An open-label trial of rituximab therapy in pulmonary alveolar proteinosis.

Rituximab, a monoclonal antibody directed against the B-lymphocyte antigen CD20, has shown promise in several autoimmune disorders. Pulmonary alveolar proteinosis (PAP) is an autoimmune disorder characterised by autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF). An open-label, proof-of-concept phase II clinical trial was conducted in 10 PAP patients. The intervention consisted of two intravenous infusions of rituximab (1,000 mg) 15 days apart. Bronchoalveolar lavage (BAL) fluid and peripheral blood samples were collected. The primary outcome was improvement in arterial blood oxygenation. Both arterial oxygen tension and alveolar-arterial oxygen tension difference in room air improved in seven out of the nine patients completing the study. Lung function and high-resolution computed tomography scans, which were secondary outcomes, also improved. Peripheral blood CD19+ B-lymphocytes decreased from mean ± sem 15 ± 2% to <0.05% (n = 10) 15 days post-therapy. This decrease persisted for 3 months in all patients; at 6 months, CD19+ B-cells were detected in four out of seven patients (5 ± 2%). Total anti-GM-CSF immunoglobulin (Ig)G levels from baseline to 6 months were decreased in BAL fluids (n = 8) but unchanged in sera (n = 9). In this PAP cohort: 1) rituximab was well-tolerated and effectively ameliorated lung disease; and 2) reduction in anti-GM-CSF IgG levels in the lung correlated with disease changes, suggesting that disease pathogenesis is related to autoantibody levels in the target organ.

Polarity of human parainfluenza virus type 3 infection in polarized human lung epithelial A549 cells: role of microfilament and microtubule.

Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects the epithelial cells of the respiratory tract. In the present study we investigated the interaction of HPIV-3 with the type II alveolar human lung polarized epithelial A549 cells. Although HPIV-3 entry and budding were bidirectional from both the apical and the basolateral domains, HPIV-3 exhibited preferential entry and release from the apical pole. While disruption of the cellular actin microfilament and microtubule by cytochalasin D and nocodazole, respectively, had no effect on virus entry, disruption of the microtubule but not the microfilament inhibited HPIV-3 release.

Interleukin 10 (IL-10)-mediated inhibition of inflammatory cytokine production by human alveolar macrophages.

Alveolar macrophages are an important source of inflammatory cytokines in the lung. IL-10 has been shown to inhibit inflammatory cytokine production by human alveolar macrophages, but mechanisms are unclear. The purpose of the present study was to investigate whether IL-10 modified cytokine production by interference with transcriptional pathways. Alveolar macrophages were obtained from healthy controls by fiberoptic bronchoscopy and incubated with LPS+/-IL-10. Results indicated that steady state mRNA levels of tumour necrosis factor-alpha (TNF) and interleukin 1-beta (IL-1) decreased in the presence of IL-10. Consequently, electrophoretic mobility shift assays were performed using end-labelled nuclear factor-kappa B (NF-kappa B) or activator protein-1 (AP-1) probe. NF-kappa B binding was decreased in extracts from macrophages incubated for 4 h with LPS+IL-10 in comparison to those incubated with LPS alone. IL-10 also inhibited TNF secretion and NF-kappa B activation induced by another stimulus, staphylococcal toxin. Supershift assays revealed the presence of both p50 and p65 subunits of NF-kappa B. AP-1 was not affected by IL-10. Further examination of mechanisms indicated that IL-10 delayed the LPS-mediated degradation of the inhibitor protein I kappa B, thus delaying the nuclear translocation of the p65 subunit. These observations provide the first evidence that IL-10 antagonizes cytokine transcription in human alveolar macrophages by impeding the nuclear translocation of NF-kappa B by delaying the degradation of I kappa B.

Bacterial lipoprotein based expression vectors as tools for the characterisation of African swine fever virus (ASFV) antigens.

