Immunohistochemical characterization of rat thymic non-lymphoid cells. II. Macrophages and granulocytes defined by monoclonal antibodies.

A panel of monoclonal antibodies (mAb) raised to antigens of rat thymic non-lymphoid cells (predominantly macrophages and granulocytes) was immunohistochemically characterized. Based on their staining patterns on cryostat thymic sections and double labellings using acid phosphatase activity, anti-cytokeratin mAb to exclude binding to epithelium or ED1 and ED2 mAb, specific for rat macrophages, antibodies were subdivided into four groups: (i) R-MC 39 mAb strongly reactive with macrophages in the cortex and cortico-medullary zone (CMZ) and weakly with some scattered macrophages in the medulla, blood vessels and thymocytes; (ii) R-MC 40, 41 and 42 mAb specific for cortical macrophages and most CMZ macrophages; (iii) R-MC 43 and 44 mAb predominantly recognizing CMZ and medullary macrophages; (iv) R-MC 45 mAb strongly labelling granulocytes and weakly a subset of macrophages throughout the thymus and isolated cells in the medulla. The obtained results show considerable heterogeneity within mobile thymic non-lymphoid cells and the presence of specific or common antigens in macrophages of particular topographic localization in the rat thymus.     

Expression and function of intercellular adhesion molecule 1 (ICAM-1) on rat thymic macrophages in culture

The expression of intercellular adhesion molecule 1 (ICAM-1) was studied on freshly isolated rat thymic macrophages (TMF) and after their cultivation in serum-free medium using monoclonal antibody (mAb) 1A 29 and a streptavidin-biotin immunoperoxidase technique. ICAM-1 was expressed on about 80% of freshly isolated TMF. Upon cultivation, the percentage of ICAM-1+ TMF decreased to about 30-40% in 12-day-old culture. Using double immunofluorescence staining it was found that ICAM-1 was expressed both on cortical (R-MC 40+) and CMZ/medullary (R-MC 43+) macrophage subsets. ICAM-1 was up-regulated on TMF in culture by recombinant IFN-gamma, IL1 and TNF-alpha and was down-regulated by dexamethasone. Syngeneic thymocytes bound to cultivated TMF in a rosette form at both 37 degrees C and 4 degrees C. IFN-gamma treatment did not increase the binding formation. The binding between thymocytes and IFN-gamma-stimulated TMF at 37 degrees C was inhibited by pretreatment of TMF with anti-ICAM-1 mAb or pretreatment of thymocytes with anti-LFA-1 mAb, indicating that ICAM-1 on TMF is one of the ligands involved in TMF/thymocyte adhesion and subsequent direct cell-cell communication.     

Immunohistochemical characterization of rat thymic non-lymphoid cells. I. Epithelial and mesenchymal components defined by monoclonal antibodies.

Molecular microenvironmental heterogeneity within rat thymus was studied by a panel of 13 monoclonal antibodies (mAbs) raised to thymic epithelial (TE) cells and mesenchymal stroma. Based on their anatomical distribution patterns observed with immunohistological techniques on frozen sections and double immunostaining using anti-keratin antibodies to identify epithelium, they were subdivided into five groups: (i) pan TE cells antibodies (R-MC 2, 3 and 8); (ii) cortical TE cells antibodies (R-MC 13-17); (iii) antibodies detecting subcapsular and subtrabecular TE cells and most medullary TE cells (R-MC 18-20); (iv) antibody to Hassall's corpuscles (HC) and a small subpopulation of medullary TE cells (R-MC 22); (v) mesenchymal stroma antibody (R-MC 23). The obtained results show phenotypic heterogeneity of rat thymic epithelium and its distinction compared to mesodermal-derived compartment.     

Anti-RMA: a murine monoclonal antibody that activates rat macrophages. I. Distribution and characterization of the RMA antigen.

