statistically significant (P = 0·21).

Chemiluminescence response with opsonized P. carinii

We next examined the effect of CMV on the CL response elicited by opsonized P. carinii organisms. As seen with opsonized zymosan, treatment of mononuclear cells with infectious CMV for 7 days significantly reduced the CL response (Fig. 2a), both relative to cells treated with medium only (P < 0·001) and with heat inactivated CMV (P = 0·005). Contrary to what was seen with opsonized zymosan, heat-inactivated CMV did not reduce the CL response of cells triggered with opsonized P. carinii (P = 0·37).

Fig. 2.

Fig. 2

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Effect of CMV on P. carinii-induced respiratory burst of mononuclear cells. Twenty-four h after seeding the cells were infected with CMV at a multiplicity of infection of 0·5 or were treated with an equivalent amount of inactivated virus or pure medium. After 7 days of infection, the cells were assayed for chemiluminescence response to opsonized P. carinii. (a) represents the results from all examined donors (n = 14); (b) the stratified results from CMV seronegative (n = 6) and (c) the stratified results from CMV seropositive (n = 8) donors. All examinations were performed with triplicate cultures. Columns represent the mean results from the indicated experiments, and bars indicate the S.E.M. of the means from each experiment.

When the data were analysed according to the CMV antibody status of the donors (Fig. 2b,c) we found that the effect of infectious CMV on the CL response was highly significant in seropositive donors, both as compared to the response of control cells and to the response of cells treated with heat-inactivated CMV (P < 0·001 and 0·002, respectively). Although a similar effect of infectious CMV was apparently seen in seronegative donors, this difference was not statistically significant (P = 0·34 and 0·25, respectively). The effect of heat-inactivated virus was more elusive with apparent opposite effects in seronegative and seropositive donors.

Effect of dexamethasone on activation of the respiratory burst

Steroids are often used in the treatment of PCP and we therefore examined, whether dexamethasone would also by itself or together with CMV interfere with the CL response of mononuclear cells to opsonized P. carinii. In preliminary expts 1 μm dexamethasone was shown significantly to reduce the zymosan-induced CL response in untreated mononuclear cells and no further decrease was seen, when higher concentrations were used (data not shown). We therefore chose to use this dose in further experiments with P. carinii. It is seen in Fig. 3 that 1 μm dexamethasone significantly reduced the maximal P. carinii-induced CL response in untreated cells (P < 0·001; Fig. 3) as well as in cells treated with heat-inactivated CMV (P = 0·004). In cells treated with infective CMV, in which the CL response was already severely impaired, dexamethasone also tended to decrease the response (P = 0·10). However, infective CMV and dexamethasone together could not lower the responses further than what was seen with inactivated CMV.

Fig. 3.

Fig. 3

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Effect of dexamethasone and IFN-γ on the P. carinii-induced respiratory burst of CMV-infected and uninfected mononuclear cells. Cells were treated with dexamethasone (1 µm) during all 8 days of culture or treated with IFN-γ (100 IU/ml) for the last 48 h of culture. Furthermore, cells were either left uninfected (a), were infected with CMV at a multiplicity of infection of 0·5 (c), or were treated with an equivalent amount of inactivated virus as indicated (b). After 7 days of infection the cells were assayed for chemiluminescence response to opsonized P. carinii. All experiments were performed with triplicate cultures. Columns represent the mean results from 7 experiments, and bars indicate the S.E.M. of the means from each experiment.

Effect of IFN-γ on the chemiluminescence response

Since IFN-γ is known to possess both immunomodulatory and antiviral effects, we wanted to study the influence of this proinflammatory cytokine on the CL response elicted by P. carinii and to examine whether it would be able to counteract the inhibitory effect of CMV infection.

Priming of the cells with 100 IU/ml IFN-γ added once daily for the last two days of incubation significantly increased the P. carinii-induced CL response in untreated mononuclear cells (P = 0·03; Fig. 3). The same trend was seen in cells incubated with heat-inactivated CMV, but the difference was smaller and not statistically significant (P = 0·16). In cells treated with infective CMV, IFN-γ did not have any effect (P = 0·89).

