INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The generation of a pulmonary cell-mediated immune response requires antigen recognition, processing, and presentation to T lymphocytes (1, 2). Pulmonary dendritic cells (DCs) show diminished antigen-presenting cell (APC) activities when low doses of heat-killed Listeria (HKL) are introduced via the airways (3). The APC activities of DCs are upregulated by large numbers of HKL or when liposome-encapsulated clodronate, which eliminates resident alveolar macrophages (AMs) in vivo (4), is delivered to the lungs. These interventions also promote the pulmonary T cell-mediated immune response to HKL in vivo.

Resident AMs antagonize the activities of pulmonary interstitial DCs by sequestering particulate antigen within the air spaces, and via the release of transforming growth factor  (TGF-), interleukin 10 (IL-10), prostaglandin E₂, and nitric oxide (NO), which effectively suppress the accessory activities of DCs (5, 6). The immunosuppressive effects of AMs limit inflammation of the gas-exchange surface in response to low doses of bacterial antigens. However, in the pulmonary response to large numbers of bacteria, competent APCs are required to promote antigen-specific immunity.

A variety of proinflammatory stimuli elicit the influx of activated monocytes from the blood into the lung. Exudate macrophages are smaller than resident AMs, show fewer intracytoplasmic organelles, and differ in their immune phenotype and functional activities (7). A murine anti-rat macrophage antigen (RMA) monoclonal antibody (MAb), raised against interferon  (IFN-)-activated normal rat alveolar macrophages, binds an ~ 120-kD surface membrane antigen that is strongly expressed by resident AMs, but weakly expressed by exudate macrophages, pulmonary interstitial macrophages, and blood monocytes (8). The RMA antigen is lineage specific and absent on granulocytes and lymphocytes. The molecular characteristics of the RMA antigen have not yet been fully evaluated; however, its surface expression is upregulated by tumor necrosis factor  (TNF-) and phorbol esters, and anti-RMA stimulates mitogenesis and multikaryon formation by normal AMs (9).

In the present studies, Lewis rats were challenged intratracheally with PKH-26-labeled HKL (3), and macrophages from the bronchoalveolar (BAL) fluid were purified by fluorescence-activated cell sorting (FACS), on the basis of their relative intensity of RMA staining, into RMA-bright and RMA-dim (RMA^bright/dim) macrophages. The RMA^bright/dim macrophages were compared for their cytokine expression, NO release, and ability to serve as accessory cells for T lymphocytes.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Inbred, pathogen-free, 6- to 8-wk-old female Lewis rats (150-250 g) were obtained from Charles River Laboratories (Kingston, MA). Rats were housed in a restricted access animal care facility and permitted access to food and water ad libitum.

Heat-killed Listeria monocytogenes

HKL was obtained from the Bacteriology Laboratory of the Massachusetts General Hospital (Boston, MA). The concentration of organisms was determined in a McFarland nephelometer and bacteria were heat inactivated in a 63° C water bath for 90 min; viability was assessed by the failure of the bacteria to grow on blood agar plates. Aliquots (10⁹ bacteria/ml in saline) were stored at 20° C.

Complete Medium and Culture Conditions

Cells were cultured in complete medium (CM): RPMI 1640 (Mediatech, Herndon, VA) with 10% heat-inactivated fetal bovine serum (FBS; Sigma, St. Louis, MO), gentamicin (50 µg/ml; GIBCO-BRL, Gaithersburg, MD), 0.5% 1 M HEPES buffer (GIBCO-BRL), and 2-mercaptoethanol (5 × 10⁵ M; Sigma), at 37° C in a humidified chamber of 95% air and 5% CO₂.

