Activation of Humoral Immunity during the Pathogenesis of Experimental Chronic Lung Allograft Dysfunction.

Alloreactive and autoreactive antibodies have been associated with the development of chronic lung allograft dysfunction (CLAD), but their pathogenic role is disputed. Orthotopic left lung transplantation was performed in the Fischer-344 to Lewis rat strain combination followed by the application of ciclosporine for 10 days. Four weeks after transplantation, lipopolysaccharide (LPS) was instilled into the trachea. Lungs were harvested before (postoperative day 28) and after LPS application (postoperative days 29, 33, 40, and 90) for histopathological, immunohistochemical, and Western blot analyses. Recipient serum was collected to investigate circulating antibodies. Lung allografts were more strongly infiltrated by B cells and deposits of immunoglobulin G and M were more prominent in allografts compared to right native lungs or isografts and increased in response to LPS instillation. LPS induced the secretion of autoreactive antibodies into the circulation of allograft and isograft recipients, while alloreactive antibodies were only rarely detected. Infiltration of B cells and accumulation of immunoglobulin, which is observed in allografts treated with LPS but not isografts or native lungs, might contribute to the pathogenesis of experimental CLAD. However, the LPS-induced appearance of circulating autoreactive antibodies does not seem to be related to CLAD, because it is observed in both, isograft and allograft recipients.

ß-Nicotinamide Adenine Dinucleotide (ß-NAD) Inhibits ATP-Dependent IL-1ß Release from Human Monocytic Cells.

While interleukin-1ß (IL-1ß) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1ß maturation, is released from damaged cells along with ß-nicotinamide adenine dinucleotide (ß-NAD). Here, we tested the hypothesis that ß-NAD controls ATP-signaling and, hence, IL-1ß release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')-O-(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of ß-NAD. IL-1ß was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca2+-independent phospholipase A2 (iPLA2ß, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous ß-NAD signaled via P2Y receptors and dose-dependently (IC50 = 15 µM) suppressed the BzATP-induced IL-1ß release. Signaling involved iPLA2ß, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that ß-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular ß-NAD that suppresses ATP-induced release of IL-1ß by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

Surfactant inhibits ATP-induced release of interleukin-1ß via nicotinic acetylcholine receptors.

Interleukin (IL)-1ß is a potent pro-inflammatory cytokine of innate immunity involved in host defense. High systemic IL-1ß levels, however, cause life-threatening inflammatory diseases, including systemic inflammatory response syndrome. In response to various danger signals, the pro-form of IL-1ß is synthesized and stays in the cytoplasm unless a second signal, such as extracellular ATP, activates the inflammasome, which enables processing and release of mature IL-1ß. As pulmonary surfactant is known for its anti-inflammatory properties, we hypothesize that surfactant inhibits ATP-induced release of IL-1ß. Lipopolysaccharide-primed monocytic U937 cells were stimulated with an ATP analog in the presence of natural or synthetic surfactant composed of recombinant surfactant protein (rSP)-C, palmitoylphosphatidylglycerol, and dipalmitoylphosphatidylcholine (DPPC). Both surfactant preparations dose-dependently inhibited IL-1ß release from U937 cells. DPPC was the active constituent of surfactant, whereas rSP-C and palmitoylphosphatidylglycerol were inactive. DPPC was also effective in primary mononuclear leukocytes isolated from human blood. Experiments with nicotinic antagonists, siRNA technology, and patch-clamp experiments suggested that stimulation of nicotinic acetylcholine receptors (nAChRs) containing subunit α9 results in a complete inhibition of the ion channel function of ATP receptor, P2X7. In conclusion, the surfactant constituent, DPPC, efficiently inhibits ATP-induced inflammasome activation and maturation of IL-1ß in human monocytes by a mechanism involving nAChRs.

Adaptive and innate immune responses in a rat orthotopic lung transplant model of chronic lung allograft dysfunction.

Acute rejection and respiratory infections are major risk factors for chronic lung allograft dysfunction (CLAD) after lung transplantation. To shed light on the enigmatic etiology of CLAD, we test the following hypotheses using a new experimental model: (i) Alloimmune-independent pulmonary inflammation reactivates alloimmunity. (ii) Alloimmunity enhances the susceptibility of the graft toward pathogen-associated molecular patterns. Pulmonary Fischer 344 to Lewis rat allografts were treated with lipopolysaccharide (LPS), which consistently results in lesions typical for CLAD. Grafts, local lymph nodes, and spleens were harvested before (day 28) and after LPS application (days 29, 33, and 40) for real-time RT-PCR and immunohistochemistry. Mixed lymphocyte reactions were performed on day 33. Four weeks after transplantation, lung allografts displayed mononuclear infiltrates compatible with acute rejection and overexpressed most components of the toll-like receptor system. Allografts but not secondary lymphoid organs expressed increased levels of Th1-type transcription factors and cytokines. LPS induced macrophage infiltration as well as mRNA expression of pro-inflammatory cytokines and effector molecules of innate immunity. Unexpectedly, T-cell reactivity was not enhanced by LPS. We conclude that prevention of CLAD might be accomplished by local suppression of Th1 cells in stable grafts and by controlling innate immunity during alloimmune-independent pulmonary inflammation.

Expression of nestin after renal transplantation in the rat.

