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Infection and Immunity, March 1999, p. 1347-1352, Vol. 67, No. 3
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Gamma Interferon Stimulates Rat Alveolar Macrophages To Kill Pneumocystis carinii by L-Arginine- and Tumor Necrosis Factor-Dependent Mechanisms

James F. Downing,dagger Diane L. Kachel, Rajamouli Pasula, and William J. Martin II*

Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Department of Medicine, Indiana University Medical Center, Indianapolis, Indiana 46202-2879

Received 30 July 1998/Returned for modification 24 August 1998/Accepted 15 December 1998


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Pneumocystis carinii pneumonia remains a serious complication for immunocompromised patients. In the present study, P. carinii organisms interacted with gamma interferon (IFN-gamma )-stimulated alveolar macrophages (AMs) to activate the L-arginine-dependent cytocidal pathway involving reactive nitrogen intermediates (RNI) that were assayed as nitrite (NO2-). Unstimulated cultures of AMs produced negligible quantities of RNI. Addition of P. carinii organisms to IFN-gamma -primed AMs resulted in greatly enhanced production of RNI. NO2- levels increased from 0.8 ± 0.4 to 11.1 ± 3.8 µM as early as 6 h after P. carinii organisms were incubated with IFN-gamma -stimulated AMs and to 35.1 ± 8.9 µM after a 24-h incubation, a near-maximum level. High levels of NO2- were produced by AMs primed with as little as 10 U of IFN-gamma per ml in the presence of P. carinii, and a 20-fold increase in IFN-gamma concentration resulted in only a further 65% increase in NO2- production. RNI-dependent killing of P. carinii was demonstrated by both a 51Cr release assay and a [35S]methionine pulse immunoprecipitation assay. Addition of either monoclonal tumor necrosis factor alpha (TNF-alpha ) neutralizing antibody or 200 µM NG-monomethyl-L-arginine (L-NGMMA), a competitive inhibitor of the L-arginine-dependent pathway, significantly decreased NO2- production and reduced P. carinii killing. TNF-alpha alone had no effect on P. carinii viability. These results suggest that (i) the specific interaction of P. carinii organisms with IFN-gamma -primed AMs triggers the production of RNI, (ii) RNI are toxic to P. carinii, and (iii) TNF-alpha likely plays a central role in mediating P. carinii killing by IFN-gamma -stimulated AMs.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Pneumocystis carinii pneumonia remains a significant cause of morbidity and mortality in immunocompromised patients (15, 40). Individuals with AIDS seem to be particularly susceptible to infection with this pathogen. The mechanisms which underlie this susceptibility to P. carinii organisms remain unclear. Despite recent advances in both the diagnosis and the treatment of P. carinii pneumonia, the full range of immunologic responses of the host to the organism is not understood (13, 39).

Alveolar macrophages (AMs) provide active immune surveillance against many potentially infectious microbes (23, 48) including P. carinii (29, 49). The function of AMs is regulated, in part, by interactions with T lymphocytes and cytokines. A major function of gamma interferon (IFN-gamma ), which is primarily a product of CD4+ lymphocytes, is macrophage activation (30). The progressive loss of CD4+ lymphocytes in AIDS is associated with a significant decrease in the release or synthesis of cytokines such as IFN-gamma (33). Thus, AMs from patients with AIDS are fully capable of directed microbicidal activity but probably lack appropriate activation signals mediated by T-cell-secreted lymphokines (12, 32).

Activated macrophages possess several distinct cytotoxic mechanisms including O2-derived radicals (3), reactive nitrogen intermediates (RNI) (17, 18, 44, 46), tumor necrosis factor alpha (TNF-alpha ) (9, 19), and cytolytic proteases (1, 2). It has been suggested elsewhere that both TNF-alpha and oxygen intermediates are capable of directly killing P. carinii (37), and yet, the relevance of these findings to macrophage-mediated killing of P. carinii is uncertain. As an example, the effect of TNF-alpha in P. carinii pneumonia may not relate to its proposed direct toxic effect on the organism (37); rather, it may be secondary to TNF-alpha 's ability to stimulate AMs to generate RNI which can be lethal to P. carinii organisms. TNF-alpha has recently been demonstrated to be released by mononuclear cells in direct response to either P. carinii or a specific P. carinii protein (6, 8). Furthermore, anti-TNF antibody can significantly reduce clearance of P. carinii organisms in vivo (7). TNF is upregulated in vivo in both healthy and immunodeficient mice; however, immunodeficient mice develop P. carinii pneumonia, suggesting that other immune factors are required to control the infection (21). Recently, a study used in vivo gene transfer of a TNF inhibitor and demonstrated significant exacerbation of P. carinii pneumonia (22).