Antibodies

Anti-rat MAbs were used to purify and characterize cells in these studies. These antibodies included W3/25 (CD4), OX-8 (CD8), OX-39 (CD25), OX22 (CD45RC), and OX-6 (Ia) (all from Accurate Chemical & Scientific, Westbury, NY); OX-62 (Serotec/Harlan Bioproducts, Indianapolis, IN); B7-1 (CD80), B7-2 (CD86), biotinylated anti-granulocyte (HIS-48) (all from PharMingen, San Diego, CA); and RMA-1 [anti-rat macrophage; R. Kradin (8)]. Other antibodies included purified rabbit anti-human factor XIIIa (Calbiochem-Behringwerke, La Jolla, CA), which cross-reacts with rat factor XIIIa, and murine anti-iNOS (inducible nitric oxide synthase) (Transduction Laboratories, Lexington, KY). Monoclonal antibodies were prepared either as ascites or supernatants and used at predetermined optimal concentrations. For antigen localization in situ lung tissues were stained by an avidin-biotin immunoperoxidase technique, as previously described (10). Surface immune phenotype was examined with a Becton Dickinson (Mountain View, CA) FACS 440 cytofluorimeter after direct staining with fluorescein isothiocyanate (FITC)-conjugated anti-rat MAbs or indirect staining with goat F(ab')₂ anti-mouse IgG-FITC (Tago Immunologicals, Camarillo, CA) or steptavidin red 670 (GIBCO-BRL).

Generation of Antigen-Specific Immune T Cells

Rats were immunized with an emulsion of 100 µg of HEL (Sigma) or 1 × 10⁷ HKL in Freund's complete Adjuvant (Difco, Detroit, MI). The emulsion (0.1 ml) was injected bilaterally at the base of the tail; inguinal lymph nodes were harvested at 10-14 d, mechanically dispersed, and separated on Isolymph (Gallard Schlesinger Industries, Carle Place, NY). Lymph node mononuclear cells (2 × 10⁶) were incubated in 24-well culture plates with HEL (100 µg/ml) or HKL (10⁷/ ml) antigen in a humidified chamber of 95% air and 5% CO₂. IL-2 (100 U/ml; Cetus, Emeryville, CA) was added to the medium on Day 5 and then two or three times weekly. Every 3-4 wk, the cultures were restimulated with HEL or HKL, respectively, in the presence of normal irradiated (3000 cGy; Mark I model 30 cesium-137 source; Sheperd and Associates, San Fernando, CA) syngeneic spleen cells at a 10:1 ratio of spleen cells to T cell blasts. Immune phenotyping by cytofluorimetry showed that the antigen-specific lymphoblasts were > 95% W3/25⁺ (CD4)OX22 (CD45RC). The specificity of the response was judged by a greater than threefold difference in the magnitude of [³H]thymidine incorporation by T cell blasts proliferating in response to specific antigen as compared with irrelevant antigen.

Intratracheal Instillation

Rats were lightly anesthetized with chloral hydrate (400 mg/kg), and the trachea was surgically exposed. HKL (10⁶-10¹⁰/rat) with or without PKH-26 label (Sigma) was injected intratracheally in 100 µl of normal saline via a 25-gauge needle. Indocyanine green (2.5 mg/ml; Becton Dickinson Microbiology Systems, Mountain View, CA) was coadministered intratracheally as a marker of bacterial distribution. Controls received intratracheally a comparable volume of sterile saline. The rats were subsequently sacrificed at 24-72 h; leukocytes were applied to glass slides and stained with modified Wright stain (Leukostat; Fisher Scientific, Pittsburgh, PA) for differential counts of cells in the BAL fluid and for further analysis as described.

Isolation of RMA⁺ Macrophage Subsets

At 24 h after intratracheal injection of PKH-26-HKL, rats were sacrificed and the lungs were subjected to bronchoalveolar lavage with saline-0.6 mM EDTA. The BAL cells were resuspended in 0.5% bovine serum albumin (BSA)-phosphate-buffered saline (PBS) and stained with optimal dilutions of mouse anti-rat RMA and F(ab')₂ goat anti-mouse IgG-FITC (Tago). Subsequently the cells were incubated briefly in 1% normal mouse serum, followed by treatment with biotinylated HIS-48 (PharMingen) and steptavidin red 670 (GIBCO-BRL). Cells were then resuspended in sterile PBS and sorted in a Coulter (Hialeah, FL) EPICS 753 equipped with a 488-nm argon laser. Forward versus side light scatter was used to identify macrophages and positive staining with HIS 48 was used to exclude granulocytes. RMA^bright/dim-positive cells were sorted separately on the basis of their distinct FITC staining intensities. Each population was simultaneously analyzed for uptake of PKH-26-HKL. Approximately 2 × 10⁶ purified cells were collected, some of which were run back through the instrument after sorting to verify purity.