Activated macrophages participate in inflammation by eliminating foreign cells, promoting wound healing, and modulating the immune response. A murine monoclonal antibody, designated anti-rat macrophage activator (RMA), was raised against alveolar macrophages (AM) activated with interferon-gamma (IFN-gamma) and phorbol myristate acetate (PMA). The RMA antigen is expressed by resident macrophages but not by other cells. Binding to AM by anti-RMA is not competitively inhibited by the murine monoclonal antibodies MRC OX-41, OX-42, and OX-43. Surface membrane expression of RMA antigens is upregulated by lipopolysaccharide, PMA, and tumor necrosis factor-alpha but not by IFN-gamma. Stimulation of AM with anti-RMA yields distinct ultrastructural alterations, as well as de novo protein and DNA synthesis. Immunoprecipitation of [35S]methionine metabolically labeled AM yields a 120 kD protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that is not altered by chemical reduction. We conclude that the RMA antigen is macrophage specific and that binding of anti-RMA to AM promotes functional activities in a subset of these cells.     

In vitro characterization of rat thymic macrophages.

Thymic macrophages have been isolated from Wistar rats and maintained in long-term culture. Day 1-isolated thymic macrophages expressed MHC class I and II antigens, Thy-1, CR3, CD4, ED1, ED2, as well as acid phosphatase and non-specific esterase activities. Whereas cultured macrophages were positive for the activation antigen recognized by the mAb OX48, which was not expressed after isolation, MHC class II and Thy-1 expression were down-regulated during the culture, but could be induced with human rIL-2 or with Con A-SCM, which are also inducers of IL-2R, a cell-surface marker not expressed on Day 1 or on cultured macrophages.     

Epithelial cells and macrophages in myasthenia gravis thymus culture

Monolayer culture of thymic nonlymphoid cells derived from female patients with myasthenia gravis (MG) and individuals who underwent heart surgery was established to investigate the cellular composition of the thymic microenvironment and the interaction of nonlymphoid cells with autologous thymocytes. Thymic epithelial cells were identified by immunoperoxidase staining using monoclonal antibodies (mAbs) specific for cytokeratin and MR6 and MR19 antigens expressed on cortical and medullary epithelial cells, respectively. Macrophages were characterized by determination of alpha-naphthyl acetate esterase activity and detection of M1 antigen by mAb. It was demonstrated that in MG thymus cultures the number of cortical MR6+ epithelial cells is significantly reduced, and the ability of the remaining MR6+ cells to bind autologous thymocytes is markedly affected. On the other hand, the number of macrophages and the interaction of those cells with thymocytes were similar in MG and control thymus cultures. Since MR6+ epithelial cells are numerically and functionally affected in MG, maturational events of T cells occurring in the inner cortex may be altered. The mechanisms underlying the induction and expansion of T helper clones in MG are discussed.     

Rat thymic cultures: morphological and phenotypical characterization

Rat thymic cultures have been established in order to analyse the morpho-functional characteristics of thymic epithelial and non-epithelial cells in vitro. Stromal cultures, originating from implanted thymic fragments, consist of fibroblasts occupying most of the culture surface, epithelial cells forming discrete colonies, thymocytes and bone marrow-derived cells. Epithelial cells show a low class II MHC antigen expression, which is highly increased in semi-adherent cells, and do not interact with thymocytes. Thymocytes proliferate extensively at the beginning of the culture, but almost disappear at the end of the first week; however, restarting of thymocyte proliferation occurs during the second week of culture. Bone marrow-derived cells include ED- Ia+ CR3- IL-2R- dendritic cells (DC), ED+ Ia+ CR3+ IL-2R+ non-adherent thymic phagocytic cells (PTR) and ED+ Ia- CR3- IL-2R- adherent type 1 and 2 macrophages, derived from PTR. Both PTR and DC establish lympho-stromatic complexes with thymocytes present in the cultures. These results suggest that PTR and DC present in rat thymic cultures belong to different cell lineages, and that they are, respectively, the in vitro equivalents of intrathymic macrophages and interdigitating cells.     