CMV replication and cytopathogenicity

In order to examine whether CMV replicated productively in mononuclear cells we titrated the amounts of supernatant-and cell-associated virus at various time points after the infection. As seen from Fig. 4, virus in the amounts of 103−104 pfu/ml could be recovered 24 h after infection, and even though this seemed marginally increased in the cell-associated fraction at day 3 of infection, no signs of a permissive virus replication in the mononuclear cells were recorded.

Fig. 4.

Fig. 4

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Infectious CMV in cultures of mononuclear cells. Cells were seeded and after 24 h infected with CMV at a multiplicity of infection of 0.5. (▪). After 1 h of adsorption the virus-containing medium was removed, and fresh culture medium was added (indicated by the arrow). At the indicated time after infection, cultures were assayed for extracellular (•) and cell-associated (○) infectious CMV. Each point represents the titre of CMV in one culture, and the lines are drawn between the mean log-titre in each group.

During culture, cells infected with CMV or treated with heat-inactivated virus showed morphological changes distinct from what was seen in cultures receiving medium only. The changes consisted mostly of a tendency of the monocytes/macrophages to aggregate in large clumps (Fig. 5). The effect was seen equally pronounced in cultures treated with infective and inactivated virus and irrespective of the serological status of the donor. The phenomenon was observed from day 2 and remained unchanged during culture. No signs of a CMV-like cytopathic effect with cytomegalic changes were observed.

Fig. 5.

Fig. 5

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Morphology of CMV-infected and uninfected mononuclear cells. Cells were seeded and after 24 h infected with CMV at a multiplicity of infection of 0·5 (a), or treated with an equivalent amount of heat-inactivated virus (b) or with pure medium (c). After 6 days of infection micrographs were taken. The bar indicates 100 µ m.

DISCUSSION

It is well-known that some viral infections may predispose to infections with other microorganisms [10]. Part of this disposition has been demonstrated to be due to impairment of phagocyte functions [11]. CMV is known to have immunosuppressive properties affecting T-lymphocytes [12,13], whereas the effect on phagocytic cells is less well known.

In the present study we have demonstrated that CMV can reduce activation of the respiratory burst in human monocyte-derived macrophages. Reduction of superoxide production was seen both when opsonized zymosan – a potent activator of the alternative complement pathway [14]– and P. carinii opsonized with human serum were used to stimulate superoxide production. The effect was more pronounced in mononuclear cells from CMV antibody-positive persons; than from CMV seronegative donors.

Several effects by CMV may explain our results. First a direct cytopathic effect could be the underlying cause, but was not demonstrated. In cultures infected with CMV, the monocytes/macrophages tended to aggregate in clusters. However these changes were also seen in cells incubated with heat-inactivated CMV, thus it is not due to CMV infection. Second, an immunological recall reaction of specific anti-CMV lymphocytes present in the cultures might be involved in the phenomenon, either by reducing the number of virus-infected macrophages by a cytotoxic T cell reaction or in a less conspicuous way impairing the ability of phagocytic cells to produce superoxide. The latter possibility is more likely since cultures derived from CMV seropositive and –negative donors looked alike micoscopically. However cytokine production from lymphocytes present in cell cultures might affect macrophages in CMV seropositive donors and reduce activation of the respiratory burst. The intracellular pathway leading to activation of the respiratory burst is far from clarified, however, Th2 type cytokines like IL-4 and IL-10 have been shown to decrease the respiratory burst in human macrophages [15,16]. Furthermore IL-4 production has been demonstrated by human blood lymphocytes from CMV seropositive donors when exposed to CMV [17] and a Th2 orientated profile has been found in kidney transplanted patients with CMV infection when compared with uninfected transplanted controls [18]. Against the hypothesis involving a cytotoxic T cell reaction speaks the similarity between cultures derived from CMV sero-positive and -negative donors. However the more pronounced effect seen in CMV antibody-positive donors suggests that an immune mechanism might be a contributing factor. A third, and also likely possibility would be a direct effect of viral glycoprotein-binding to cellular surface receptors, or a blocking effect of immediate-eraly (IE) or early CMV gene-products on the pathway leading to activation of the respiratory burst. Also supporting this is the report that purified CMV glycoproteins can induce production of IL-1 and activation of the intracellular transcription nuclear factor (NF-κ B) [19]. Different results have been reported on the ability of the Ad 169 CMV stain to infect monocyte-derived macrophages. Thus Minton et al. [20] were unable to detect IE antigen, whereas Söderberg et al. [21] using a cultivating system including lymphocytes, as in this study, were able to detect both IE and the late pp65 antigen in monocyte-derived macrophages infected with the Ad 169 strain.

