INTRODUCTION

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Mice lacking granulocyte/macrophage colony stimulating factor (GM-CSF) generated by homologous recombination develop PAP (1, 2). Surfactant proteins (SP-A, -B, and -C) were all significantly increased in BAL from these GM-CSF knockout mice compared with wild-type mice, and their lungs contained eosinophilic acellular material in alveolar spaces accompanied by increased tubular myelin. Interestingly, levels of surfactant mRNAs in mutant mice were indistinguishable from that in wild-type mice but alveolar macrophages from GM-CSF-deficient mice contained a marked increase of surfactant protein and lipid. The derangement in PAP was suggested to be related to defective macrophage surfactant catabolism or clearance. In addition, the authors postulated a regulatory role for GM-CSF in surfactant uptake, perhaps by regulating collectin receptors. Another murine model of PAP resulted from homozygous mutation of the GM-CSF c receptor gene demonstrated the necessity of GM-CSF signalling in macrophages to process surfactant (3). Correction of PAP in this model was accomplished by bone marrow transplantation from wild type mice to c deficient mice demonstrating the central role alveolar macrophages play in this disorder (4). Furthermore, Huffman and colleagues recently reported that pulmonary epithelial expression of GM-CSF using a SP-C-GM-CSF construct in GM-CSF deficient mice corrected the alveolar proteinosis and lymphocytic infiltrates (5). In a further animal model, PAP occurred in severe combined immunodeficient (SCID) mice that had nonfunctional T and B lymphocytes supporting a role for these cells in surfactant homeostasis (6).

We investigated the function of the GM-CSF gene in four consecutive whole lung lavages from a PAP patient and demonstrate that the GM-CSF gene in BAL cells could be induced by LPS in vitro, but that GM-CSF protein in the BAL cell supernatants was nondetectable with or without LPS. We found that the GM-CSF deficiency following LPS stimulation of BAL cells could be neutralized by anti-IL-10 antibody but not anti-TGF- antibody. These data represent a novel comparison of a disease model where GM-CSF deficient mice develop PAP, but in an adult patient with PAP there is inducibility of the gene but absence of protein. An additional case report of PAP in an Australian patient had improvement in symptoms, alveolar arterial oxygen gradient, and chest radiographs following subcutaneous injections of recombinant GM-CSF (7).

	METHODS

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Patient Summary

Our patient is a 29-yr-old Vietnamese female who had resided in the U.S. for 6 yr. She complained of dyspnea on exertion progressing to less than one block's distance before stopping. She was a lifelong nonsmoker with no occupational inorganic dust exposure or evidence of malignancy. Her chest radiograph revealed bilateral basilar alveolar and interstitial infiltrates. A diagnostic BAL demonstrated that recovered fluid was turbid and cream colored. Electron microscopy identified characteristic tubular myelin within macrophages and lavage fluid.

Whole lung lavage was performed December 1994, with a double lumen endotracheal tube. Procedure 1 was terminated after 13 L of lavage at which point the returned fluid was almost clear. The patient did well until August 1995 when she experienced increasing dyspnea and her chest radiograph again revealed bilateral infiltrates. A second whole lung lavage was performed with 21 L, of normal saline. The BAL cell differential was 58% macrophages, 36% lymphocytes and 6% neutrophils. Flow cytometry revealed 35% CD4+ cells and 56 CD8+ cells for a CD4+/CD8+ ratio of 0.6. The third whole lung lavage performed in October 1995 was on the opposite side and the cell differential was 81% macrophages, 14% lymphocytes and 5% neutrophils. A fourth whole lung lavage was performed October 1996, and the cell differential was 47% macrophages, 29% lymphocytes and 24% neutrophils. Flow cytometry revealed 51% CD4+ cells and 44% CD8+ cells for a CD4+/CD8+ ratio of 1.2.

As a comparison group, four normal volunteers (ages 27-32) who were nonsmokers had a research BAL with a fiberoptic bronchoscope. Under local anesthesia, five, 20 ml aliquots of normal saline were instilled sequentially followed by suction in the right middle lobe, right lower lobe, and lingula. None had occupational exposure to inorganic dust and all were in good health. The New York University Human Subjects Committee approved the protocol. The BAL cell differential means were macrophages 83 ± 2%, lymphocytes 15 ± 2%, and neutrophils 2 ± 2%.