Role of β2 Integrins in the Binding of Thymocytes to Rat Thymic Macrophages

A role of β2 integrins and one of their ligands, ICAM-1, in thymic macrophage (TMF)/thymocyte interactions was studied. TMF were isolated as adherent cells from 4-day old culture of thymic-cell suspensions either from normal or hydrocortisone-treated rats. Adherent cells were 94-98% positive with ED1 (a pan-macrophage marker). The majority of them (75-95%) expressed the CD11b and CD18 molecules, and 60-70% expressed CD54 (ICAM-1). A low proportion of TMF (10-20%) expressed CDlla (LFA-1). The expression of all these antigens was upregulated by IFN-α and TNF-α. The effect of these mAbs on TMF/thymocyte binding was studied using a simple rosette assay by incubating unstimulated or IFN-γ or TNF-α stimulated TMF, grown on microscopic slides with resting or ConA +IL-2 activated thymocytes. It was found that LFA-1/CD18 and ICAM-1 play a significant role in the TMF/thymocyte adhesion. In addition, a LFA-l-dependent/ICAM- 1-independent adhesion pathway was observed, suggesting that LFA-1 might use another ligand. The inhibitory effect of anti-CD18 mAb (WT-3) was higher than the effect of anti-LFA-1 mAb (WT-1) and was a consequence of blocking the CD18 chain both on thymocytes and TMF. No significant difference in the expression and function of adhesion molecules was found between TMF obtained from normal or hydrocortisone-treated rats. The involvement of CD1 1b in these processes was of lesser importance than the role of the CD11a molecule. By using mAbs to different epitopes of the CD11b molecule, such as OX-42 (anti-CD11b/CD11c), ED7, and ED8 (anti-CD11b), it was found that they were either slightly or moderately inhibitory under certain experimental conditions or did not significantly modulate TMF/thymocyte binding. Oχ-42 was slightly stimulatory in some experiments. Cumulatively, these results show that 2 integrins play a significant role in TMF/thymocyte interactions and probably contribute to T-cell development in vivo.     

Organotin-induced thymus atrophy concerns the OX-44+ immature thymocytes. Relation to the interaction between early thymocytes and thymic epithelial cells?

After single oral application of the organotin compound di-n-butyltindichloride (DBTC) to rats, a reversible dose-dependent thymus weight reduction is observed. This is maximal at day 4 and recovers to the control value approximately at day 9 after administration. In this study the changes in thymocyte subpopulations after a single oral dose of 15 mg DBTC/kg body weight were analysed by immunohistology. Thymus glands of exposed rats were collected at day 1,2,3,4,5,7 and 9 after DBTC dosing and frozen sections were screened for various thymocyte differentiation antigens. Staining by mAb HIS-44 that labels a subset of cortical thymocytes showed that the thymus atrophy was restricted to the cortex. Here a time-dependent decrease of labelling by CD2 (OX-34), CD8 (OX-8), CD4 (ER-2), and CD5 (OX-19) was observed. In contrast, the number of cortical OX-44+ cells increased from day 2 to day 5. This increase can reflect an increase of CD4-CD8- double-negative thymocytes or of macrophages. However, most of these OX-44+ cells were negative for acid phosphatase, which is present in most macrophages. We concluded that these OX-44+ cells were mainly CD4-CD8- thymocytes and that the thymocyte subpopulation of this phenotype, i.e. CD4-CD8-OX-44+, may be the target cell for DBTC. It is discussed whether DBTC might disturb the interaction of early thymocytes and thymic epithelium, probably by an interaction with the CD2 antigen.     