In the present study, IFN-gamma -primed AMs release cytotoxic levels of RNI in response to P. carinii organisms. Furthermore, the IFN-gamma -primed AMs are directly cytotoxic to P. carinii organisms, and the killing can be largely abrogated by an anti-TNF-alpha neutralizing antibody or NG-monomethyl-L-arginine (L-NGMMA), a potent inhibitor of the L-arginine-dependent generation of RNI. These studies support the hypothesis that the loss of priming by cytokines such as IFN-gamma may be a critical immune deficit preventing an effective response to P. carinii organisms. Further, the response of the IFN-gamma -primed AMs is largely dependent on TNF-alpha to augment L-arginine-dependent production of RNI for effective killing of P. carinii.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

AM isolation. AMs were obtained from pathogen-free Harlan Sprague Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, Ind.) as previously described (38). Rats were briefly ether anesthetized prior to the intraperitoneal injection of euthanasia solution. A 14-gauge angiocatheter was inserted into the trachea proximal to the bifurcation, and the lungs were lavaged six times with 8-ml aliquots of Hanks balanced salt solution without Ca2+, Mg2+ supplemented with 0.6 mM EDTA, penicillin (100 U/ml), and streptomycin (100 µg/ml). The lavage fluid was centrifuged (600 × g) for 10 min to pellet inflammatory cells. Cytopreparation smears were prepared and stained as previously described (38) and revealed >95% of the cells obtained to be viable AMs. Cells were resuspended in Hanks balanced salt solution and incubated in 24-well tissue culture plates at a density of 106 AMs/well in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum, glutamine (0.6 mg/ml), penicillin (100 U/ml), and streptomycin (100 µg/ml). AMs were allowed to adhere at 37°C for 2 h prior to removal of nonadherent cells. Throughout the 24- to 48-h incubation, polymyxin B (100 U/ml) (Calbiochem, San Diego, Calif.) was included in all samples to minimize any effects of contaminating endotoxin.

P. carinii isolation. P. carinii pneumonia was induced in pathogen-free, female Lewis rats (Harlan Sprague Dawley, Inc.) by steroid-induced immunosuppression and transtracheal inoculation of P. carinii organisms as previously described (4). Briefly, animals were given water supplemented with dexamethasone (2 µg/ml), tetracycline (500 µg/ml), and nystatin (200 U/ml) ad libitum. Rats were transtracheally administered 106 P. carinii organisms 5 to 7 days after initiation of immunosuppression. P. carinii organisms were harvested 6 to 8 weeks after inoculation when the animals were moribund with P. carinii pneumonia. Rats were sacrificed as described above, and the lungs were lavaged. P. carinii organisms were isolated by differential centrifugation as previously described (4). After centrifugation (600 × g, 10 min) to pellet inflammatory cells, the lavage fluid containing P. carinii was centrifuged (1,400 × g, 30 min), and the pellet was resuspended in DMEM at a concentration of 107 to 108 P. carinii organisms per ml.

Assay for nitrite. Nitrite (NO2-) concentrations in the medium of cultured AMs represent an accurate measure of RNI. NO2- was quantified by a microplate assay method according to the work of Ding et al. (10). Briefly, 100-µl aliquots of conditioned medium were removed from AM cultures and incubated with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine hydrochloride, and 2.5% H3PO4) at room temperature for 10 min. The absorbance at 540 nm was determined with a Titertek microplate enzyme-linked immunosorbent assay reader (Flow Laboratories, McLean, Va.). NO2- concentrations were determined from a standard curve with sodium nitrite (Eastman Kodak, Chicago, Ill.) from 0 to 200 µM.

