Aerosol granulocyte-macrophage colony-stimulating factor for pulmonary alveolar proteinosis.

Recently, granulocyte-macrophage colony-stimulating factor (GM-CSF) auto-antibodies have been found in many patients with pulmonary alveolar proteinosis (PAP). The present study reports a retrospective case series of patients who used aerosolised GM-CSF in the treatment of idiopathic PAP. Between 1999 and 2003, 12 patients elected to receive aerosolised GM-CSF (250 microg b.i.d. every other week) in lieu of whole-lung lavage or observation. Patient characteristics, pulmonary function tests, arterial blood gas analysis, laboratory values and chest radiographs were extracted from the patient's medical records. Of the six patients tested, all had GM-CSF neutralising antibodies. Additionally, abnormalities in GM-CSF gene expression (one patient), receptor expression (two patients) and ability to upregulate adhesion molecules (one patient) were found. All patients except one had a positive response (mean improvements in arterial oxygen tension, alveolar-arterial oxygen gradient, carbon monoxide diffusing capacity of the lung and forced vital capacity were 17.1 mmHg, 18.4 mmHg, 16.6% pred and 13.5% pred, respectively). Two patients made a complete recovery and were disease free 1 and 2 yrs after discontinuing treatment. Four patients showed complete response to both the initial course or when treated again for recurrence after discontinuation of treatment. One patient required dose escalation (500 microg b.i.d.) with complete response. GM-CSF was well tolerated without late toxicity after median (range) follow-up of 30.5 (3-68) months. In conclusion, aerosolised granulocyte-macrophage colony-stimulating factor is safe and effective in treating pulmonary alveolar proteinosis providing an alternative to whole-lung lavage or subcutaneous granulocyte-macrophage colony-stimulating factor.

Interleukin-2 liposomes for primary immune deficiency using the aerosol route.

This is the first report of aerosol interleukin 2 (IL-2) liposome administration to individuals with immune deficiency. Parenteral IL-2 therapy has shown beneficial effects in some patients with cancer, common variable immunodeficiency (CVID), and human immunodeficiency virus (HIV) but is problematic because of side effects including fever and malaise as well as local swelling (delayed type hypersensitivity like reaction) after each subcutaneous IL-2 injection. Provision of an IL-2:human albumin liposome formulation via the aerosol route had few side effects in a recent clinical trial in cancer patients. Details of good manufacturing practice (GMP) synthesis and analysis of IL-2 liposomes (N= 6 lots) made without albumin carrier protein and placebo liposomes (three lots) are presented. After centrifugation, IL-2 was closely associated with the liposome pellet (99%). Mean diameter of liposomes was 1.1 microm. Patient acceptance, safety, toxicity, and immune effects of IL-2 liposomes were studied in individuals with primary immune deficiency (N = 15) and subsequently, a larger cohort of patients with hepatitis C. Experience in the immune deficient patients is the subject of this report. Placebo liposomes (12 weeks) and IL-2 liposomes (12 weeks) were provided using a nebulizer. Aerosol placebo liposomes and IL-2 liposomes were well tolerated. No changes in chest X-ray or pulmonary function were seen. Since biologic activity of aerosol IL-2 liposomes has been seen in viral disease (hepatitis C), additional studies of aerosol IL-2 liposomes in individuals with hepatitis C and HIV are planned.

The signal transduction pathway of CD23 (Fc epsilon RIIb) targets I kappa B kinase.

Alveolar macrophages play a crucial role in initiating the inflammatory response in allergic asthma through the cross-linking of the low affinity IgE receptors (Fc epsilon RIIb or CD23) by IgE-allergen immunocomplexes. We have previously shown that CD23 cross-linking in monocytes and U937 cells targets I kappa B alpha, leading to the activation of the transcription factor NF-kappa B. We demonstrate in this paper that CD23-initiated signaling in U937 cells leads to hyperphosphorylation of I kappa B alpha at Ser32/Ser36 residues. Overexpression of a dominant-negative I kappa B alpha transgene containing mutations at Ser32/Ser36 completely inhibits degradation of I kappa B alpha, NF-kappa B activation, and gene transcription that follows CD23 cross-linking. Investigation of the second messengers mediating the CD23-dependent activation of NF kappa B demonstrates that I kappa B kinases (IKKs) but not p90rsk are selectively activated following CD23 cross-linking and mediates the phosphorylation of I kappa B alpha. Cotransfection experiments with an IKK beta negative dominant completely inhibit CD23 induced NF kappa B activation. Furthermore, the activation of tyrosine kinase(s) by CD23 is required for the induction of IKK activity, I kappa B alpha degradation, and NF-kappa B nuclear translocation. Taken together, our results show that CD23 cross-linking in the monocytic lineage induces tyrosine kinase activation followed by activation of IKK, which phosphorylates I kappa B alpha at the N-terminal domain (Ser32/Ser36), inducing its degradation, NF-kappa B activation and gene transcription.

Signal transduction pathways triggered by the FcepsilonRIIb receptor (CD23) in human monocytes lead to nuclear factor-kappaB activation.