Microglia/macrophages responses to kainate-induced injury in the rat retina

The present study was aimed to elucidate how retinal microglia/macrophages would respond to neuronal death after intravitreal kainate injection. An increased expression of the complement receptor type 3 (CR3) and an induction of the major histocompatibility complex (MHC) class II and ED-1 antigens were mainly observed in the inner retina after kainate injection. Prominent cell death revealed by Fluoro Jade B (FJB) staining and ultrastructural examination appeared at the inner border of the inner nuclear layer (INL) at 1 day post-injection. Interestingly, some immunoreactive cells appeared at the outer segment of photoreceptor layer (OSPRL) at different time intervals. Our quantitative analysis further showed that CR3 immunoreactivity was drastically increased peaking at 7 days but subsided thereafter. MHC class II and ED-1 immunoreactivities showed a moderate but steady increase peaking at 3 days and declined thereafter. Double labeling study further revealed that retinal microglia/macrophages expressed concurrently CR3 and ED-1 antigens (OX-42+/ED-1+) or MHC class II molecules (OX-42+/OX-6+) and remained branched in shape at early stage of kainate challenge. By electron microscopy, microglia/macrophages with CR3 immunoreactivity displayed abundant cytoplasm containing a few vesicles and phagosomes. Other cells ultrastructurally similar to Müller cells or astrocytes could also engulf exogenous substances. In conclusion, retinal microglia/macrophages responded vigorously to kainate-induced neuronal cell death that may also trigger the recruitment of macrophages from neighboring tissues and induce the phagocytotic activity of cells other than retinal microglia/macrophages.     

MHC class II antigens on canine bronchoalveolar cells

To evaluate the expression of MHC (major histocompatibility complex) antigens on canine bronchoalveolar cells (BAC), bronchoalveolar lavages (BAL) were performed in mongrel and German shepherd dogs. MHC class II antigens on canine BAC and peripheral blood mononuclear cells (PBMC) were detected by monoclonal antibodies (mAbs) B1F6, 7.5.10.1 and Q5/13 recognising canine MHC class II antigens, using cytofluorometry. These mAbs reacted with more than 20% of BAC and PBMC in both breeds of dog. The percentage of MHC class II positive cells in BAC were lower than those in PBMC. There was no significant difference in the percentages of MHC class II positive BAC and PBMC in mongrel and German shepherd dogs. To further identify the expression of MHC class II antigens on BAC, the cells were separated into adherent and nonadherent cells by petri dish adherence. The percentages of MHC class II positive cells in adherent and non-adherent cell populations were similar. Nearly half the lymphocytes in normal BAC were T cells detected by mAbs F3-20-7 and 1A1; B cells were scarce and represented less than 10% of nonadherent cells. Immunoprecipitation by anti-MHC class II mAbs, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed MHC class II-like molecules on canine BAC and PBMC. After stimulation with phytohaemagglutinin (PHA), the percentages of class II positive cells in BAC and PBMC were significantly increased. Thus, these anti-MHC class II mAbs may prove to be of advantage in experiments designed to evaluate the changes in class II antigen expression on canine BAC during the course of immune response in the lung, as in pulmonary allograft rejection.     

Cultivation, proliferation and characterization of thymic macrophages.

Successful long-term culture of murine thymic macrophages was achieved by plating adherent thymic cells, in the presence of L cell-conditioned medium, on dishes coated with an extracellular matrix. Adherent thymic cells in normal conditions of in-vitro culture do not proliferate. Those maintained on plastic tissue-culture dishes, and exposed to L cell-conditioned medium, proliferate slowly to a limited degree and form very small colonies. In contrast, when cultured in dishes coated with an extracellular matrix formed by corneal endothelial cells, in the presence of L cell-conditioned medium, adherent thymic cells proliferate rapidly and after 12-21 days in culture form large colonies (about 3-5 mm in diameter). The proliferating cells were identified to be mononuclear phagocytes by their morphological appearance, their ability to ingest both bacteria and antibody-coated erythrocytes and by their nonspecific esterase activity. These cells were also shown to exhibit cell surface antigens that are characteristic of differentiated macrophages, e.g. Fc receptors and the specific macrophage cell surface marker F4/80. A high percentage of these cultured cells were found to bear I-A antigens. The adherent thymic mononuclear phagocytes could be trypsinized and passaged while maintaining both their ability to proliferate and their specific macrophage characteristics for a period of 70 days. Thus, monocyte-macrophage stem cells ae present in the thymus, and under appropriate in-vitro conditions, can be made to proliferate and mature to I-A-bearing macrophages.     