Cytotoxic effect of acidified nitrate on P. carinii. Toxicity of RNI to P. carinii organisms was quantified by two independent methods. First, a 51Cr release assay was utilized as previously described (27, 39, 50). P. carinii organisms were incubated in DMEM with or without fetal bovine serum containing 1 µCi of [51Cr]sodium chromate (New England Nuclear, Boston, Mass.) per ml for 18 h. Organisms were washed four times in DMEM to remove unincorporated 51Cr. Approximately 2 × 106 P. carinii organisms were incubated at 37°C for 24 h in 1.5 ml of DMEM titrated to pH 4.5 and containing varying concentrations of sodium nitrite. NO2- was not added to control samples. Following the incubation, P. carinii organisms were centrifuged at 12,000 × g for 10 min in an Eppendorf 5415C microcentrifuge (Brinkmann Instruments, Inc., Westbury, N.Y.). The cell pellet was washed twice with an equal volume of DMEM. Cytotoxicity was expressed as percent 51Cr release.

To determine if RNI interfered with the biological activity of P. carinii organisms, a second method, which examined [35S]methionine incorporation into P. carinii proteins and monitored P. carinii injury as a function of a decrease in newly synthesized 35S-labeled immunoprecipitated P. carinii proteins, was used. P. carinii organisms were incubated with acidified NO2- as described above. After an 18-h incubation, each sample was pulsed with 1 µCi of [35S]methionine (Amersham, Arlington Heights, Ill.) for an additional 6-h incubation. Samples were solubilized by repeated freeze-thaw, and nonsoluble debris was removed by brief centrifugation at 1,000 × g for 5 min. Aliquots of supernatant (50 µl) were incubated with 20 µg of monoclonal anti-P. carinii antibody (Accurate Scientific, Westbury, N.Y.) for 2 h at room temperature. This antibody recognizes a P. carinii-specific 116-kDa protein and does not significantly cross-react with any proteins produced by AMs. Pulse-labeled P. carinii proteins were then immunoprecipitated by the addition of protein A-agarose and incubated for an additional 2 h prior to centrifugation at 12,000 × g for 5 min. The pellet was extensively washed four times before quantification of P. carinii-specific radiolabeled proteins as described elsewhere (47). Autoradiography of sodium dodecyl sulfate-polyacrylamide gels and Western blot analysis confirmed that a single major P. carinii-specific 116-kDa protein was isolated by this method. The inhibitory effect of acidified nitrate on P. carinii-specific protein synthesis was expressed as follows: 100 × [1 - (counts per minute of NO2- treated)/(counts per minute of control)].

AM-mediated killing of P. carinii. AMs were isolated by adherence and incubated in DMEM with or without polymyxin B for 2 h prior to addition of recombinant rat IFN-gamma (Gibco, Grand Island, N.Y.). Following incubation, 51Cr-labeled P. carinii or unlabeled P. carinii organisms were added to AM cultures at a ratio of 10 P. carinii organisms to 1 AM. Following an additional 24-h incubation, cytotoxicity to P. carinii was determined by 51Cr release and [35S]methionine pulse immunoprecipitation. The inhibitory effect of activated macrophages on P. carinii was measured by 51Cr release and by percent inhibition of [35S]methionine incorporation into P. carinii-specific proteins.

Inhibition of P. carinii killing by L-NGMMA and anti-TNF-alpha antibody. AMs in DMEM containing polymyxin B were incubated with either 200 µM NGMMA (Calbiochem) or 50 µg of monoclonal TNF-alpha neutralizing antibody (Genzyme, Chicago, Ill.) for 2 h. After IFN-gamma was added to AM cultures at a final concentration of 0 to 200 U/ml (200 µl), the incubation continued for an additional 24 h. Following the incubation, P. carinii organisms (107) were added to the cultures, and the effects were assessed by the 51Cr release assay and the [35S]methionine pulse-label immunoprecipitation as described above.

Effect of TNF-alpha alone on P. carinii killing. P. carinii organisms were incubated in the presence of 100 U of rat TNF-alpha (Genzyme, Cambridge, Mass.) per ml in DMEM for 6 h at 37°C. Mouse L929 cells (American Type Culture Collection, Manassas, Va.) were utilized as a positive control in the standard 51Cr release cytotoxicity assay (29).