African swine fever virus (ASFV) is the causative agent of an important pig disease for which protective mechanisms are still poorly understood. The present work was aimed at the characterisation of ASFV antigens using previously reported vectors that allow their expression as fusion proteins with the bacterial lipoprotein OprI. Several recombinant clones induced SLA-restricted, ASFV-specific lymphoproliferation and one (A2) was demonstrated to stimulate ASFV-specific CTL activity in vitro, in opposition to the effect of UV inactivated virus. The nucleotide sequence of the fragment cloned in A2 showed 99% identity with a portion of the G1340L ORF of the BA71V isolate, and the expressed fusion lipoprotein induced specific antibodies in vivo. Blood mononuclear leukocytes from a pig immunised with outer membrane preparations from A2 showed to reduce strongly (99.6%) the ASFV yield in cultures of autologous macrophages. However, after inoculation with virulent virus the pig developed acute fatal ASF. Overall our results show that OprI based expression vectors are valuable tools to screen viral antigens in terms of their capacity to trigger immune competent cells.

Pulmonary alveolar proteinosis is a disease of decreased availability of GM-CSF rather than an intrinsic cellular defect.

Granulocyte-macrophage colony stimulating factor (GM-CSF) deficient mice develop a pulmonary alveolar proteinosis (PAP) syndrome which is corrected by the administration/expression of GM-CSF. These observations implicate GM-CSF in the pathogenesis of human PAP. We hypothesized that human PAP may involve an intrinsic cellular defect in monocytes/macrophages with an inability to produce GM-CSF and/or respond to GM-CSF. Thus, we investigated the cytokine responses to GM-CSF and LPS from peripheral blood monocytes and alveolar macrophages from patients with idiopathic PAP and healthy controls. Macrophage inflammatory protein-1-alpha (MIP) was measured from GM-CSF-stimulated cells and GM-CSF was measured from LPS-stimulated cells by ELISA. The MIP and GM-CSF production by monocytes and alveolar macrophages did not differ between PAP patients and healthy controls. Growth of the GM-CSF-dependent human myeloid cell line TF-1 was inhibited by serum from all patients studied (n = 10) and all patients had anti-GM-CSF antibody in their serum. The BAL from PAP patients had less detectable GM-CSF by ELISA than healthy controls (P = 0.05); in contrast, the inhibitory cytokine, interleukin-10 (IL-10), was increased in PAP compared to controls (P = 0.04). IL-10 is a potent inhibitor of LPS-stimulated GM-CSF production from healthy control alveolar macrophages. These studies are the first to demonstrate that circulating monocytes and alveolar macrophages from PAP patients are able to synthesize GM-CSF and respond to GM-CSF, suggesting no intrinsic abnormalities in GM-CSF signaling. In addition, these observations suggest that PAP in a subset of patients is the result of decreased availability of GM-CSF due to GM-CSF blocking activity and reduced GM-CSF production by IL-10.

Increased alveolar macrophage nuclear factor-kappa B activation and macrophage inhibitory protein-1alpha levels in lung transplant patients.

BACKGROUND: Lung transplantation is increasingly used as the treatment for many end-stage pulmonary diseases. A major cause of morbidity and mortality in patients who undergo lung transplantation is rejection of the allograft. Proinflammatory macrophage-derived cytokines may sustain and/or enhance the immunological response to lung allograft antigens. Nuclear factor-kappa B (NF-kappaB) is a transcription factor that regulates the production of many of these cytokines and growth factors in alveolar macrophages (AMs). The aim of our study was to evaluate the activation of NF-kappaB in AMs and the levels of one of the proinflammatory cytokines whose production it controls, macrophage inhibitory protein-1alpha (MIP-1alpha), in AMs from transplanted lungs compared to those from healthy controls. METHODS: Twenty-eight (28) transplant recipients were included in the study. NFkappaB activation was evaluated by electrophoretic mobility shift assay of whole cell extracts and by immunohistochemical analysis on cytospin preparations. Concentrated bronchoalveolar lavage fluid was analyzed by enzyme-linked immunosorbent assay for MIP-1alpha levels. RESULTS: NF-kappaB was activated in alveolar macrophages from transplant patients as compared to healthy controls. MIP-1alpha levels in epithelial-lining fluid were elevated in transplant patients as compared to healthy controls. Increased MIP-1alpha levels correlated with viral infections in the transplant patients. Neither finding was found to correlate with acute rejection by transbronchial biopsy. CONCLUSIONS: These results demonstrate that NF-kappaB activation and MIP-1alpha levels are increased in transplanted lungs and may play a role in the inflammatory cytokine cascade that leads to the long-term tissue damage and allograft rejection in these patients.