Chronic allograft injury (CAI) limits the long-term success of renal transplantation. Nestin is a marker of progenitor cells, which probably contribute to its pathogenesis. We hypothesize that nestin is induced by ischemia/reperfusion injury and acute rejection, main risk factors for CAI. Syngeneic renal transplantation was performed in Lewis rats and allogeneic transplantation in the Fischer 344 to Lewis strain combination, which results in reversible acute rejection and in CAI in the long-run. The Dark Agouti to Lewis rat strain combination was used to study fatal acute rejection. In untreated kidneys, nestin immunoreactivity was detected in glomeruli and in very few interstitial or microvascular cells. Syngeneic transplantation induced nestin expression within 4 days, which decreased until day 9 and returned to control levels on day 42. Nestin expression was strong during acute rejection and still detected during the pathogenesis of CAI on day 42. Nestin-positive cells were identified as endothelial cells and interstitial fibroblast-like cells co-expressing alpha-smooth muscle actin. A sub-population of them expressed proliferating cell nuclear antigen. In conclusion, nestin is induced in renal grafts by ischemia/reperfusion injury and acute rejection. It is expressed by proliferating myofibroblasts and endothelial cells and probably contributes to the pathogenesis of CAI.

Neuropeptide Y is expressed by rat mononuclear blood leukocytes and strongly down-regulated during inflammation.

Neuropeptide Y (NPY), a classical sympathetic comediator, regulates immunological functions including T cell activation and migration of blood leukocytes. A NPY-mediated neuroimmune cross-talk is well conceivable in sympathetically innervated tissues. In denervated, e.g., transplanted organs, however, leukocyte function is not fundamentally disturbed. Thus, we hypothesized that NPY is expressed by blood leukocytes themselves and regulated during inflammation. NPY mRNA and peptide expression were analyzed in mononuclear leukocytes isolated from the blood vessels of healthy rat kidneys, as well as from the blood vessels of isogeneic and allogeneic renal grafts transplanted in the Dark Agouti to Lewis or in the Fischer 344 to Lewis rat strain combination. Depending on the donor strain, acute allograft rejection is either fatal or reversible but both experimental models are characterized by massive accumulation of intravascular leukocytes. Leukocytes, predominantly monocytes, isolated from the blood vessels of untreated kidneys and isografts expressed high amounts of NPY mRNA and peptide, similar to expression levels in sympathetic ganglia. During acute allograft rejection, leukocytic NPY expression drastically dropped to approximately 1% of control levels in both rat strain combinations. In conclusion, NPY is an abundantly produced and tightly regulated cytokine of mononuclear blood leukocytes.

Donor B cells in splenic follicles of experimental pulmonary allograft recipients.

BACKGROUND: After transplantation, passenger leukocytes move to lymphoid organs of the recipient. These cells appear to initiate allograft rejection, but they also might be involved in tolerance induction. MATERIALS AND METHODS: Orthotopic left lung transplantation was performed in the Dark Agouti to Lewis rat strain combination with no immunosuppression. Recipient spleens were removed at intervals of 24 h until day 6 after transplantation. For comparison, spleens from renal allograft recipients were analysed. Donor-derived major histocompatibility complex (MHC) class II antigens were detected by monoclonal antibody OX76. In double-staining experiments with antibodies specific for leukocyte subpopulations, their localisation and identity was analysed. RESULTS: OX76-positive leukocytes were already detected in recipient spleens on day 1 post-transplantation. They increased in number until day 3 and decreased in number thereafter. Most of them were localised in splenic follicles and expressed the B cell variant of CD45R and IgG. Cell surface antigens typical for other leukocyte subpopulations were not detected. In the spleens of renal allograft recipients, only few donor-derived cells were seen. CONCLUSION: After lung transplantation, numerous MHC class II-positive B cells migrate to the splenic follicles of the recipient. These cells might, in part, be responsible for immunologic differences observed between renal and pulmonary allografts.

Macrophages in experimental rat lung isografts and allografts: infiltration and proliferation in situ.

Alveolar macrophages (AMs) and peribronchial/perivascular macrophages are probably involved in lung allograft damage. We investigate leukocyte infiltration into graft tissue and address the question whether proliferation in situ contributes to macrophage homeostasis and accumulation. Lung transplantation was performed in the Lewis (LEW)-to-LEW and in the Dark Agouti-to-LEW rat strain combination. Graft infiltration by ED1+ and ED2+ (CD163) macrophages was analyzed by immunohistochemistry (IHC) and compared with infiltration by lymphocytes. Cells in the S-phase of the cell cycle were pulse-labeled with BrdU and detected immunohistochemically. Finally, the donor or recipient origin of AMs was determined by IHC and in situ hybridization. ED1+ AMs in allogeneic transplants increased by more than 25-fold from Days 1 to 5. In addition, large, peribronchial/perivascular infiltrates developed containing numerous ED1+ cells. Although AMs in normal rat lungs are CD163-, AMs up-regulated CD163 between Days 4 and 5, reaching maximum values on Day 6. Lymphocytes were less numerous than macrophages. About 16% of the AMs and 10% of the peribronchial/perivascular macrophages were in the S-phase of the cell cycle on Day 2 post-transplantation. No differences in the frequency of BrdU+ macrophages were obvious between isografts and allografts. AMs of donor origin increased in number considerably during allograft rejection. In conclusion, the cellular infiltrate in lung allografts is dominated by macrophages, which exhibit an unusual phenotype and a strong capacity for mitotic self-renewal.