Statistics. Results are expressed as means ± standard deviations (SD). Data analysis was performed by two-way Student's t test or by analysis of variance (ANOVA) for multiple comparisons. Significance was accepted for P < 0.05. All experiments were performed in triplicate.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

P. carinii-stimulated production of RNI by IFN-gamma -primed AMs. Initial studies were performed to determine the optimal time of incubation and IFN-gamma concentration to stimulate the production of RNI in AM cultures in the presence of P. carinii. By utilizing IFN-gamma at 100 U/ml, NO2- production was determined to be time dependent from 0 to 36 h (P < 0.001, ANOVA) (Fig. 1a). By using IFN-gamma at 100 U/ml, NO2- in AM culture supernatants from P. carinii-infected AMs increased from 0.8 ± 0.4 µM NO2- to a maximum of 44.4 ± 9.7 µM in the presence of P. carinii at 36 h (Fig. 1a). In the absence of IFN-gamma , negligible amounts of NO2- were detectable. With IFN-gamma stimulation, a significant amount of NO2- was detectable as early as 6 h of incubation with P. carinii. This is consistent with previous studies in our laboratory which have demonstrated that P. carinii attachment to AMs occurs within the first 4 h of incubation (38). After 24 h of incubation, 35.1 ± 8.9 µM NO2- was present, which is near the maximal levels produced by AMs as a function of time.


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  		FIG. 1.   Time course and IFN-gamma concentration dependence of NO2- production by IFN-gamma -primed AM cultures exposed to P. carinii. Cultured AMs were treated with recombinant IFN-gamma for 24 h in the presence of polymyxin B prior to the addition of P. carinii trophozoites at a ratio of 10 P. carinii organisms to 1 AM. NO2- production increased as a function of time (*, P < 0.001, ANOVA) (a). Priming AMs with increasing IFN-gamma concentrations prior to addition of P. carinii resulted in increased NO2- production (*, P < 0.001, ANOVA) (b). Data represent the means ± SD.

The induction of NO2- in AM cultures by P. carinii was dependent on the length of incubation and also on the concentration of IFN-gamma . In the absence of IFN-gamma stimulation, measurable NO2- was negligible (0.8 ± 0.4 µM). Over a concentration range of 0 to 200 U/ml, IFN-gamma -induced NO2- synthesis increased from control levels of 0.8 ± 0.4 to 41.1 ± 10.6 µM with 200 U of IFN-gamma /ml (Fig. 1b) (P < 0.001, ANOVA). NO2- generation (17.2 ± 1.1 µM) was significantly inhibited in the presence of L-NGMMA (11.3 ± 0.8 µM, P < 0.05) but not in the presence of the stereoisomer D-NGMMA (16.8 ± 0.5 µM). Neither L-NGMMA nor D-NGMMA was toxic to AMs by a 51Cr assay (data not shown). These data demonstrate the sensitivities of AMs to IFN-gamma regarding RNI synthesis. Near-maximum levels of NO2- were produced in AM cultures exposed to P. carinii when primed with as little as 10 U of IFN-gamma per ml. However, a 20-fold increase in IFN-gamma resulted in only a 65% further increase in NO2- production by AMs.

Effect of NO2- on viability of P. carinii. Chemically generated RNI were directly cytotoxic to P. carinii organisms. Assay of P. carinii injury by both the 51Cr release assay and the [35S]methionine immunoprecipitation method produced similar results. For example, at 1 mM acidified nitrate, (46.9 ± 7.5)% 51Cr was released (Fig. 2a); and the [35S]methionine immunoprecipitation assay (Fig. 2b) resulted in a (48.8 ± 13.8)% suppression of protein synthesis as assessed by a decrease in immunoprecipitable [35S]methionine counts (P < 0.05, both comparisons). These results clearly demonstrate that the P. carinii organisms are sensitive to toxic RNI.