Isolation and Culturing of BAL Cells

The recovered bronchoalveolar lavage fluid was filtered through sterile gauze. A total cell count was done in a hematocytometer, and cell differentials were performed on cytocentrifuge slides stained with Diff-Quick and 500 cells were counted. Cell viability was determined by trypan blue exclusion, and recovered cells were > 95% viable. Cells were washed in PBS 3× and resuspended in RPMI 1640 containing penicillin (100 U/ml) and streptomycin (100 µg/ml) at 1 × 10⁶ cells/ml in polypropylene tubes and cultured in the presence or absence of LPS (2 µg/ml) at 37° C for 24 h.

After culturing, BAL cells were spun down and supernatants collected and stored at 80° C. The amount of cytokines secreted into the supernatants were measured using commercial ELISA kits according to the manufacturer's instructions.

Reagents

Commercial kits for quantitative sandwich enzyme-linked, immunosorbent assays (ELISA) for GM-CSF (limit of detection 8 pg/ml) and IL-10 (limit of detection 15 pg/ml) were purchased from Biosource International (Camarillo, CA) and the IL-1-specific ELISA kit (limit of detection 2 pg/ml) was purchased from Cistron Biotechnology (Pine Brook, NJ). ELISA assays were performed in triplicate with the mean ± standard deviation presented.

Lipopolysaccharide (LPS) 055:B5 was purchased from Sigma (St. Louis, MO). Neutralizing antibody directed against IL-10 and control rat IgG antibody were purchased from Pharmingen (San Diego, CA) and pan-specific, neutralizing anti-TGF- antibody was purchased from R & D Systems (Minneapolis, MN).

RNA Analysis

Cytokine gene expression was analyzed by Northern blot analysis. Total RNA was isolated by guanidinium isothiocyanate lysis and cesium chloride centrifugation as described (8). Equal amounts of RNA were fractionated by electrophoresis through a 1% agarose-formaldehyde gel, and transferred to Hybond-N nylon filter (Amersham). cDNA inserts were labeled with [-³²P]dCTP by random priming. cDNAs for GM-CSF and IL-10 were obtained from Genetics Institute and ATCC, respectively. Filters were hybridized with labeled probes in Church buffer (7% SDS, 1% bovine serum albumin, 1 mM EDTA, 0.25 M Na₂HPO₄, pH 7.2) at 68° C overnight. Filters were washed in 2× SSC (1× SSC is 0.15 M NaCl, 0.015 M sodium citrate) 0.5% SDS at room temperature for 5 min, 2× SSC-0.1% SDS at room temperature for 15 min and then in 0.1× SSC-0.5% SDS at 65° C for 30 min. Autoradiography was performed at 80° C. To control for RNA loading, expression of the -actin gene was examined.

Statistics

Comparisons of unstimulated BAL cells to LPS-stimulated BAL cells for GM-CSF for the four normal controls used the Wilcoxon rank sum test.

	RESULTS

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Release of GM-CSF from BAL Cells from PAP and Normal Controls

GM-CSF was not spontaneously released by BAL cells obtained from any of the four whole lung lavages from our PAP patient (Sensitivity: 8 pg/ml), whereas GM-CSF release the four normal controls was only minimally present in one individual (Table 1). Upon stimulation with LPS, high levels of GM-CSF were secreted by BAL cells from normal controls (271 ± 160 pg/ml, p < 0.05). In contrast, no GM-CSF was secreted by LPS-stimulated BAL cells recovered from any of the four whole lung lavages of the PAP patient (Table 1).