Accessory Cell Activities

FACS-sorted RMA^bright/dim cells from the HKL-challenged lungs were enumerated in a hemocytometer by trypan blue exclusion and resuspended in CM. To assay for APC activity, cells were irradiated (3000 cGy) and plated (1 × 10⁴ cells/well) into 96-well flat-bottom micro- titer plates (Falcon/Fisher Scientific, Pittsburgh, PA). HEL-immune T cells (4 × 10⁴) were added to wells as responders with specific HEL antigen (200 µg/ml) or irrelevant antigen (10⁷ HKL/ml). An allogeneic mixed lymphocyte response (MLR) was established by incubating 1 × 10⁴ irradiated RMA^bright/dim macrophages post-HKL treatment (10⁸ HKL, intratracheal) (Lewis rat) with normal allogeneic Brown-Norway spleen cells (1 × 10⁵) for 6 d. Lewis rat spleen cells served as syngeneic responder controls. To assay for immune suppression, unsorted BAL macrophages were added in graded doses to wells containing irradiated syngeneic spleen cells (2 × 10⁵/well), HKL-immune T cells, and HKL. BAL cells from saline-treated rats served as controls. The culture plates were incubated for 72 h (or 6 d for the MLR) at 37° C; wells were pulsed with [³H]thymidine (1 µCi/well, specific activity 80 Ci/mmol; DuPont/NEN, Boston, MA) for 6 h prior to harvesting. The pulsed wells were harvested in a semiautomatic cell harvester (Skatron, Sterling, VA) and counted in a Tri-Carb liquid -scintillation spectrometer (Packard, Sterling, VA).

RNase Protection Assay for Cytokine Gene Expression

To detect cytokine mRNA expression, FACS-sorted RMA^bright/dim pulmonary macrophages were lysed in Trizol (GIBCO-BRL) to extract total cellular RNA. Concentration was determined by OD₂₆₀ readings, and purity by A_260/280 ratio and examination of 28S and 18S bands by agarose gel electrophoresis. A ³²P-labeled antisense RNA probe of high specific activity was synthesized from a commercially developed multitemplate cDNA set (PharMingen), specifically designed to detect cytokines (TNF, IFN-, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-10) using T7 polymerase. Excess probe and target RNA (1-20 µg) were hybridized at 56° C overnight; remaining free probe and RNA were digested with RNase. Protected probe-RNA hybrids were purified by standard phenol-chloroform extraction and ethanol precipitation methods, and resolved on a 5% polyacrylamide-urea denaturing gel (40 × 0.4 mm) at 50° C until the dye front reached 30 cm. The gel was dried and radioactivity quantified by autoradiography or phosphoimaging; inequities in sample loading were normalized by constructing densitometric ratios of the cytokine band of interest to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene.

Nitric Oxide Production

RMA^bright/dim macrophages were purified from BAL fluid after intratracheal challenge with HKL. Conditioned supernatants were prepared from RMA^bright/dim macrophages (2 × 10⁵/well) cultured in CM ± HKL (10⁷/ml) and/or recombinant murine IFN- (500 U/ml; Genentech, San Francisco, CA) for 48 h. In some wells, macrophages were treated with an NOS inhibitor (N'-monomethyl-L-arginine [L-NMMA], 10 mM; Sigma). Nitrite in the conditioned supernatants was measured as previously described (11). Briefly, 50 µl of conditioned supernatant was incubated in an equal volume of Griess reagent at 25° C for 20 min. Chromophore absorbance at 562 nm was determined in a microtiter plate reader (Biotek Instruments, Winooski, VT). Nitrite concentration was determined by using graded concentrations of sodium nitrite (Sigma) as a standard.