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  		FIG. 2.   RNI generated by acidified NO2 have P. carinii inhibitory activity. Freshly isolated P. carinii organisms were first labeled for 18 h with 51Cr prior to incubation for 24 h in DMEM adjusted to pH 4.5 and containing varying concentrations of sodium nitrate. 51Cr released from P. carinii was determined, and P. carinii injury was expressed as 51Cr release (a). Alternatively, P. carinii organisms were incubated in acidified NO2 solution and pulsed with [35S]methionine during the last 6 h of incubation. Inhibition of protein synthesis was then quantified from immunoprecipitation of a specific P. carinii protein by using a monoclonal anti-P. carinii gp116 antibody (b). Data represent the means ± SD.

IFN-gamma -primed AMs inhibit P. carinii by L-arginine-dependent RNI. To examine the role of L-arginine-dependent RNI production in the killing of P. carinii by IFN-gamma -primed AMs, the IFN-gamma -primed AM-mediated cytotoxicity against P. carinii was determined in the presence or absence of L-NGMMA. IFN-gamma -primed AM cultures directly injured P. carinii organisms with 51Cr release of (52.2 ± 6.6)% as assessed by a 51Cr release assay (Fig. 3A). In the presence of L-NGMMA, the injury was reduced to (16.7 ± 5.1)% (P < 0.01). Similar results were obtained by the [35S]methionine pulsed immunoprecipitation method. IFN-gamma -primed AMs inhibited P. carinii-specific protein synthesis by (47.4 ± 7.8)%. In the presence of the inhibitor L-NGMMA, this level of protein synthesis inhibition was significantly reduced to (5.4 ± 2.1)% (P < 0.01) (Fig. 3B). L-NGMMA alone did not affect P. carinii protein synthesis or viability (data not shown). These data demonstrate that (i) IFN-gamma -primed AMs are cytotoxic to P. carinii and (ii) the L-arginine-dependent pathway to RNI is responsible for a significant portion of this cytotoxicity.


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  		FIG. 3.   Inhibitory activity toward P. carinii by IFN-gamma -primed AMs is primarily dependent on RNI generation. Adherent AMs were cultured in the presence of IFN-gamma (100 U/ml) for 24 h prior to the addition of P. carinii. Incubation was continued for an additional 24 h. The dependence on RNI synthesis for killing was demonstrated by addition of 200 µM L-NGMMA, a competitive inhibitor of the L-arginine pathway. L-NGMMA prevented AM-mediated killing of P. carinii as determined by either 51Cr release (a) or inhibition of [35S]methionine incorporation into P. carinii proteins (b). Data represent the means ± SD.

TNF-alpha mediates P. carinii cytotoxicity by IFN-gamma -primed AMs. Inclusion of neutralizing TNF-alpha antibody with the IFN-gamma -primed AMs incubated with P. carinii demonstrated that TNF-alpha is important for the mediation of P. carinii injury by IFN-gamma -primed AMs. P. carinii organisms stimulated IFN-gamma -primed AMs to generate significant amounts of RNI (Fig. 4). However, in the presence of anti-TNF-alpha antibody, RNI production was significantly reduced at each concentration of IFN-gamma (10 to 200 U/ml) tested (P < 0.01, all comparisons). This strongly suggests that TNF-alpha may be an important mediator of P. carinii-stimulated generation of RNI by the IFN-gamma -primed AMs.


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  		FIG. 4.   Antibody to TNF-alpha reduces P. carinii-stimulated RNI production by IFN-gamma -primed AMs. AMs were cultured in the presence of increasing concentrations of IFN-gamma (0 to 200 U/ml) for 24 h prior to the addition of P. carinii. Incubation was continued for an additional 24 h in the presence or absence of 50 µg of monoclonal TNF-alpha neutralizing antibody. The TNF-alpha antibody significantly reduced NO2- production at each concentration of IFN-gamma tested (P < 0.01, each comparison). Data represent means ± SD.

