Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Pneumocystis carinii is an opportunistic, pulmonary pathogen which causes pneumonia in individuals suffering from a variety of immunodeficiencies (1). Although infection has been observed in humans and animals with genetic B cell defects (2, 3), the majority of cases have been associated with AIDS (Acquired Immune Deficiency Syndrome) or other conditions which affect CD4⁺ T-cell number and function (4). Animal studies have demonstrated that mice lacking CD4⁺ lymphocytes due to either passive antibody treatment (5) or targeted gene disruption (6) are susceptible to P. carinii infection. In addition, adoptive transfer studies in severe combined immunodeficient (scid) mice have specifically demonstrated that CD4⁺ T-lymphocytes are critical for natural host resistance (7). The scid mouse model provides a unique and defined system for studying the host response to P. carinii. Scid mice have functional macrophages, but lack lymphocytes of T- and B-cell lineage and are susceptible to a wide variety of opportunistic infections, including P. carinii (10). Importantly, spleen cells or fractionated CD4⁺ T-cells from immunocompetent mice can be adoptively transferred into these mice to reconstitute their immune system, and mediate clearance of P. carinii from the lung (8, 11). In the absence of CD4⁺ T-cells, scid mice develop a progressive P. carinii infection which eventually causes death. Despite the large number of organisms present in the alveoli, and the presence of alveolar macrophages, there is a remarkable lack of pulmonary inflammation in these scid mice until the very late stages of disease. However, when spleen cells or CD4⁺ T-cells from immunocompetent mice are used to immunologically reconstitute infected scid mice, an intense inflammatory cascade is initiated (8, 11). Infammatory cell infiltration begins at day 7 and peaks at day 12 post-reconstitution (PR) and clears the organisms from the lung over a 21-day period (11). This phenomena has been demonstrated to be CD4⁺ T cell-dependent, indicating that these helper T lymphocytes function at some level to mediate the inflammatory cascade necessary to clear infection (5, 8).

Cytokines are important peptide mediators of inflammation, which are involved in host resistance to a variety of pulmonary bacterial (12), viral (13), and fungal (14) infections. Interleukin-1 (IL-1) and tumor necrosis factor (TNF)- are potent inflammatory cytokines which are critical for the clearance of P. carinii from the lungs of reconstituted scid mice (15, 16). In addition, roles for interferon- (IFN-) (17), interleukin-6 (IL-6) (18), and granulocyte macrophage-colony stimulating factor (GM-CSF) (19) in the host response to P. carinii have been suggested. Cytokines are produced and secreted by many cells of the lung, including those types which directly interact with P. carinii. These include CD4⁺ T-lymphocytes (20), alveolar macrophages (21, 22), and the pulmonary epithelium (23). Although it is unlikely that CD4⁺ T-cells directly kill P. carinii and clear the alveoli, they are known to mediate inflammatory and immune processes through local regulation of the complex cytokine network (20). CD4⁺ T-cells are not only capable of producing and secreting several cytokines themselves, but can also activate other cell types, such as macrophages, for the expression of these proinflammatory mediators (24). Despite these findings, the effect of CD4⁺ T-cells on the temporal expression of proinflammatory cytokine mRNAs in the lungs of a P. carinii-infected host has yet to be examined. The scid mouse provides a valuable model to study P. carinii-induced pulmonary cytokine expression in the presence or absence of the critical CD4⁺ T-cells.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Animals

Male CB.17^+/+ and CB.17 scid/scid mice were obtained from a colony at the Trudeau Institute Animal Breeding Facility (Saranac Lake, NY). The mice were bred and housed in microisolator cages, and were free of common murine pathogens. The foundation stock was originally obtained from Dr. Leonard Schultz of the Jackson Laboratory (Bar Harbor, ME). In order to induce infection, 3-wk-old scid mice were cohoused with P. carinii-infected scid mice for 5 wk. At 8 wk of age, 30 P. carinii-infected scid mice were immunologically reconstituted as previously described (15). Briefly, spleens were asceptically removed from 6-wk-old male CB.17^+/+ donor mice, gently pushed through stainless steel screens into Hanks' balanced salt solution (HBSS), and then triturated with a Pasteur pipette. The cells were washed twice with phosphate-buffered saline (PBS) (pH 7.2), counted, and then resuspended in PBS at a concentration of 3.5 × 10⁷ splenocytes/ml. P. carinii-infected scid mice were reconstituted with a 1-ml tail vein injection of the cell suspension.