Cellular composition of peripheral lymph and skin of sheep defined by monoclonal antibodies

The surface phenotype of cells in peripheral lymph collected from afferent lymphatics leading to the popliteal lymph node of sheep was determined using a panel of monoclonal antibodies (mAbs). The majority of lymphocytes (83.5%) expressed the sheep pan-T cell antigen and only 13.3% bore surface immunoglobulin molecules. All peripheral T cell subsets occurring in sheep were detected; 50.2% of lymphocytes were positive for mAb SBU-T4 (T helper), 7.3% were positive for mAb ST-8 (T cytotoxic), and 8.4 and 43.0% expressed T subset markers recognized by mAbs 197 and T-80, respectively. Major histocompatibility complex (MHC) class I antigens were detected on 71.1% of lymphocytes and MHC class II antigens on 21.8%. The macrophage/veiled cells found in peripheral lymph did not express lymphocyte subset markers but were positive for MHC class I and II antigens, the sheep homologue of T6 antigen, leukocyte common antigen and mAb 175 (myeloid/erythroid). Macrophage-like cells occurring in the epidermis of skin taken from the lower hindleg gave positive staining reactions to the same mAbs which stained the macrophage/veiled cells in peripheral lymph. These results illustrate differences between the migration of lymphocyte subsets through nonlymphoid as compared to lymphoid tissues and point to a possible developmental or migratory relationship between the macrophage-like cells in skin and those in afferent popliteal lymph.     

Mechanisms Involved in the Binding of Thymocytes to Rat Thymic Dendritic Cells

The effects of monoclonal antibodies (mAbs) to cell-surface molecules, divalent cations, and various cell-signaling and metabolic inhibitors on the binding of thymocytes to rat thymic dendritic cells (TDC) were studied using a rosette assay. It was found that TDC/thymocyte adhesion was stronger and faster at 37°C than at 4°C. Flow cytometric analysis demonstrated that bound thymocytes were predominantly CD4⁺CD8⁺ and CD4⁺CD8^-, but in comparison to the phenotype of whole thymocytes, they were enriched in the mature TCRαβ^hi subset. The binding of thymocytes to TDC at 37°C was almost completely dependent on Ca²⁺ and Mg²⁺ and partly on an intact cytoskeleton and calmodulin-dependent protein kinase. The adhesion was independent of new protein synthesis and the activities of protein kinases A and C, tyrosine kinases, as well as phosphotyrosine protein phosphatases. The TDC/thymocyte adhesion at 37°C was partly blocked by anti-LFA-1 (WT.1), anti-CD18 (WT.3), and anti-ICAM-1 (1A29) mAb. MAbs to class II MHC (OX-3 and OX-6), CD4 (W3/25), CD8 (OX-8), and αβTCR (R73) stimulated the adhesion via an LFA-1-dependent pathway, whereas an anti-CD45 mAb (G3C5) stimulated the rosette formation independently of LFA-1. MAbs to CD2 (OX-34), CD11b (ED7), CD11b/c (OX-42), and class I MHC (OX-18) were without significant effects on the adhesion process.     

Rat thymic epithelial cells in culture constitutively secrete IL-1 and IL-6.