Immunohistochemistry

Macrophages from the lungs of rats treated intratracheally with 10⁸ HKL were plated in Lab-Tek chambers and allowed to adhere at 37° C for 2 h. The nonadherent cells were washed away and the wells were treated separately with selected antibodies and stained by an indirect immunoperoxidase technique, as previously reported (8).

Statistical Analysis

Data are expressed as means ± standard deviation (SD). The Student unpaired t test was used to assess differences between groups.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Changes in Macrophage Representation in BAL

Intratracheal instillation of > 10⁷ HKL yielded an increase in BAL leukocytes (p < 0.01) (Figure 1). The percentages of granulocytes and lymphocytes increased with the dose of HKL (Figure 2). RMA^dim macrophages were smaller and displayed less internal granularity than RMA^bright macrophages, as judged by diminished forward and right-angle light scatter, respectively (Figure 3A). The immunostaining intensity of RMA^bright macrophages post-HKL treatment and normal AMs was comparable (Figure 3B), consistent with the speculation that resident AMs were the immediate precursors of RMA^bright macrophages post HKL treatment.

View larger version (7K): [in this window] [in a new window]   Figure 1.   Leukocytes are increased in BAL fluid 24 h after the intratracheal delivery of HKL. Lewis rats received graded doses of HKL (106-1010) intratracheally; controls received saline. At 24 h the number of leukocytes in the BAL fluid was enumerated. Data represent the mean ± SD of at least three separate experiments. p < 0.01 for all doses > 1 × 107 HKL compared with saline control.

View larger version (21K): [in this window] [in a new window]   Figure 2.   Differential representation of leukocytes in BAL fluid after intratracheal HKL challenge. Lewis rats received graded doses of HKL (107-1010) intratracheally; controls received saline. At 24 h, leukocytes in the BAL fluid were applied to glass slides and treated with modified Wright stain, and a differential count was determined. Data represent lymphocytes, granulocytes, and macrophages as a percentage of the total number of leukocytes in the BAL fluid. Data are from one of three representative experiments.

View larger version (29K): [in this window] [in a new window]   Figure 3.   Cytofluorimetric analysis of RMA+ macrophages. Rats received 108 PKH-26-labeled HKL intratracheally and were subjected to a saline BAL at 24 h. The cells in the BAL fluid were stained in suspension with FITC-anti-RMA and analyzed for intensity of RMA staining. RMA+ macrophages were electronically back-gated in order to assess the light scatter characteristics of the RMAbright/dim cells. (A) RMAdim macrophages showed less forward and side angle light scatter than did RMAbright cells. (B) Histograms of RMAbright/dim macrophage staining post-HKL treatment (open histograms) are superimposed over the RMA staining of normal AMs (black histogram).

RMA^bright macrophages accounted for > 95% of the RMA⁺ macrophages in the BAL fluid at intratracheal doses of < 10⁸ HKL (Figure 4). After the intratracheal delivery of 10⁸ HKL, approximately 20% of macrophages were RMA^dim and their percentage increased to ~ 60% with the largest HKL challenge (10¹⁰).

View larger version (17K): [in this window] [in a new window]   Figure 4.   The percentage of RMAdim macrophages increases in BAL fluid with dose of HKL delivered in vivo. The percentage of RMAbright/dim macrophages was analyzed by cytofluorimetry 24 h after HKL challenge. There was a marked increase in the representation of RMAdim macrophages at doses of HKL > 107 (all p < 0.01 compared with saline or HKL doses of 1 × 106, 1 × 107). Data represent the means of at least three experiments.