Nitric oxide regulation of asthmatic airway inflammation with segmental allergen challenge.

BACKGROUND: Despite evidence of increased nitric oxide (NO) in asthmatic compared with healthy individuals, the role of NO in airway inflammation is unclear. OBJECTIVE: The purpose of the study was to determine the in vivo effects of localized allergen challenge on airway NO levels and transcription factor activation. METHODS: In this study localized allergen challenge was used as a model of asthmatic exacerbation to determine the relationship of NO to airway inflammation. RESULTS: With allergen challenge, asthmatic patients had a rise in airway NO levels, whereas NO levels in healthy controls did not change. The increased NO in asthma with allergen challenge compared with healthy control subjects was associated with an increase in inflammatory cytokines (GM-CSF and macrophage inflammatory protein-1) in epithelial lining fluid and eosinophilic infiltrate in bronchoalveolar lavage fluid (BAL) and biopsy specimens. To investigate the mechanisms of cytokine gene expression, activation of the transcription factors activator protein-1 and nuclear factor-kappaB (NF-kappaB) in cells from BAL were evaluated. Activator protein-1 was not activated before or after local allergen challenge. In contrast, NF-kappaB activation was less in BAL cells from asthmatic patients with increased NO in comparison with controls. CONCLUSION: Our studies are the first to suggest an inverse correlation between NF-kappaB and airway NO in a localized segmental allergen challenge model in allergic asthmatic patients. The current study demonstrates that activation of the inflammatory response (eg, cytokines, cellular infiltrate) in allergic asthmatic patients is temporally associated with increased airway NO. We propose that NO that is up-regulated by cytokines is part of an autoregulatory feedback loop (ie, allergen challenge stimulates inflammatory cytokine production, which in turn stimulates NO production, and NO down-regulates cytokine production).

Nitric oxide blocks nuclear factor-kappaB activation in alveolar macrophages.

Nitric oxide (NO) is an important endogenous regulatory molecule implicated in both proinflammatory and antiinflammatory processes in the lung. Previously, we demonstrated that in human alveolar macrophages (AM), NO decreased inflammatory cytokine production, including that of interleukin-1beta, tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha. One mechanism by which NO could regulate such diverse cytokine production is through effects on the transcription factor nuclear factor-kappaB (NF-kappaB), which controls the expression of the genes for these inflammatory cytokines and growth factors. We therefore investigated whether NO affects NF-kappaB activation in AM in vitro and in vivo. In vitro studies with AM showed that NF-kappaB activation by lipopolysaccharide (LPS) is decreased by NO in a dose-dependent manner. NO prevented an LPS-mediated decrease in the NF-kappaB inhibitory protein IkappaB-alpha. In asthma, airway NO levels are increased, whereas in primary pulmonary hypertension (PPH), airway NO levels are lower than in healthy lungs. In vivo investigations were conducted with freshly isolated AM from healthy controls, asthmatic individuals, and PPH patients. Healthy individuals had airway NO levels of 8 +/- 2 ppb (mean +/- SEM), which is associated with low NF-kappaB activation. Asthma patients with airway NO levels > 17 ppb showed minimal NF-kappaB activation, whereas asthmatic individuals with NO levels

Differential regulation of human blood monocyte and alveolar macrophage inflammatory cytokine production by nitric oxide.