Lung Tissue Preparation

At predetermined timepoints mice were killed by cervical dislocation. The chest cavity and surrounding connective tissue were cut open to expose the lungs and trachea. The lower left lung lobe was tied off at the bronchial airway with surgical string, and then removed with sterile scissors. The isolated lung tissue was immediately snap frozen in liquid nitrogen, and stored at 80°C for subsequent RNA isolation. For tissue fixation, a 20-gauge, 1.25 inch intravenous catheter unit was then inserted into the trachea, and tied in place with surgical string. The lungs were inflated with 30 cm gravity flow pressure of 2% gluteraldehyde, 100 mM cacodylic acid fixative (Sigma, St. Louis, MO). The lungs were fixed for 10 min under gravity flow pressure, and then removed from the animal and placed in fixative for 16 h at 4°C. The lungs were stored at 4°C in 100 mM cacodylic acid, pH 7.4. Prior to embedding, the fixed lungs were dehydrated in sequential 15-min washes of 30%, 50%, 70%, 80%, 90%, 95%, and 99% ethanol. At this time, the lower right lung lobe of each animal was removed, snapped into a tissue cassette, and then placed in xylene. Each lung lobe was embedded in parafin, and 4-µM sections were cut from the tissue blocks.

Ribonuclease Protection Assay

Total RNA was isolated from lung tissue using TRIzol Reagent (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. Each frozen lung lobe (50-100 mg) was homogenized in 1 ml of TRIzol Reagent. Each final RNA pellet was resuspended in 50 µl of diethylpyrocarbonate-treated water. The RNA concentration and purity was quantified using the GeneQuant RNA/ DNA Calculator (Pharmacia Biotech, Piscataway, NJ). Quantitation of steady-state cytokine mRNA levels was performed using a previously described multi-cytokine ribonuclease protection assay (RPA) (25). The mL-11 template set (a gift from Dr. Monte V. Hobbs) was used to transcribe radiolabeled, antisense riboprobes for murine IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6, TNF-, TNF-, IFN-, and the murine ribosomal protein, L32. The riboprobe synthesis reaction consisted of 60 ng mL-11 template set; 120 mCi [-³²P]UTP (uridine triphosphate) (3,000 Ci/mmol; Dupont NEN, Wilmington, DE); 5 nmol ATP (adenosin triphosphate); 5 nmol GTP (guanosine triphosphate); 5 nmol CTP (cytidine triphosphate); 150 pmol UTP; 2.5 µg yeast tRNA; 100 nmol dithiothreitol; 1 µg bovine serum albumin; 20 nmol spermidine; 10 U RNase inhibitor; and 50 U T7 RNA polymerase (Life Technologies) in 1× transcription buffer (40 mM Tris-HCl, pH 7.5, 6 mM MgCl₂). The reaction was incubated for 90 min at 37°C, and then diluted to 100 µl with DNase I buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 0.02 U/µl RQ1 DNase I [Promega, Madison, WI]). After a 30-min incubation at 37°C, the riboprobes were purified by phenol/chloroform extraction and ethanol precipitation. The dried pellet was then resuspended in 50 µl of hybridization buffer (400 mM NaCl, 40 mM PIPES, pH 6.7, 1 mM EDTA [ethylenediaminetetraacetic acid], pH 8.0, 80% formamide [Sigma]), analyzed by scintillation counting, and then diluted to a final concentration of 2.6 × 10⁵ counts per minute (cpm)/µl in hybridization buffer. Five-microgram samples of each RNA assayed were dried in a Speed-Vac Concentrator (Savant, Farmingdale, NY), and resuspended in 8 µl of hybridization buffer. Two microliters of the diluted riboprobe were added to each RNA sample, heated to 80°C for 3 min, and then immediately placed at 56°C for 16 h. After solution hybridization, 100 µl of RNase cocktail (0.2 µg/ml RNase A [Sigma], 600 U/ml RNase T1 [Life Technologies], 10 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM EDTA, pH 8) was added to each sample, and incubated 45 min at 30°C. Eighteen microliters of proteinase K cocktail (0.67 mg/ml proteinase K [Life Technologies], 3.5% sodium dodecyl sulfate, 100 µg/ml yeast tRNA) was then added to each sample, and incubated for 15 min at 37°C. The protected RNA duplexes were purified by phenol/chloroform extraction and ethanol precipitation, and the pellets were resuspended in 5 µl of RPA loading buffer (80% formamide, 0.5× TBE, 0.05% bromphenol blue [Sigma]). The protected, radiolabeled RNA fragments were electrophoresed on a 5% acrylamide/8 M urea sequencing gel, and the dried gel was used to expose X-AR film (Eastman Kodak, Rochester, NY) at 80°C with intensifying screens (Quanta III; Dupont, Wilmington, DE).