BACKGROUND: Alveolar macrophages play a key role in the initiation of the inflammatory reaction of allergic asthma. Alveolar macrophages and peripheral blood monocytes are activated when IgE/allergen immune complexes bind to the CD23 receptor, which leads to the production of inflammatory cytokines. OBJECTIVE: We sought to investigate the molecular mechanisms regulating this early inflammatory response. We have focused on the study of the signal transduction pathways triggered by CD23 in human monocytes and the promonocytic cell line U937. METHODS: CD23 was cross-linked in human monocytes and U937 cells with IgE immune complexes. Surface expression of CD23 was determined by FACS analysis. Transcription factor activation and gene transcription were studied by gel-shift assays and Northern blot analysis, respectively. IkappaBalpha phosphorylation and degradation was analyzed by Western blot. RESULTS: Nuclear factor (NF)-kappaB is the main transcription factor involved in the gene activation that follows CD23 cross-linking in monocytes. CD23-induced NF-kappaB is a heterodimer composed of p65/p50 subunits. NF-kappaB nuclear translocation is secondary to the phosphorylation and subsequent degradation of the NF-kappaB inhibitory molecule IkappaBalpha. Tyrosine kinase-dependent, and not protein kinase C-dependent, pathways mediate CD23-triggered NF-kappaB activation but do not participate in the direct phosphorylation of IkappaBalpha. IkappaBalpha degradation and NF-kappaB nuclear translocation correlate with transcriptional activation of the inflammatory cytokines TNF-alpha and IL-1beta. CONCLUSIONS: NF-kappaB is the main transcription factor involved in the signal transduction pathway of CD23 in monocytes.

Mannose-binding lectin deficiency associated with neutrophil chemotactic unresponsiveness to C5a.

BACKGROUND: Mannose-binding lectin (MBL) plays an important role in host defense by activating the complement cascade. OBJECTIVE: Three children with a history of recurrent infections since infancy were found to have MBL deficiency associated with a neutrophil chemotactic unresponsiveness specific to C5a. We have studied the genomic sequence of the C5a receptor (C5aR) in 2 of the subjects to determine whether this unresponsiveness was due to a genetic mutation or to aberrant complement activation associated with the MBL deficiency. METHODS: MBL genotype analysis was performed by PCR-based methods with use of specific primers and restriction enzymes to detect the 3 previously reported mutations. Expression of C5aR was analyzed by flow cytometry. The C5aR gene was amplified from the patient's genomic DNA by PCR and sequenced by standard procedures. RESULTS: C5aR was found to be expressed normally on the neutrophils of one of the subjects. Sequence analysis of the C5aR gene revealed a point mutation that substituted threonine at position 261 for alanine in one patient but no abnormality in the other, suggesting gene polymorphism. Treatment of 2 patients with granulocyte-colony stimulating factor corrected the neutrophil chemotactic abnormality in vitro and induced a significant clinical improvement. CONCLUSION: MBL deficiency can be associated with neutrophil chemotactic unresponsiveness to C5a and it is clinically manifested by recurrent and chronic infections. Treatment of these patients with granulocyte colony-stimulating factor results in normalization of neutrophil chemotaxis against C5a and significant clearing of infections.

The anti-androgen hydroxyflutamide and androgens inhibit interleukin-6 production by an androgen-responsive human osteoblastic cell line.

While androgens clearly have significant skeletal effects, the paracrine mediators of androgen action on bone are at present unclear. Interleukin-6 (IL-6) is a candidate cytokine that is produced by osteoblastic lineage cells and promotes osteoclastogenesis and bone resorption. Here, we assessed constitutive as well as IL-1beta- and tumor necrosis factor-alpha (TNF-alpha)-stimulated IL-6 mRNA expression by Northern analysis and protein secretion by immunoassay in a human androgen-responsive osteoblastic cell line (hFOB/AR-6) which contains approximately 4000 androgen receptors (ARs)/nucleus. Treatment with 5alpha-dihydrotestosterone (DHT) dose-dependently inhibited constitutive and TNF-alpha/IL-1beta-stimulated IL-6 mRNA steady-state levels in hFOB/AR-6 cells by 70-80% at 10-7 M. In addition, testosterone also suppressed TNF-alpha/IL-1beta-stimulated IL-6 mRNA levels by 57%, while the adrenal androgen dehydroepiandrosterone had no effect. Of note, the specific AR antagonist, hydroxyflutamide, also inhibited IL-6 mRNA levels by 70%. Consistent with the Northern analyses, treatment with 5alpha-DHT, testosterone, and hydroxyflutamide also inhibited IL-6 protein production by 79%, 62%, and 71%, respectively (p < 0.001), while these agents had no effect on IL-6 soluble receptor levels. Finally, we demonstrated that hydroxyflutamide treatment of hFOB/AR-6 cells markedly inhibited the activation and binding of NF-kappaB (a known stimulator of IL-6 gene transcription) to its response element, thus providing a potential mechanism for its effect on IL-6 production by osteoblasts. These data are consistent with the hypothesis that suppression of osteoblast IL-6 production by androgens may mediate, at least in part, the antiresorptive effects of androgens on bone. Moreover, our findings also indicate that hydroxyflutamide, which is a known AR antagonist in most tissues, may function as a selective AR modulator for effects on IL-6 production by osteoblasts.