BACKGROUND: Nitric oxide (NO) has been associated with airway inflammation in asthma. Our previous work suggests that NO functions in an anti-inflammatory capacity through downregulation of stimulated cytokine secretion by normal human alveolar macrophages. Functional differences between alveolar macrophages and blood monocytes are thought to be related to maturation. OBJECTIVE: The purpose of this study was to determine the effect of NO on stimulated cytokine production by monocytes from asthmatics and normal healthy controls. METHODS: Monocytes and alveolar macrophages were obtained from normal volunteers (n = 13) and asthmatics with atopy (n = 7). Monocyte and alveolar macrophage cultures were stimulated with 0.5 microgram/mL lipopolysaccharide +/- 1.0 mM DETA NONOate (releases NO in culture with t1/2 = 20 hours at 37 degrees C) and incubated for 24 hours. Cell-free supernatants were collected and assayed by ELISA for tumor necrosis factor-alpha (TNF) and granulocyte macrophage colony stimulating factor (GM-CSF). RESULTS: Nitric oxide did not inhibit TNF production in monocytes of asthmatics and normals (mean +/- SEM % TNF stimulation = 19.6 +/- 9.7). Similar to previous results, NO did inhibit alveolar macrophages (% TNF suppression = 60.6 +/- 4.4). To determine whether this differential effect of NO on the two cell populations was related to maturation, monocytes were matured by culture for 7 days. The in vitro matured monocytes demonstrated 51.7 +/- 7.9% suppression of TNF. For each cell population, the responses of the asthmatics and healthy controls were not different. The differential effect is not cytokine specific since similar results were obtained with GM-CSF. CONCLUSION: These results demonstrate a differential effect of NO on monocyte and alveolar macrophages cytokine regulation and this effect may be related to the state of maturation.

Identification of a 25-aminoacid sequence from the major African swine fever virus structural protein VP72 recognised by porcine cytotoxic T lymphocytes using a lipoprotein based expression system.

Identification of African swine fever virus (ASFV) proteins recognised by cytotoxic T lymphocytes (CTL) from swine surviving ASFV/NH/P68 infection was assessed using expression vectors based on the Pseudomonas aeruginosa outer membrane lipoprotein I gene (oprI). Viral antigens expressed as fusion lipoproteins were shown to be taken efficiently by porcine blood-derived macrophages incubated with outer membrane protein preparations from transformed E. coli. To assess recognition by CTL the fusion lipoprotein-treated macrophages were used as targets in 51Cr release microcytotoxicity assays. Using this approach it was shown that the aminoacid sequence HKPHQSKPILTDENDTQRTCSHTNP from the major structural ASFV protein (VP72), encoded by a recombinant clone (pVUB72) is presented by macrophages, which are lysed under restriction of SLA class I antigens. Overall, the results demonstrate that the oprI based vectors are valuable tools to study ASFV-specific CTL activity.

Human neutrophils express GH-N gene transcripts and the pituitary transcription factor Pit-1b.

Since GH stimulates the development and function of granulocytes, we investigated the expression of GH in granulocyte subsets. By immunocytochemistry, 25 +/- 7% of the human neutrophils were shown to express immunoreactive GH, whereas eosinophils were negative. Reversed transcription (RT)-PCR analysis demonstrated GH mRNA in neutrophils. Restriction analysis revealed that neutrophils express the GH-N gene but not the GH-V gene. Furthermore, we demonstrated by western blot analysis that neutrophils express an alternatively spliced variant of the pituitary transcription factor Pit-1, designated Pit-1b.

Growth hormone expression in murine bone marrow cells is independent of the pituitary transcription factor Pit-1.

GH has been shown to promote the development and function of leukocytes. The expression of both GH and GH-receptors in lymphoid cells has led to the hypothesis that GH acts in an autocrine or paracrine fashion. The described effects of GH on hematopoiesis and B cell development, led us to investigate GH expression in bone marrow cells. By immunocytochemistry, we show that bone marrow-derived granulocytes and macrophages contain immunoreactive GH. We found that 65 +/- 24% of the granulocytes were stained with anti-GH, whereas 5.8 +/- 1.5% of the granulocytes contained detectable amounts of GH mRNA as assessed by in situ hybridization. To address a possible alternative regulation mechanism in bone marrow and to establish whether locally derived GH might still play a role in pituitary-deficient dwarf mice, we also addressed GH expression in bone marrow from hypopituitary Snell dwarf mice. These mice have a mutated gene for the pituitary transcription factor Pit-1 that is deficient in DNA binding. Our finding that GH expression (immunoreactive protein and mRNA) in bone marrow cells from dwarf mice is similar to that in normal mice points to a Pit-1 independent regulation of GH in mouse bone marrow.