For quantitation, the dried gels were placed against phosphorimager screens (Molecular Dynamics, Sunnyvale, CA). The intensity of each specific cytokine band was measured using a computer-linked phosphorimager using the ImageQuant software (Molecular Dynamics). To correct for RNA loading, each intensity score was normalized to the intensity of hybridization for the L32 gene. A one-way analysis of variance (ANOVA) was performed using the SigmaStat 2.0 software (Jandel, San Rafael, CA) to determine the significance of observed variations in cytokine mRNA levels at the different timepoints.

In Situ Hybridizations

Murine cDNA clones for IL-1 and TNF- were subcloned into the plasmid vector, pBluescript II SK⁺ (Stratagene, La Jolla, CA), for the in vitro transcription of RNA (26). Sense and antisense orientations were confirmed on Northern blot analysis. IL-1 and TNF- antisense RNAs were transcribed from 1 µg of linearized plasmid template according to the procedure described above. After ethanol precipitation, the riboprobes were dissolved in diethyl pyrocarbonate (DEPC)-treated water. Full-length transcripts were approximately 1.4 kb for IL-1 and 1.5 kb for TNF-. Prior to hybridization, limited alkaline hydrolysis was performed to create riboprobes ranging in length from 0.1-0.3 kb. Hydrolyzed transcripts were sized by denaturing agarose gel electrophoresis.

Tissue sections were treated according to the method of Angerer and colleagues with modifications (27). Briefly, the sections were treated for 30 min at 37°C with 1 µg/ml proteinase K (Life Technologies). The slides were washed and then dipped in 0.25% acetic anhydride, 0.1 M triethanolamine, pH 8 for 10 min. After dehydration through a graded series of ethanol washes, the slides were dried and hybridized overnight at 56°C in 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1× Denhardt's solution (Life Technologies), 10% dextran sulfate, 0.5 mg/ml yeast tRNA, and 0.3 mg/ml antisense riboprobe. After hybridization, the slides were washed twice for 10 min and once for 40 min in 1× standard saline citrate (SSC). The slides were treated with 20 µg/ml RNase A (Sigma) diluted in RNase buffer (0.5 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) for 30 min at 37°C. The slides were then subjected to 30-min washes in RNase buffer at 37°C, 0.1× SSC at 22°C, 0.1× SSC at 68°C, and 0.1× SSC at 22°C. The slides were dehydrated by passing through a graded series of ethanol washes, dried, then exposed to NTB-2 emulsion (Kodak) for 21 days at 4°C. Slides were counterstained with hematoxylin and eosin to visualize lung architecture. After photodocumentation of the in situ hybridization slides, the coverslips were removed by soaking in xylene. The slides were then subjected to Gomori's methenamine silver (GMS) staining with fast green counterstain to visualize P. carinii organisms. Microscope coordinates were recorded for all photographs taken, so that identical lung regions could be examined for hybridization signal, as well as for P. carinii infection.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Pulmonary Cytokine Expression in scid Mice

Groups of three P. carinii-infected scid mice were killed at 8, 10, and 13 wk of age. In addition, three pathogen-free scid mice were killed as controls. Lung sections from these animals were stained with GMS. The control animals were found to be free of infection, while the exposed animals demonstrated increasing P. carinii infection with increasing age (data not shown). To determine whether pulmonary cytokine mRNA levels were induced by P. carinii infection in the absence of CD4⁺ T-cells, steady-state levels of cytokine mRNAs were measured in these animals using an RPA (Figure 1). Despite the presence of P. carinii organisms in the lungs of 8- and 10-wk-old scid mice, there was no significant increase in the steady-state mRNA levels of any of the cytokines measured (Table 1). However, IL-1 and IL-1 mRNA levels were significantly elevated above control values in the more heavily infected 13-wk-old scid mice. The mRNA levels for IL-1 and IL-1 were increased 2.1- and 4.4-fold, respectively, as compared with the P. carinii-free scid controls (Table 1). There was also a 3.3-fold increase in the level of TNF- mRNA in the 13-wk-old scid mice as compared with controls. This increase did not reach statistical significance in this experiment, but given the nature of immune reconstitution studies in scid mice, this increase in TNF- may be a significant observation (Table 1).