To study the in vitro interactions between rat thymic non-lymphoid cells and thymocytes, we established a system for long-term cultivation of thymic epithelial cells (TEC). TEC were cultivated and successfully propagated for over 8 months in RPMI 1640 medium containing 15% FCS, dexamethasone, insulin, epidermal growth factor, and poly-L-lysin as an adhesive matrix. Their epithelial nature has been confirmed using monoclonal anti-cytokeratin (CK) antibodies. More than 95% of these cells were reactive with K 8.13 and CK 8 mAbs, which are pan-epithelial markers for rat TEC in situ. An epithelial cell clone (TE-R 2.5) established from a long-term TEC culture was 100% reactive with these anti-CK antibodies. Phenotypic analysis of TEC cultures was performed by a large panel of mAbs reactive with a subset of rat TEC or CK polypeptides as well as UIex europaeus agglutinin I using a streptavidin-biotin immunofluorescence assay. Although the results obtained demonstrated phenotypic heterogeneity among these cells, most cultures, including the TE-R 2.5 clone, were of subcapsular/medullary phenotype. Medium conditioned by TEC cultures exhibited IL-1 and IL-6 activities when tested on D10S and B9 sensitive cell lines, respectively. Cytokine activities were neutralized (IL-1) or significantly inhibited (IL-6) by specific polyclonal antibodies. In addition, both anti-IL-1 and anti-IL-6 antibodies reacted with TEC in culture and epithelial (CK-positive) cells on thymic cryostat sections, indicating that thymic epithelium provides an important intrathymic source for molecules contributing to T cell activation.     

Flow cytometric analysis reveals unexpected shared antigens between histologically defined populations of thymic stromal cells

Utilizing flow cytometry, the expression of antigens recognizedby six thymic stromal cell (TSC) reactive mAbs was investigatedon fresh TSCs and TSC lines. It was found that some thymic epithelialcells and dendritic cells share antlgenlc phenotypes, and thatmost TSC reactive mAbs have a more extensive distribution thanwould have been predicted from immunohlstology. While thesefindings illustrate the higher sensitivity of flow cytometricanalysis, they more Importantly emphasize the great complexityof TSC that direct T cell development. In order to Identifythe molecular parameters that define the various steps involvedin T cell differentiation, TSC antigens (non-TCR/MHC/co-receptor)that are functional will have to be Identified. This study representsthe initial steps in characterizing such antigens.     

Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts

Mouse monoclonal antibodies (MAbs) have been prepared against rat T cell blasts. One MAb called MRC OX-40 recognized an antigen that differed from any previously described in that its expression was detected only on T blasts that also expressed the CD4 antigen. The OX-40 MAb did not detect an activation determinant of CD2 or CD4 molecules but recognized a distinct chain of mol. wt 50,000. The OX-40 MAb augmented T cell proliferation at late stages on in vitro responses. Other MAbs without obvious counterparts in other species were MRC OX-48 and MRC OX-49,50 which recognized cell surface molecules of mol. wts of about 95,000 and 90,000, respectively. The OX-48 antigen was not expressed on resting lymphocytes but was found on a subset of T and B blasts and also on other leucocytes. The OX-49,50 antigen was found on most haemopoietic cells but was expressed at greatly increased levels after lymphocyte activation and this was also the case for MRC OX-47 antigen which is of unknown M_r. The MRC OX-39 MAb was found to bind the rat IL-2 receptor; expression of this antigen was detected on thymic dendritic cells as well as on T blasts. The phenotype of rat T blasts compared to resting cells was also examined and changes in expression of L-CA, Thy-l, OX-2 and CD8 antigens were seen in addition to the changes found with the above MAbs.     