Induction of GM-CSF mRNA Expression in PAP and Normals

To determine whether deficient GM-CSF production was due to the lack of GM-CSF gene induction, expression of GM-CSF mRNA was examined by Northern blot analysis. As shown in Figure 1A, expression of GM-CSF mRNA was strongly induced by LPS at 4 h but declined by 24 h. The Northern blot was performed on three of the four PAP whole lung lavages with LPS stimulation over unstimulated cells increasing 3.8-fold, 8.8-fold, and 5.6-fold. The kinetics of GM-CSF mRNA accumulation induced by LPS-stimulated BAL cells from the PAP patient were consistent with that of LPS-stimulated peritoneal mouse macrophages, with a maximal induction between 3-5 h (9). Induction of GM-CSF mRNA expression by LPS was also seen in normal BAL cells (Figure 1B).

View larger version (37K): [in this window] [in a new window]   Figure 1.   Northern blot analysis of GM-CSF mRNA induction by LPS. BAL cells isolated from (A) the PAP patient and (B) a normal control (NL) were induced with LPS (2 µg/ml) 4 and 24 h. As a control for RNA loading, expression of the -actin gene was also analyzed.

Inhibitory Role of IL-10 on GM-CSF Production

To gain insight into the mechanism of deficient GM-CSF production in the PAP patient, the role of inhibitory molecules was investigated. IL-10, also known as cytokine synthesis inhibitory factor, has been shown to inhibit the production of cytokines such as IL-1, IL-1, IL-6. IL-8. TNF-, GM-CSF and G-CSF at the transcriptional level (10). The role of transforming growth factor- (TGF-), a potent macrophage deactivator (12), was also investigated.

IL-10 mRNA was strongly induced at both 4 h and 24 h upon stimulation with LPS in BAL cells from the PAP patient (Figure 2A). In contrast, IL-10 mRNA was not inducible in fresh BAL cells from the normal controls studied after LPS stimulation for 24 h (Figure 2B). BAL cells from the PAP patient released IL-10 spontaneously on three out of four whole lung lavages, and IL-10 release could be further stimulated by LPS each time (Table 1). In contrast, IL-10 production by BAL cells from four normal controls was not induced following LPS stimulation (Table 1).

View larger version (40K): [in this window] [in a new window]   Figure 2.   Northern blot analysis of IL-10 mRNA induction by LPS. (A) PAP Patient. Total BAL cells from the PAP patient were cultured in the presence or absence of LPS (2 µg/ml) for 4 h and 24 h. As a control for RNA loading, expression of the -actin gene was analyzed. (B) Normal Control. Total BAL cells were untreated or treated with LPS (2 µg/ml) for 24 h.

To determine the role of IL-10 in suppressing GM-CSF production, total BAL cells from the PAP patient were stimulated with LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies. As demonstrated in Figure 3A, neutralization of IL-10 resulted in the release of GM-CSF stimulated by LPS (136 pg/ml) in amounts similar to normal controls (130-494 pg/ml) (Table 1). The specificity of IL-10 inhibition of GM-CSF production was further demonstrated by the fact that neutralizing anti-TGF- antibodies and control IgG antibodies failed to restore GM-CSF production (Figure 3A).

View larger version (32K): [in this window] [in a new window]   Figure 3.   Inducibility of GM-CSF and IL-1 release by BAL cells from PAP. Total BAL cells were stimulated with LPS in the absence or presence of 10 µg/ml of control IgG antibody, neutralizing anti-IL-10 antibody or anti-TGF- antibody. GM-CSF (A) and IL-1 (B) production was quantitated by ELISA. Results are representative of three experiments from three whole lung lavages and data is the mean of paired samples.

As a control for LPS inducibility, IL-1 production by total BAL cells from the PAP patient was analyzed (Figure 3B). The release of IL-1 by total BAL cells stimulated with LPS was slightly enhanced by treatment with anti-IL-10 antibody (73 pg/ml) compared with LPS alone (38 pg/ml). The production of IL-1 was strongly enhanced by anti-TGF- antibody (123 pg/ml) which had no effect on GM-CSF release.