Uptake of HKL by RMA⁺ Macrophage Subsets

The uptake of PKH-26-labeled HKL by RMA⁺ macrophages was determined by dual-color cytofluorimetry and confirmed directly by epifluorescence microscopy. RMA^bright macrophages accounted for virtually all of the PKH-26⁺ macrophages at intratracheal doses of < 10⁸ HKL (Table 1). The percentage of PKH-26⁺ RMA^bright/dim macrophages increased directly with the size of the HKL challenge; however, higher percentages of RMA^bright macrophages showed bacterial uptake in vivo.

At doses of < 10⁸ HKL, ~ 1.0-5.0 × 10⁵ RMA^bright macrophages showed uptake of labeled bacteria. For all intratracheal doses of HKL  10⁸, the numbers of PKH-26⁺ RMA^bright macrophages at 24 h were increased but did not exceed ~ 2 × 10⁶. A dose-dependent increase in PKH-26⁺ RMA^dim macrophages was observed for doses  10⁸ HKL, and the numbers of PKH-26⁺ RMA^dim and PKH-26⁺ RMA^bright macrophages in the BAL fluid were comparable at the 10¹⁰ HKL challenge.

When RMA^dim and RMA^bright macrophages were incubated with PKH-26⁺ HKL ex vivo, > 95% of both RMA⁺ macrophage subsets showed ingestion of HKL; however, RMA^bright macrophages incorporated larger numbers of HKL than did RMA^dim macrophages (6 ± 2 versus 2 ± 0.5 HKL/ cell; p = 0.02).

Cytokine Gene Expression and Nitric Oxide Production

At 4 h after intratracheal administration of 10⁸ HKL, > 95% of the macrophages in the BAL were RMA^bright. They yielded an ~ 8-fold increase in IL-1 expression and an ~ 2-fold increase in TNF, compared with saline controls, as judged by RNase protection assay (not shown). At 24 h after intratracheal HKL challenge, RMA^bright/dim macrophages expressed both TNF and IL-1, and the RMA^dim subset was responsible for the majority of their expression (Figure 5).

View larger version (12K): [in this window] [in a new window]   Figure 5.   Cytokine expression by RMAbright/dim macrophages. RMAbright/dim macrophages were purified by FACS 24 h after HKL challenge. RNA was extracted and analyzed by RNase protection assay. Data represent the normalized densitometric ratio of RNA expression by RMAdim and RMAbright cells and represent one of three comparable experiments.

RMA^bright macrophages produced more NO than did RMA^dim cells under all of the experimental conditions (Figure 6). In the absence of additional stimulation in vitro, only RMA^bright macrophages spontaneously released NO after intratracheal HKL administration. Whereas a subset (20 ± 3%) of RMA^bright macrophages showed strong intracytoplasmic staining for iNOS after intratracheal HKL challenge (10⁷-10⁹), RMA^dim cells were either iNOS negative or showed dim intracytoplasmic staining (not shown). Additional ex vivo stimulation with HKL (10⁷), IFN- (500 U/ml), or IFN- plus HKL yielded about two times more release of NO by RMA^bright than RMA^dim cells; the addition of the NOS inhibitor N-monomethyl-L-arginine (L-NMMA) abrogated the release of NO (not shown).

View larger version (16K): [in this window] [in a new window]   Figure 6.   Nitric oxide production by RMA+ macrophages. Macrophages harvested from BAL fluid 24 h after intratracheal challenge with 1 × 109 HKL were purified into RMAbright/dim subsets by FACS and plated (2 × 105) in wells containing medium alone or medium with IFN- (500 U/ml); HKL (107/ml); or IFN- plus HKL. Data represent means + SD of three experiments; all p < 0.05.

Suppression of Antigen-driven Lymphocyte Proliferation

The effect of RMA^bright/dim macrophages on the proliferation of HKL-immune T lymphocytes incubated with normal irradiated (3000 cGy) splenic APCs was examined. Wells that received unfractionated RMA⁺ macrophages from saline-treated controls showed strong suppression of responses at all doses of added macrophages. Macrophages harvested from lungs that received 10⁷ HKL intratracheally suppressed this response; however, macrophages from lungs that received 10⁹ HKL intratracheally yielded substantially less suppression at added doses of < 5 × 10⁴/well (Figure 7).