View larger version (77K): [in this window] [in a new window]   Figure 1.   Pulmonary cytokine mRNA abundance in P. carinii-infected scid mice. Steady-state mRNA levels in the lungs of infected 8-, 10-, and 13-wk-old scid mice, and from P. carinii-free scid mice were measured using a multi-cytokine RPA. The migration position of each cytokine-specific protected fragment, as determined from a standard curve based on the migration of RNA of known molecular weight, is denoted to the left. Lanes 1-3: P. carinii-free scid mice; lanes 4-6: 8-wk-old scid mice; lanes 7-9: 10-wk-old scid mice; lanes 10-12: 13-wk-old scid mice.

Pulmonary Cytokine Expression in Reconstituted P. carinii-infected scid Mice

Moderately infected 8-wk-old scid mice were reconstituted with a single tail-vein injection of unfractionated spleen cells from an immunocompetent donor animal. Groups of three mice were killed at 3, 4, 5, 7, 10, 12, 15, 18, 22, and 27 days PR. GMS staining demonstrated a moderate P. carinii infection up to 12 days PR, but no organisms were detected at days 22 and 27 PR (data not shown). To determine whether proinflammatory cytokines played a role in the pulmonary resolution of P. carinii infection in reconstituted scid mice, RPAs were performed (Figure 2). No significant changes in the mRNA levels of any of the cytokines assayed were observed prior to day 10 PR. Levels of TNF-, TNF-, IL-1, IFN-, IL-1, IL-6, and IL-3 mRNAs were all increased at day 10 PR, maximal at day 12 PR, and returned to control levels by day 22 PR when the P. carinii organisms had been cleared (Table 2). IFN-, IL-1, and TNF- demonstrated the largest increases in mRNA levels of 7.5-, 7.5-, and 7.3-fold above controls, respectively. IL-6 and TNF- were increased approximately 4.5-fold, while IL-3 and IL-1 were increased 3.1- and 2.4-fold above controls, respectively (Table 2). The cytokine mRNA profiles in the lungs of reconstituted P. carinii- infected scid mice undergoing resolution of infection were strikingly different from that observed over the same time period in infected scid mice which had not been immunologically reconstituted. The mRNA profiles for IL-1 and TNF- through either the resolution of infection, or the progression of disease are represented graphically in Figure 3. In reconstituted scid mice, the mRNA levels for TNF- and IL-1 increased sharply at days 10, 12, and 15 PR, then returned to baseline coincident with clearance of the organism from the lungs. In contrast, the unreconstituted scid mice did not demonstrate increased TNF- and IL-1 levels in response to P. carinii infection until late in the disease.

View larger version (81K): [in this window] [in a new window]   Figure 2.   Pulmonary cytokine mRNA abundance in immunologically reconstituted, P. carinii-infected scid mice. Eight-wk-old, infected scid mice were reconstituted with spleen cells from a normal donor, and steady-state mRNA levels in the lungs at various times post-reconstitution (PR) were measured using a multi-cytokine RPA. The migration position of each cytokine-specific protected fragment, as determined from a standard curve based on the migration of RNA of known molecular weight, is denoted to the left. Lanes 1-3: 4 days PR; lanes 4-6: 5 days PR; lanes 7-9: 7 days PR; lanes 10-12: 10 days PR; lanes 13-15: 12 days PR; lanes 16-18: 15 days PR; lanes 19-21: 18 days PR; lanes 22- 24: 22 days PR.

View larger version (11K): [in this window] [in a new window]   Figure 3.   Time course of TNF- and IL-1 mRNA abundance in immunologically reconstituted (closed diamond) and unreconstituted (open circle) P. carinii- infected scid mice. The relative mRNA abundance at each time point is a ratio of each cytokine: mL32. The time points are expressed as days post-reconstitution (DPR) for both groups of animals because they are age matched.