The effect of CMV on the respiratory burst in human macrophages has to our knowledge not previously been studied. In lung transplanted patients CMV has been associated with invasive aspergillosis [22], the control of which depends, at least in part, on superoxide production in phagocytic cells [23,24]. Furthermore, in an animal model Miller et al. [25] have demonstrated a decreased H2O2 production in guinea pigs infected with CMV.

Hydrocortisone has previously been shown to increase production of IE gene products of CMV and also to induce unrestricted replication in monocyte-derived macrophages [26] which might explain the reduced respiratory burst in cells incubated with dexamethasone. In our study preincubation of cells with dexamethasone reduced the respiratory burst. If dexamethasone was given together with CMV, both infectious and heat-inactivated, a more severe reduction of the respiratory burst was seen, although the response was not completely abolished.

The putative ability of CMV to aggravate the clinical outcome of pneumonia caused by P. carinii in HIV-infected patients has been the subject of several clinical studies reaching different conclusions. Thus Stover et al. [1] found a higher mortality in PCP patients from whom CMV was isolated, whereas Jakobsen et al. [3] found no association between concomitant CMV infection and PCP. These studies were performed before the widespread use of corticosteroids to treat moderate to severe PCP in AIDS patients. More recent studies by Hylander et al. [4] and Jensen et al. [5] have related coinfection of CMV and P. carinii to a worse prognosis. Administration of corticosteroids to such patients infected with CMV leads to increased viral production. Thus, the results presented in this study may offer some explanation to the clinical data presented above. Among transplanted patients CMV infection has also been correlated to the occurrence of PCP [26], an association that could be related to a suppressive effect of the virus infection on the respiratory burst.

IFN-γ has previously been shown to increase superoxide production in monocyte-derived macrophages stimulated with P. carinii [27]. These results were confirmed in this study using chemiluminescence to study activation of the respiratory burst. Interestingly, this effect was not seen in cells treated with either heat-inactivated or infectious CMV. Thus, a more prolonged or irreversible block of superoxide production seems to have taken place. When opsonized zymosan was used to stimulate macrophages instead of P. carinii, no effect of IFN-γ on the respiratory burst of mononuclear cells was demonstrated (Laursen et al. unpublished). IFN-γ is known to promote expression of the Fcγ I receptor binding monomeric IgG and mediating phagocytosis [28] and responsible for activation of the respiratory burst, whereas the C3b complement receptor, on the other hand, is actually downregulated by IFN-γ[29]and since zymosan is known to be a potent activator of the alternate complement pathway [14], the different results obtained with IFN-γ depending on the triggering stimulus, most likely reflect the different receptors used by opsonized zymosan and P. carinii to stimulate the phagocytic cells.

In conclusion we have demonstrated a suppressive effect of CMV on the respiratory burst in human monocyte-derived macrophages when stimulated with P. carinii and zymosan opsonized with human serum. Preincubation with dexamethasone by itself reduced superoxide production, a reduction which was aggravated in cells infected with CMV. IFN-γ was unable to reverse the effect of CMV. Our results thus suggest that uncritical and long-term use of corticosteroids, especially in CMV-infected patients, may impair the defence against P. carinii in accordance with some clinical observations.

Acknowledgments

The skilful technical assistance of Mr Erik H. Nielsen, Ms Birthe Søby, Ms Maria Moussavi, and Ms Margit Aagaard is greatly acknowledged. This study was supported by grants from The Danish Lung Association, The Danish Health Science Research Council, and The Danish Foundation for the Advancement of Medical Science, Institute of Clinical Experimental Research (University of Aarhus).

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