The intrathymic microenvironment: expression of lectin receptors and lectin-like molecules of differentiation antigens and MHC gene products

The expression of MHC products, differentiation antigens and lectin receptors has been investigated in the various cell types populating different compartments of the thymus. The ultrastructural classification of suspended thymic epithelial cells was facilitated by using a technique that preserves cortical nursing cells or medullary epithelial cell clusters. A subset of peanut lectin positive lymphocytes could be distinguished by their ability to bind soybean lectin also. This subset corresponds to the large proliferating lymphocytes that populate the area between the thymic capsule and the cortex. Ia and H-2 D/K antigens could be detected on nearly all epithelial and lymphoid cells. Expression of H-2 antigens, however, is more pronounced on medullary epithelial cells. T-cell differentiation antigens such as Thy-1 and Lyt-1 could be demonstrated not only on lymphocytes, but, interestingly enough, on cortical epithelial cells as well. These latter cells, in addition, exhibit a cell membrane-bound lectin with a specificity for D-galactose which might well be the structure responsible for binding the galactosyl residues of the peanut lectin receptor of thymic lymphocytes. Binding sites for a large set of lectins could be demonstrated on both, thymic lymphocytes and epithelium. The intrathymic differentiation pathway of T-lineage cells is discussed with regard to those lymphocytic and epithelial cell surface structures considered to enable cellular interaction.     

Immunosuppression with cyclosporin A alters the thymic microenvironment

The effect of cyclosporin A (CyA) immunosuppression on the murine thymic microenvironment and T lymphocyte development has been analysed using monoclonal antibodies to epithelial and lymphocyte subpopulations, macrophages and major histocompatibility complex (MHC) class II antigens in immunohistochemistry and flow cytometry. The major microenvironmental target for CyA-induced damage was the thymic medulla, where a reduction in all epithelial cell subsets, dendritic cells and macrophages was observed. In contrast, the thymic cortex appeared essentially normal. CyA had no detectable effect on the intensity of microenvironmental expression of MHC class II molecules in either cortex or medulla, although the number of MHC class II positive medullary cells was reduced after CyA treatment. CyA also had a differential effect on the thymic lymphocyte populations where there was little change in the Thy-1 bright, CD5 dull, CD4+, CD8+ cortical thymocytes but a depletion of the Thy-1 dull, CD5 bright, CD4 or CD8 single-positive medullary cells. This lymphocyte loss may be due partly to increased migration from thymus to spleen and other peripheral lymphoid organs, and partly to a block in the differentiation stage from cortical to medullary lymphocyte. The thymic microenvironment and lymphocyte subpopulations recover rapidly after cessation of CyA treatment, although there may be longer term functional defects resulting from the CyA-induced injury.     

Accessory cells of the lung. II. Ia+ pulmonary dendritic cells display cell surface antigen heterogeneity

In earlier studies, we had determined that class II (Ia) major histocompatibility complex (MHC) antigen expression in the normal rat lung was limited to dendritic cells and type II alveolar cells. In order to characterize the Ia+ pulmonary dendritic cells of the lung parenchyma, Lewis rat lungs were dissected free of their major airways, enzymatically digested, and serially subjected to density centrifugation on bovine serum albumin, overnight adherence, and immunopanning with a murine anti-rat monoclonal antibody (anti-OX-6) that reacts specifically with class II (Ia) MHC antigens. The purified Ia+ pulmonary cells displayed the morphologic and functional features of dendritic accessory cells, including extended cell processes, absence of nonspecific esterase staining, minimal phagocytosis of latex beads, rapid clustering with T lymphocytes, and co-stimulation of T-cell mitogen responses. Detailed immunophenotyping by cytofluorimetry and immunohistology showed that the purified dendritic cells were Ia (OX-6)+, CD45R (OX-1)+, CD45Rb (OX-22)-, ICAM-1+, and OX-43-. As many as 50% of the cells bound heat-aggregated IgG, while a smaller percentage expressed the CD43 sialophorin antigens (W3/13) expressed by a variety of blood-derived cells, and/or the OX-41 and RMA macrophage antigens. We conclude that Ia+ dendritic cells of lung are heterogeneous with respect to their expression of surface membrane differentiation antigens and may prove to be functionally distinct with respect to their accessory activities.