IL-1 and TNF- mRNA Expression In Situ

To identify areas of increased cytokine expression in the lungs of P. carinii-infected mice, in situ RNA:RNA hybridization was performed. Using radiolabeled IL-1 and TNF- antisense riboprobes, lung sections from pathogen-free scid mice, 8-wk-old P. carinii-infected scid mice, and 8-wk-old reconstituted P. carinii-infected scid mice (12 days PR) were analyzed. In the P. carinii-free control and moderately infected 8 wk-old scid mice, light, background hybridization was observed throughout the lung with both probes (Figures 4A, 4B, 5A, and 5B). This finding was in agreement with the RPA data demonstrating that there is minimal background expression of these cytokines in the lungs of P. carinii-free or moderately infected scid mice. GMS staining of the same lung sections after hybridization demonstrated the presence of P. carinii organisms in focal alveolar regions that were devoid of inflammation or cytokine expression (data not shown). In contrast, intense, focal hybridization was localized to specific alveolar regions of inflammation in the reconstituted P. carinii- infected mice (Figures 4C, 4D, 5C, and 5D). GMS staining of the same lung sections after hybridization indicated that regions of inflammation and increased cytokine expression corresponded to sites of P. carinii infection. Many host cells that were expressing elevated levels of IL-1 and TNF- were observed in direct contact with intact organisms, or contained degraded organisms within their cytoplasm (Figures 4D, 4E, 5D, and 5E). Although it was difficult to specifically identify the cell types responsible for cytokine expression in these inflammatory regions, many cells gave the appearance of activated macrophages (Figures 4F and 5F). Neither the airway epithelium nor the endothelium appeared to be involved in P. carinii-induced IL-1 and TNF- expression. Furthermore, not all of the alveoli were involved in the response. Certain regions of the alveoli appeared normal and unaffected by the infection, and were not sites of TNF- or IL-1 expression.

View larger version (128K): [in this window] [in a new window]   View larger version (128K): [in this window] [in a new window]   View larger version (140K): [in this window] [in a new window]   View larger version (108K): [in this window] [in a new window]   View larger version (92K): [in this window] [in a new window]   View larger version (102K): [in this window] [in a new window]   Figure 4.   IL-1 mRNA expression at focal regions of P. carinii colonization in reconstituted scid mice. In situ hybridization with an IL-1 antisense riboprobe was performed on lung sections from P. carinii-free (panel A), 8-wk-old P. carinii-infected (panel B), and reconstituted 8-wk-old P. carinii-infected (panels C, D) scid mice. Gomori's methenamine silver stain (panels E, F) was performed on the identical tissue section and microscope field as photographed in panel D. Panels A, B, C: original magnification ×100; panels D, E: original magnification ×400; panel F: original magnification ×1,000. Arrows denote cells containing elevated levels of IL-1 mRNA; arrowheads denote intact and degraded P. carinii organisms.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

CD4⁺ T-cells are important for resistance to P. carinii (5) as well as for mediating protective inflammatory responses against a variety of pulmonary pathogens through the regulation of local cytokine production (12). These observations led us to examine the effect of these lymphocytes on cytokine mRNA expression in P. carinii-infected scid mice. Our findings indicated that in the absence of CD4⁺ T-cells, 8- and 10-wk-old scid mice neither demonstrated elevated levels of cytokine mRNAs nor displayed signs of pulmonary inflammation in response to P. carinii infection. Elevated levels of IL-1 , IL-1, and TNF- mRNAs were observed in heavily infected 13-wk-old scid mice which had suffered severe lung damage and were nearing death. At this time, the elevated cytokine levels were likely a consequence of lung injury, were not protective for the host, and were possibly even exacerbating the disease. In contrast, when moderately infected 8-wk-old scid mice were reconstituted, they expressed elevated levels of several cytokine mRNAs, displayed signs of focal inflammatory cell infiltration, and resolved the infection. Levels of TNF-, TNF-, IFN-, IL-1, IL-1, IL-3, and IL-6 mRNAs were all elevated at day 10 PR, maximal at day 12 PR, and returned to baseline by day 22 PR. In addition, in situ hybridization demonstrated that at day 12 PR, IL-1 and TNF- mRNAs were highly abundant at focal regions of inflammation which corresponded to alveolar sites of P. carinii infection. The increased cytokine levels correlated temporally and spatially with the inflammatory cell infiltration observed in this study and by others also using the scid mouse model (11). Previous studies have demonstrated that P. carinii-free scid mice do not exhibit pulmonary inflammation after immune reconstitution, indicating that these results were caused by the organism, not the reconstitution (5). Thus, these data suggest that CD4⁺ T-cells mediate immune recognition of P. carinii and induce elevated levels of proinflammatory cytokine mRNAs at sites of infection. However, in the absence of CD4⁺ T-cells, scid mice are unable to respond to P. carinii infection with either increased cytokine mRNA levels or pulmonary inflammation.