Development of new cloning vectors for the production of immunogenic outer membrane fusion proteins in Escherichia coli.

The Pseudomonas aeruginosa lipoprotein gene (oprI) was modified by cloning an in-frame polylinker in both orientations at the end of oprI. The resulting plasmids pVUB1 and pVUB2 allow high lipoprotein production in E. coli after IPTG induction. The modified lipoproteins are present in the outer membrane and surface-exposed. Outer membrane-bound fusion proteins of different sizes were produced and used to generate antibodies without use of adjuvant. An 87 bp DNA fragment from the vp72 capsid protein gene of African Swine Fever virus (ASFV) and the entire Leishmania major glycoprotein gp63 gene were expressed in this system. Finally, a fusion lipoprotein containing a 16 amino acid epitope from the pre-S2b region of Hepatitis B virus (HBV) was presented by an antigen-presenting cell line to a T-cell hybridoma while the corresponding cross-linked S2b peptide was not. The results suggest that OprI-based fusion proteins can be used to generate both humoral and cellular immune responses.

Pit-1/GHF-1 expression in pituitary adenomas: further analogy between human adenomas and rat SMtTW tumours.

Adenomas can develop from each cell type of the anterior pituitary. In the normal pituitary, three of these cells types, the GH-, prolactin- and TSH-secreting cells, express the transcription factor Pit-1/GHF-1 which is responsible for prolactin and GH (and probably TSH) cell commitment, differentiation, probably proliferation and gene expression. We have analysed the expression of Pit-1/GHF-1 in a panel of human pituitary adenomas. All GH-, prolactin- and TSH-expressing adenomas studied expressed the Pit-1/GHF-1 factor, as demonstrated by in-situ hybridization and immunocytochemistry. The expression was higher in adenomas than in normal human pituitary. In contrast, ACTH- and LH-FSH-secreting and non-secreting adenomas were negative. Seven transplants of the spontaneous rat prolactinoma SMtTW were also investigated and all were found to be positive. This further stresses the analogy between these tumours and human prolactinomas. Taken together, the data confirm that Pit-1/GHF-1 expression is restricted to GH-, prolactin- and TSH-expressing cells, and the increased expression in adenomas is compatible with a role of Pit-1/GHF-1 in cell proliferation.

Growth hormone and prolactin are paracrine growth and differentiation factors in the haemopoietic system.

The pituitary secretory proteins growth hormone (GH) and prolactin (PRL) do not, as yet, have major roles in haematology or immunology. Recent evidence indicates that these hormones are haemopoietic growth factors and exert immunomodulatory functions at physiological concentrations. Here Robert Hooghe and colleagues discuss the significance of these hormones on different aspects of the immune system.

The transcription factor Pit-1/GHF-1 is expressed in hemopoietic and lymphoid tissues.

The expression of the Pit-1/GHF-1 transcription factor (hereafter Pit-1), which controls the expression of growth hormone (GH) and prolactin (PRL) in the pituitary gland, has been documented in human and rat hemopoietic and lymphoid tissues and cell lines. Pit-1 mRNA was detected by in situ hybridization in about 1% of rat bone marrow cells and in the spleen red pulp and marginal zone. Pit-1 was also expressed in human tonsils (mantle zone), in the thymus (rat and human, non-lymphoid cells), in lipopolysaccharide-stimulated rat peritoneal cells and in non-hepatocyte cells in the liver (rat and human). A detailed investigation of the rat spleen showed a very similar distribution for Pit-1, GH and PRL mRNA and Pit-1, GH and PRL proteins (detected by immunocytochemistry). Using polymerase chain reaction followed by Southern hybridization, the expression of Pit-1 could be confirmed in human and rat spleen, bone marrow and thymus. HL60 and RAJI leukemic cells were also positive. The sequence of fragments amplified from rat spleen and from human bone marrow completely matched published sequences of rat and human pituitary Pit-1, respectively. Expression of GH and PRL in lymphoid tissues has been documented. The straightforward hypothesis would therefore be that Pit-1's main function in lymphoid tissues is controlling GH and PRL expression, as in the pituitary gland. GH and PRL may be hemopoietic and lymphoid growth and differentiation factors.