Furthermore, antibody to TNF-alpha reduced P. carinii injury by IFN-gamma -primed AMs. By the 51Cr release assay, anti-TNF-alpha antibody significantly reduced the percent 51Cr release by P. carinii mediated by IFN-gamma -primed AMs. IFN-gamma -primed AMs injured P. carinii with a (51.2 ± 6.6)% 51Cr release, and this was diminished to (24.8 ± 11.2)% in the presence of neutralizing anti-TNF-alpha antibody (P < 0.05) (Fig. 5A). Similar results were obtained by the [35S]methionine immunoprecipitation method. The inhibition of P. carinii-specific immunoprecipitable counts ([47.4 ± 7.8]%) by IFN-gamma -primed AMs was reduced to nearly control levels ([9.1 ± 6.4]%) in the presence of anti-TNF-alpha antibody (P < 0.01) (Fig. 5B). There was no evidence that anti-TNF-alpha antibody by itself was toxic to AMs by 51Cr release assay (data not shown). These results suggest that TNF-alpha may be necessary as a critical intermediary of P. carinii injury by IFN-gamma -primed AMs. TNF-alpha alone had no effect on P. carinii viability (data not shown).


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  		FIG. 5.   TNF-alpha has a direct regulatory role in P. carinii killing and NO2- production in IFN-gamma -primed AM cultures. Adherent AMs were cultured in the presence of IFN-gamma (100 U/ml) for 24 h prior to the addition of P. carinii. Incubation was continued for an additional 24 h. Anti-TNF-alpha antibody prevented injury to P. carinii as measured by either 51Cr release (A) or inhibition of [35S]methionine incorporation into P. carinii proteins (B). Data represent the means ± SD.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

P. carinii pneumonia is a serious complication which occurs in immunocompromised subjects and has an especially poor prognosis in AIDS (34). Due to inherent difficulties in quantifying P. carinii viability, relatively little is known regarding how the immune response to P. carinii results in clearance and killing of P. carinii. IFN-gamma , a cytokine deficient during AIDS, is effective in the treatment of P. carinii infection in vivo (5, 41). One of the primary functions of IFN-gamma is activation of AMs. Fully activated macrophages can produce a number of toxic products including RNI which are necessary for adequate host defense.

The present study provides direct evidence that (a) IFN-gamma -primed AMs produce RNI in response to the interaction with P. carinii organisms, (b) chemically generated RNI are directly cytotoxic to P. carinii, (c) cytotoxicity to P. carinii by IFN-gamma -activated AMs correlates with the induction of the L-arginine-dependent generation of RNI, and (d) TNF-alpha is likely an important regulatory cytokine in macrophage-mediated cytotoxicity to P. carinii organisms.

The interaction of P. carinii with IFN-gamma -primed AMs activates the RNI pathway. IFN-gamma 's role in priming AMs for L-arginine-dependent cytotoxicity is clearly demonstrated, as only negligible levels of NO2- are detectable in AM cultures in the absence of this macrophage-activating factor. Consistent with our results, P. carinii does not appear to stimulate NO2- in unstimulated AMs previously unexposed to P. carinii (45). However, the addition of P. carinii organisms to IFN-gamma -primed AMs provides the triggering signal for production of RNI, similar to the results in a previous report (44).

The ability of RNI to kill P. carinii or inhibit P. carinii metabolism was directly examined by use of acidified nitrate. The effector molecules mediating anti-P. carinii activity resulting from acidification of nitrate include NO, NO2-, and HNO3 (25). A low pH is required to generate these RNI species from nitrate, and this may have physiological relevance to the acidic microenvironment of the AM phagolysosome. The relevance of RNI as important products of human macrophages is less certain, as some investigators have found minimal evidence of RNI production in human AMs (36), whereas others provide evidence for its potential role in host defense (20, 35).

This study demonstrated that the L-arginine-dependent pathway was directly involved in the in vitro killing of P. carinii by IFN-gamma -primed AMs. L-NGMMA, an inhibitor of the formation of RNI, reduced NO2- synthesis and AM-mediated killing of P. carinii. Furthermore, the degree of reversal of P. carinii killing in activated AM cultures by this competitive inhibitor suggests that the arginine-dependent pathway may be the primary mechanism for eliminating P. carinii by IFN-gamma -activated AMs. Additional AM functions that are candidates for effectors induced by IFN-gamma include reactive oxygen compounds and/or tryptophan starvation (31). While it is likely that the physiological response to P. carinii by AMs involves multiple cytotoxic-cytostatic mechanisms, it appears that RNI-dependent mechanisms may be a predominant mechanism of killing of P. carinii by IFN-gamma -primed AMs.