One way in which CD4⁺ T-cells may induce proinflammatory cytokine production, and P. carinii clearance, is through the activation of alveolar macrophages. CD4⁺ T-cells are capable of activating macrophages for microbicidal killing and the release of cytokines, directly via receptor-ligand interactions (28), or indirectly through the release of cytokines such as IFN- and IL-3 (29, 30). There is substantial evidence that alveolar macrophages are important for the lung-specific phagocytosis of P. carinii (31, 32), as well as for proinflammatory cytokine production (33). Alveolar macrophages have surface receptors which can bind P. carinii directly or through host opsonins, and may facilitate phagocytosis (34). In addition, the macrophage is the principle cellular source of IL-1 and TNF- in the lung. These potent proinflammatory cytokines are secreted from macrophages after in vitro exposure to P. carinii (33, 36), and are critical for the clearance of P. carinii from the lungs of reconstituted scid mice (15, 16). Furthermore, activated CD4⁺ T-cells can induce macrophage secretion of these cytokines (37, 38). Despite these findings, alveolar macrophages appear blind and unable to respond to P. carinii organisms in the absence of CD4⁺ T-cells. Although there are functional macrophages present in 8-wk-old infected scid mice, we found no detectable cytokine or inflammatory response mounted against the organism. Furthermore, no macrophages were identified that had ingested or were in close contact with intact or degraded organisms. In contrast, reconstituted scid mice carrying a comparable burden of P. carinii demonstrated obvious histological signs of inflammation, including increased numbers of macrophages in the alveoli. They also expressed elevated levels of IFN- and IL-3, products of CD4⁺ T-cells which have been shown to play a role in macrophage activation (29, 30). In addition, these mice demonstrated highly elevated levels of IL-1 and TNF- mRNAs, which were localized to areas of P. carinii infection. The recognition of macrophages as major cellular sources of IL-1 and TNF-, and the presence of many activated macrophages in these regions, indicated that they contribute to the proinflammatory cytokine response. In addition, in situ hybridization demonstrated that many macrophages either in direct contact with intact organisms or that contained degraded organisms in their cytoplasm contained elevated levels of TNF- and IL-1 mRNAs. Although alveolar epithelial cells are also capable of producing these cytokines, the majority of hydridization signal was localized to alveolar macrophages, with only background mRNA levels observed in the epithelium. Thus, immune recognition of P. carinii by CD4⁺ T-cells induces elevated IL-1 and TNF- mRNA levels at sites of P. carinii infection. The helper T-cells may be activating macrophages, or at least providing a necessary costimulatory signal, which leads to the recognition of P. carinii and subsequently phagocytosis and/or expression of proinflammatory cytokines.

The relative ease with which P. carinii is killed in an immunocompetent host, and the number of host defense mechanisms present in the lung might indicate that this organism is susceptible to attack by a number of cell types. Yet the depletion of a single cell population, CD4⁺ T-cells, renders a host unable to kill P. carinii. Although scid mice lack CD4⁺ T-cells, they do possess functional alveolar macrophages and an intact alveolar epithelium, which both interact with P. carinii (11). These cell populations are also known to produce cytokines in response to direct interaction with a variety of stimuli, including infectious agents (22, 23). However, our findings indicate that cytokine mRNA levels are elevated at sites of P. carinii infection only after the transfer of normal CD4⁺ cells to scid mice, despite the fact that alveolar macrophages are present, and organisms are observed in direct contact with the alveolar epithelium. Thus, it appears that these cells are unable to respond to P. carinii in the absence of CD4⁺ T-cells. Previous work has demonstrated that IL-1 and TNF-, two potent mediators of inflammation, are required for the resolution of P. carinii infection in scid mice, even when functional CD4⁺ T-cells are present (15, 16). In the absence of CD4⁺ T-cells, pulmonary expression of IL-1 and TNF can be induced with heat-treated aerosols of Escherichia coli, or endotoxin alone (39). Such treatment causes acute pulmonary inflammation, which although targeted at the E. coli, can clear P. carinii from the lung in the absence of CD4⁺ T-cells (40). Thus, even though TNF and IL-1 are nonspecific mediators of pulmonary inflammation, which can be secreted by cells of the lung even in the absence of CD4⁺ T-cells, their mechanism of induction in response to P. carinii infection appears to be CD4⁺ T-cell-dependent.