View larger version (13K): [in this window] [in a new window]   Figure 7.   Suppression of splenic APC activity by RMA+ macrophages. Irradiated spleen cells (2 × 105) were added to microtiter wells containing HKL-immune T lymphocytes (4 × 104) and HKL (107/ml). The wells also received graded doses of macrophages from the BAL fluid harvested 24 h after intratracheal HKL (107-109) injection, or saline control macrophages. Macrophages from lungs that received 107 HKL or saline controls suppressed APC activity at all doses. Macrophages from lungs that received 109 HKL were substantially less suppressive at lower doses. Data represent one of three representative experiments.

Next, RMA^bright/dim macrophages were added to the culture wells and examined for their ability to serve as APCs directly in the proliferative response of HEL-immune T lymphocytes to HEL antigen (Figure 8). RMA^dim macrophages exhibited excellent APC activities, whereas RMA^bright macrophages did not support proliferation.

View larger version (9K): [in this window] [in a new window]   Figure 8.   RMAdim macrophages are excellent APCs in antigen-mediated T cell immune response. RMAbright/dim cells were sorted by FACS 24 h after intratracheal challenge with HKL (108) and used as APCs (104/well) to support the mitogen response by HEL-immune T lymphocytes (4 × 104) to HEL (100 µg/ml). Normal irradiated spleen cells (2 × 105) and macrophages (1 × 104) from the BAL fluid of lungs that received saline served as APC controls. The data represent the stimulation index of wells that received HEL versus nonspecific antigen (HKL, 107/ml). RMAdim macrophages were excellent APCs and comparable in their activities to normal spleen cells. Neither RMAbright nor saline control macrophages were able to support responses.

RMA^bright macrophages had no stimulatory effect in the allogeneic MLR; however, RMA^dim macrophages stimulated a modest MLR (~ 2.5 times that of syngeneic control), consistent with weak DC activities (not shown).

Expression of DC-related Antigens by RMA⁺ Macrophages

As RMA^dim macrophages were judged to be effective APCs for nominal antigen and capable of weakly stimulating an MLR, adherent mononuclear cells from the BAL fluid were examined 24 h after intratracheal HKL administration for expression of DC-associated antigens (Figure 9). Immunohistochemical staining of adherent saline control macrophages showed that > 95% were RMA⁺; fewer than 5% stained for class II MHC antigens (OX-6), as previously reported (12); control macrophages were negative for OX-62, B7-1 (CD80) (not shown), B7-2 (CD86), and factor XIIIa.

View larger version (77K): [in this window] [in a new window]   Figure 9.   Expression of dendritic cell-related antigens by RMA+ macrophages after HKL challenge. Rats received 108 HKL or saline intratracheally; macrophages were harvested from the BAL fluid at 24 h, allowed to adhere in Lab-Tek chambers for 2 h, washed, and stained for expression of OX-62, B7-2 (CD86), and factor XIIIa. (A) OX-62, HKL; (B) OX-62, saline control; (C ) B7-2 (CD86), HKL; (D) B7-2 (CD86), saline control; (E ) factor XIIIa, HKL; (F ) factor XIIIa, saline control. Note the increased staining for dendritic cell-associated antigens after HKL administration.

When macrophages from HKL-treated lungs were examined, > 95% stained with heterogeneous intensity for RMA and approximately half of the adherent cells were class II MHC⁺ (not shown). The majority of macrophages (50-70%) were OX-62⁺ and CD86⁺ (Figure 9), whereas a smaller percentage (15-25%) stained for CD80 (not shown); faint staining for factor XIIIa was also seen on a minority (20-30%) of cells (Figure 9).