The concentration of RNI released by activated AMs into the supernatant in response to P. carinii (Fig. 1) that resulted in P. carinii injury (Fig. 3) was a much lower concentration than that noted with the chemical generation of RNI (Fig. 2). This may be for one of two reasons: (i) intact AMs have multiple cytotoxic mechanisms available to kill P. carinii or (ii) the concentration of RNI generated by activated AMs in the direct vicinity of the P. carinii organisms may be much higher than can be accurately assayed in the supernatant of these cells. Nonetheless, data from the present study demonstrate that RNI generated either from AMs or by chemical means are directly cytotoxic to P. carinii organisms.

In contrast to our study, a recent in vivo study could not correlate the upregulation of inducible nitric oxide synthase (iNOS) in AMs with the host response to P. carinii in either healthy or CD4 lymphocyte-depleted animals (43). Clearly, the cytokine cascade responsible for triggering iNOS upregulation in response to P. carinii infection is complex and may differ depending on how the experimental conditions are structured. In our study, the role of IFN-gamma in priming AMs in vitro to kill P. carinii is much easier to define, and yet, in vitro studies such as ours suffer from eliminating the complex interaction of other immunomodulatory cytokines as occurs in vivo. Our study suggests that AMs possess the necessary armamentarium to kill P. carinii in vitro with RNI. The reasons underlying the absence of adequate upregulation of iNOS in vivo are unclear. Similarly, other studies demonstrate that, even in the presence of an adequate RNI response, P. carinii infection can progress (16). These studies underscore that an adequate RNI response is not sufficient by itself to confer resistance to P. carinii infection.

The precise role of TNF-alpha in host defense against P. carinii remains uncertain. In contrast to a prior report by Pesanti (37), TNF-alpha in our assay was not directly cytocidal to P. carinii. Our findings are consistent with another report showing no direct cytotoxic effect by TNF (28). However, neutralizing antibody to TNF-alpha reduced AM-mediated killing of P. carinii organisms in our study, suggesting that TNF-alpha is important in killing of P. carinii by AMs. Another recent report also suggests that TNF-alpha is a critical intermediary in IFN-gamma stimulation of RNI production (14). TNF-alpha may have an important indirect role in P. carinii pneumonia, as it may be the direct mediator of NO2- production in an autocrine stimulatory fashion of IFN-gamma -primed AMs. It is possible that TNF-alpha is coproduced as part of the cytokine cascade and acts synergistically with IFN-gamma to enhance NO2- synthesis.

Taken together, these data demonstrate that IFN-gamma -activated AMs effectively kill P. carinii organisms by the TNF-alpha -mediated generation of RNI. Others have also noted a synergism between IFN-gamma and TNF-alpha in the generation of RNI (26). However, effective clearance of organisms in vivo likely requires more than the simple reconstitution of a missing cytokine. For example, although AMs are likely the primary phagocytic cells responsible for the clearance of P. carinii (24), functional AMs by themselves are insufficient for clearance (16, 51). CD4+ lymphocytes are necessary for effective clearance of P. carinii organisms (16, 42, 51). A recent report indicates that the simple absence of even critical proinflammatory cytokines such as IFN-gamma and TNF-alpha does not confer susceptibility to P. carinii, nor does their presence in the absence of CD4+ lymphocytes confer resistance (16). Nonetheless, selective loss of a single cytokine can be critical, as evidenced by a subject with a mutation in the IFN-gamma receptor transducing chain who developed disseminated opportunistic infection (11). The host response to opportunistic infection requires orchestration of many humoral and cellular factors to effectively clear the organism. It is clear that the optimal clearance of opportunistic organisms such as P. carinii requires both the correct proportion of cytokines and a functional cellular response.


    ACKNOWLEDGMENTS

This work was supported in part by National Institutes of Health grants HL/A143524, HL51962, and HL61285. J.F.D. was supported by National Institutes of Health grant T32 AM07532.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, 1001 West 10th St., OPW 425, Indianapolis, IN 46202-2879. Phone: (317) 630-8445. Fax: (317) 630-6386. E-mail: wjmartin{at}iupui.edu.

dagger Present address: Indiana Nephrology, Kokomo, IN 46902.

Editor:   T. R. Kozel


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Infection and Immunity, March 1999, p. 1347-1352, Vol. 67, No. 3
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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