Although the scid mice used in this experiment were reconstituted with total spleen cells, several reports have demonstrated that CD4⁺ T-cells are responsible for resistance to P. carinii (5, 6, 8). Scid mice reconstituted with fractionated CD4⁺ T-cells are able to mount an inflammatory response against P. carinii and clear infection in the absence of B cells. However, reconstitution with spleen cells, including B cells, results in quicker resolution of the infection (8, 9). It has been suggested that antibody production by B cells may facilitate the clearance of P. carinii in this model. Alternatively, B cells may provide additional costimulatory signals to CD4⁺ T-cells by binding CD40 ligand on these cells, and amplifying the antigen-specific T-cell response. Recent studies have found that functional CD40 ligand (CD40L), transiently expressed on the surface of activated T-cells, is critical for resistance (9). Disruption of cognate CD40L-CD40 interactions interferes with antigen-specific priming of CD4⁺ T-cells, therefore diminishing T-cell expansion, cytokine secretion, and the initiation of specific T-cell immune responses (41, 42). CD40L is also required for the CD4⁺ T-cell-mediated activation of macrophages, including inflammatory cytokine production (28). Thus, a possible mechanism of resistance is that CD4⁺ T-cells mount an antigen-specific response to P. carinii, which leads to elevated expression of pulmonary cytokines by T cells and alveolar macrophages, and subsequently pulmonary inflammation. However, in the absence of CD4⁺ T-cells, or when their cognate functions are disrupted, cytokine mRNA expression is not triggered, and the host cannot generate an effective inflammatory response.

Helper T-cells mediate the generation of pulmonary immune responses against pulmonary pathogens through regulation of local cytokine production. The cytokines that show highly elevated mRNA levels in the reconstituted scid model of P. carinii infection may serve several functions in the generation of a successful cell-mediated immune response. IL-1, IL-6, TNF-, and TNF- may promote CD4⁺ T-cell proliferation and activation in response to P. carinii, thus amplifying the initial immune response (43). IL-1 and TNF- may also play a role in the recruitment of additional CD4⁺ T-cells and macrophages to sites of P. carinii infection by inducing chemokine secretion (47) and upregulating the expression of intercellular adhesion molecules (48). In addition, TNF-, IFN-, and IL-3 may activate macrophages for microbicidal killing, as well as for further inflammatory cytokine production (17, 29, 30, 49, 50). However, it is important to note that the data presented herein are steady-state levels of cytokine mRNAs found in whole lung homogenates. Although pulmonary protein levels have not been measured, many studies have found that increased steady-state mRNA levels are indicative of increased protein levels. Consistent with our findings of elevated cytokine mRNA levels, increased levels of TNF- (16), IL-1 (15), IL-6 (18), and IFN- (17) protein have been found in the lungs of reconstituted P. carinii-infected scid mice during recovery. Thus, we suggest that the observed increased mRNA levels are associated with increased cytokine protein levels in the lung during resolution of P. carinii infection.

In conclusion, this study has compared pulmonary cytokine expression in P. carinii-infected scid mice in the presence or absence of CD4⁺ lymphocytes. In the absence of CD4⁺ T-cells, moderately infected scid mice demonstrate neither increased pulmonary cytokine expression nor detectable pulmonary inflammation in response to P. carinii infection. However, when scid mice carrying a comparable burden of organisms were immunologically reconstituted, both elevated pulmonary cytokine mRNA levels and acute alveolar inflammation were observed. Alveolar sites of infection were identified as regions of focal cytokine expression and inflammation. Thus, CD4⁺ T-cells play a critical role in the induction of pulmonary inflammation in response to P. carinii infection by inducing elevated pulmonary cytokine mRNA levels at sites of infection.