Macrophages were analyzed by cytofluorimetry 24 h after HKL injection. Positive staining for OX-62, CD80, and CD86 was confined to RMA^dim macrophages (Table 2). A comparable percentage of RMA^dim cells stained for OX-62 and CD86 (~ 40%), whereas a smaller percentage (~ 10%) was CD80⁺.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study, we investigated the phenotype and functional activities of pulmonary mononuclear phagocytes in response to HKL. On the basis of light scatter properties, RMA^bright macrophages were larger than RMA^dim macrophages and displayed greater internal granularity, reflecting an increased complement of intracytoplasmic organelles and incorporation of HKL. The light scatter characteristics and intensity of RMA staining were comparable for RMA^bright macrophages after HKL challenge and normal AMs, supporting the hypothesis that RMA^bright macrophages were derived directly from resident AMs.

In contrast to RMA^bright macrophages, RMA^dim cells were small with modest internal granularity, as judged by light scatter, sharing these physical characteristics in common with blood monocytes (7). When < 10⁸ HKL were delivered intratracheally, RMA^bright macrophages accounted for the vast majority of lung macrophages in the BAL fluid and few RMA^dim macrophages were detected. These findings suggest a proinflammatory threshold for blood monocyte recruitment into the lung. This could limit inflammation and safeguard the gas-exchange surface, when the numbers of HKL are small and can be effectively eliminated by resident AMs. A threshold requirement for the recruitment of RMA^dim macrophages may reflect the in vivo regulation of chemokines that promote the migration of monocytes from blood to lung, including inducible protein 10 (IP-10), RANTES, macrophage chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1 (MIP-1) (13).

Four hours after injection of 10⁸ HKL, RMA^bright cells accounted for virtually all of the pulmonary macrophages in the BAL fluid and showed substantial IL-1 and modest TNF gene expression. At 24 h after HKL challenge, RMA^dim macrophages were detected in the BAL fluid and were the dominant producers of IL-1 and TNF. Both IL-1 and TNF have been implicated in the cascade of early proinflammatory events leading to cellular inflammation. TNF and IL-1 induce IL-8 expression by endothelial cells (1), which may have accounted for the substantially increased numbers of granulocytes in the lung after HKL challenge. In addition, TNF and IL-1 promote the expression of proinflammatory CXC and CC chemokines (13), and increase the surface membrane expression of critical endothelial adhesion molecules ICAM-1 and VCAM-1, favoring the transmigration of leukocytes into the lung.

The ingestion of bacteria is a cardinal pulmonary defense that is mediated, in part, by macrophages. Phagocytosis in vivo is determined by levels of differentiation and activation of responding phagocytes, the accessibility of antigen, and the availability of opsonic factors, e.g., complement and immunoglobulin (14), that regulate the uptake of microorganisms. When < 10⁸ HKL were delivered intratracheally, their uptake was primarily mediated by RMA^bright macrophages. The percentage of PKH-26⁺ HKL-RMA^bright macrophages increased directly with the dose of the HKL, with 97% RMA^bright macrophages showing bacterial uptake at the highest (10¹⁰) dose of HKL. At intratracheal HKL doses  10⁸, the total numbers of PKH-26⁺ HKL-RMA^bright macrophages reached a maximum and plateaued in vivo.

Increasing numbers of PKH-26⁺ HKL-RMA^dim macrophages were detected at doses of > 10⁸ HKL, and the numbers of PKH-26⁺ HKL-RMA^dim and PKH-26⁺ HKL-RMA^bright macrophages were comparable at doses of > 10⁹ HKL. However, the percentages of PKH-26⁺ HKL-RMA^dim macrophages were consistently lower for most HKL challenges. The phagocytic potency of RMA^bright macrophages was evidenced by their ability to incorporate larger numbers of HKL than did RMA^dim macrophages, on a per-cell basis in vitro, possibly reflecting the larger surface membrane area available for binding HKL (15).