Milleron and colleagues reported a significant BAL lymphocytosis in 9 patients with PAP (13). The mean percent lymphocytes was 57 with a significant increase in CD4+ cells and CD8+ cells. We also noticed increased lymphocytes in each whole lung lavage specimen from our PAP patient. IL-10 is a chemotactic factor for CD8+ T lymphocytes and enhances the activity of CD8+ cytotoxic lymphocytes (14). In one of the lavage specimens, we were able to separate adherent cells (> 80% alveolar macrophages) from nonadherent cells (> 70% lymphocytes). There was 4-fold more GM-CSF released from LPS-stimulated adherent cells than nonadherent cells, and 3-fold more IL-10 released from LPS-stimulated nonadherent cells than adherent cells. Interesting, the mouse GM-CSF knockout models had increased lymphocyte accumulation in the PAP lung.

	DISCUSSION

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We report here the first human case of PAP evaluated for GM-CSF gene expression and protein release by BAL cells. We have shown in a human study that BAL cells isolated from the lung failed to secrete GM-CSF protein, although the GM-CSF gene was expressed. The mechanism of GM-CSF deficiency was further addressed by identifying an inhibitory role for IL-10 in suppressing GM-CSF production. The murine models show that PAP results from disruption of the GM-CSF gene or the c receptor gene, whereas the mechanism of human adult PAP results from a functional deficiency of GM-CSF production possibly due to increased levels of the inhibitory cytokine IL-10. Increased IL-10 could possibly lead to secondary forms of PAP as well. Secondary PAP has been reported in accelerated or acute silicosis, Pneumocytis carinii pneumonia, and secondary to hematologic malignancies (15, 16). Macrophages upregulate GM-CSF mRNA during phagocytosis of sheep red blood cells analogous to macrophage stimulation by LPS (9). GM-CSF deficiency could reduce macrophage phagocytosis and processing of excess surfactant.

Interleukin-l0, originally identified as a cytokine synthesis inhibitory factor, is produced by CD4⁺/CD8⁺ T cells, B cells and monocytes/macrophages (10, 11). Stimulation of peripheral blood monocytes by LPS results in cytokine production including IL-1, IL-1, IL-6, IL-8, TNF-, G-CSF, GM-CSF and IL-10. The endogenously produced IL-10 is able to downregulate the production of the former cytokines as well as its own production. The IL-10-mediated inhibition of cytokine production is accompanied by reduced accumulation of mRNAs (17). Wang and colleagues have shown that IL-10 selectively inhibits NF-B translocation from the cytosol to the nucleus consequently inhibiting mRNAs for IL-1, IL-6, IL-8, and TNF- in a dose dependent manner (18). AP-1, NF-IL6, CREB, SP-1 and other transcription factors were not affected by IL-10. They demonstrated that IL-10 blocks NF-B activation in response to distinct NF-B inducers such as LPS and TNF-. In this report, we have shown that GM-CSF mRNA expression can be induced by LPS, but the release of GM-CSF is inhibited by IL-10. IL-10 is a potent inhibitor of GM-CSF, including GM-CSF production by chronic myelomonocytic leukemia cells (19). It is a direct inhibitor of T-cell activation by inhibiting IL-12 (17). GM-CSF inhibits the immunosuppressive activity of AM that downregulates local T cell responses (20) and GM-CSF deficiency would allow for increased T cell suppressor activity. We would suggest that IL-10 from nonadherent cells inhibits GM-CSF secretion by acting on translation or protein processing in alveolar macrophages.

These data derived from a patient with PAP demonstrate a deficiency in GM-CSF analogous to the GM-CSF knockout mouse. However, the mechanisms for deficient GM-CSF differs between the human and the mouse model. Our patient may represent a form of secondary PAP due to an unknown insult; further studies are indicated to determine if our case report is generalizable to secondary PAP from silica exposure, Pneumocystis carinii penumonia. PAP in remission, and congenital PAP. Studies of GM-CSF and inhibitory molecules on other patients with PAP, a rare disorder, are also indicated. Other mechanisms may also be extant, e.g., defects in expression or function of the GM-CSF receptor, and other suppressor molecules such as Th2 cytokines may be revelant. Restoring GM-CSF function in patients with PAP may be therapeutic, e.g., treating with subcutantous injections (7), or aerosolizing recombinant GM-CSF directly to the lung, or administration of anti-IL-10 antibody. Importantly, these studies demonstrate the need for patient-oriented investigations to complement gene knockout models of human disease.