In addition to their role as mediators of the innate immune response, macrophages regulate adaptive T cell and B cell immunity. Normal resident AMs suppress the mitogenic activities of DCs and T lymphocytes (5), and the elimination of resident AMs in vivo yields increased cellular (4) and humoral (16) pulmonary immune responses. Immune suppression by activated AMs is mediated by a variety of factors. Nitric oxide is a potent suppressor of DC and T lymphocyte activities (6). RMA^bright macrophages exhibited increased NO production, as judged by iNOS protein expression and the secretion of nitrite. As RMA^bright macrophages appear to be directly related to resident AMs, we speculate that they share the capacities of AMs to release large amounts of NO and free radicals of oxygen (2, 3), in keeping with their role as scavengers of bacteria and other particulates. In previous studies, AMs were demonstrated to produce larger amounts of NO than did blood monocytes or interstitial macrophages in response to HKL. This may have contributed to the limited APC activities of DCs harvested from HKL-challenged lungs (11). In contrast, RMA^dim macrophages, which are postulated to have recently differentiated from normal blood monocytes, appear to share their limited capacities to secrete NO (4).

The cellular pulmonary immune response to HKL (17) in vivo varies directly with the intratracheal dose of HKL and with the percentage of RMA^dim macrophages in the BAL fluid. Blood monocytes have been recognized to differentiate into DCs with cytokine stimulation in vitro (18). Like DCs, but unlike RMA^bright macrophages, RMA^dim cells displayed increased expression of class II MHC, OX-62, CD80, and CD86. CD80 and CD86 are highly expressed by DCs and mediate their accessory activities by binding to CD28 and CTLA-4 on the surface membrane of T lymphocytes (19). The expression of factor XIIIa by RMA^dim macrophages is additional evidence that they may be related to monocyte-derived DCs (MDDCs). Intracytoplasmic factor XIIIa is a phenotypic feature of MDDCs and dermal DCs (20) and has previously been detected in inflammatory lung macrophages (21).

RMA^dim macrophages were less suppressive than RMA^bright macrophages or normal AMs when added to culture wells that contained competent splenic APCs and immune T lymphocytes. In addition, RMA^dim macrophages functioned directly as APCs for antigen-specific T lymphocytes in vitro and weakly stimulated an MLR, supporting their possible functional differentiation toward DCs.

Pulmonary DCs can be elicited by a variety of inflammatory stimuli in vivo. McWilliam and colleagues demonstrated that DCs accumulate at pulmonary mucosal surfaces in response to intrapulmonary challenges with bacteria or lipopolysaccharide (LPS) (22, 23). Increased numbers of DCs have been reported in the airways and lung interstitium in response to either intratracheal or parenteral IL-2 and IFN- (5, 6).

The possibility that pulmonary DCs may have precursors in the blood circulation was suggested by Schneeberger and colleagues, who demonstrated Ia⁺ OX-62⁺ cells in the lung vasculature of rats (24). As blood monocytes cultured in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF (18) can develop into immature DCs, we speculate that the accumulation of pulmonary RMA^dim exudate macrophages with characteristics of DCs reflects their differentiation from circulating monocytes. The stimulus for activation may be cytokines generated in the lung during the response to HKL. Although we did not specifically address whether circulating RMA^dim monocytes with DC characteristics were increased in the blood after HKL challenge, increased numbers of MDDCs have been observed in the lung and systemic blood of mice treated intratracheally with bleomycin (25), suggesting that differentiation of MDDCs may occur in the circulation before their migration into the inflamed lung.

The presence of exudate macrophages with DC characteristics may explain why macrophages from inflamed lungs have previously been reported to have APC activities, whereas normal AMs are generally highly suppressive (26, 27). It may also explain the previously reported observation from this laboratory that pulmonary T cell-mediated responses to HKL develop in vivo only when large doses of HKL are delivered to the lungs (3).

The ability to increase the numbers of DCs to the lung via the recruitment of MDDCs from the circulating blood suggests a potentially important mechanism for expanding the immune response in vivo. Whether exudate macrophages without accessory activities and MDDCs coexist and interact within the population of RMA^dim pulmonary exudate macrophages will be a topic of future investigations.