Mannose-binding lectin (MBL) deficiency. Variant alleles in a midwestern population of the United States.

OBJECTIVES: To describe a method for the genotype analysis of mutations in the gene encoding mannose binding lectin (MBL), study the incidence of MBL gene mutations in a population of the Midwest of the United States, and compare it with previous reports in other populations. The objective of this report is also an extensive review of the literature to analyze the importance of MBL deficiency in human disease. DATA SOURCES: Blood samples were obtained from the blood bank of the Mayo Clinic. They represented a population of blood donors living in the Midwest of the United States. A review of the literature was performed by selection of articles from Medline database. STUDY SELECTION: Blood samples, 148, were randomly selected from a pool of blood donors. They included both females and males. Blood donors had been previously screened by a questionnaire and were found to be generally healthy. For the literature review, articles containing original data on MBL in humans were selected. RESULTS: Forty-five (30.4%) of the analyzed blood donors carried one variant allele, while 8 donors (5.4%) showed homozygosity or compound heterozygosity for MBL gene mutations. Allele frequency for the different MBL variants is provided. Our results are similar to those reported for the Danish population. Literature review provides evidence for a significant role of MBL deficiency in the innate immunity. The incidence of MBL mutations is higher among patients with recurrent infections and autoimmune disorders. CONCLUSIONS: Mannose binding lectin deficiency has a definite role in the pathogenesis of primary immunodeficiency in humans and screening patients with recurrent infections and autoimmunity might be beneficial. The significance of MBL deficiency among apparently healthy blood donors remains to be determined.

Primary immunodeficiencies.

The primary immunodeficiencies are congenital disorders that affect the function of the immune system. The result is an inadequate immune response to microorganisms, self-antigens, and tumor cells, which leads to increased susceptibility to infections, autoimmunity, or malignant disease. A substantial advance has been made in the understanding of the exact molecular mechanisms leading to primary immunodeficiencies; however, for some types, a specific genetic defect has not yet been determined. The life expectancy of patients with primary immunodeficiencies has increased considerably because of bone marrow transplantation and replacement therapies. Gene therapy has already been used for a particular type of immunodeficiency and is a promising alternative for the future management of many other types of primary immunodeficiencies. A better understanding of the genetic defects that lead to primary immunodeficiencies would result in the development of novel therapeutic strategies.

Functional role for Syk tyrosine kinase in natural killer cell-mediated natural cytotoxicity.

Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

Regulation of I kappa B alpha and p105 in monocytes and macrophages persistently infected with human immunodeficiency virus.

The mechanisms regulating human immunodeficiency virus (HIV) persistence in human monocytes/macrophages are partially understood. Persistent HIV infection of U937 monocytic cells results in NF-kappa B activation. Whether virus-induced NF-kappa B activation is a mechanism that favors continuous viral replication in macrophages remains unknown. To further delineate the molecular mechanisms involved in the activation of NF-kappa B in HIV-infected monocytes and macrophages, we have focused on the regulation of the I kappa B molecules. First, we show that persistent HIV infection results in the activation of NF-kappa B not only in monocytic cells but also in macrophages. In HIV-infected cells, I kappa B alpha protein levels are decreased secondary to enhanced protein degradation. This parallels the increased I kappa B alpha synthesis secondary to increased I kappa B alpha gene transcription, i.e., increased RNA and transcriptional activity of its promoter-enhancer. Another protein with I kappa B function, p105, is also modified in HIV-infected cells: p105 and p50 steady-state protein levels are increased as a result of increased synthesis and proteolytic processing of p105. Transcriptional activity of p105 is also increased in infected cells and is also mediated by NF-kappa B through a specific kappa B motif. These results demonstrate the existence of a triple autoregulatory loop in monocytes and macrophages involving HIV, p105 and p50, and MAD3, with the end result of persistent NF-kappa B activation and viral persistence. Furthermore, persistent HIV infection of monocytes and macrophages provides a useful model with which to study concomitant modifications of different I kappa B molecules.

Two factors, IRF1 and KBF1/NF-kappa B, cooperate during induction of MHC class I gene expression by interferon alpha beta or Newcastle disease virus.

The expression of class I genes of the Major Histocompatibility Complex is stimulated by IFN. The promoter of these genes contains an interferon response sequence (IRS) which overlaps the major enhancer. These elements are recognized by several protein factors, including IRF-1, which binds the IRS, and KBF1/NF-kappa B, which binds the enhancer. We demonstrate here that infection by Newcastle Disease Virus (NDV) results in an increased expression of class I genes, by a mechanism partially different from that of IFN, but that in both cases the cooperative action of IRF1 and KBF1/NF-kappa B is required. In F9 embryonal carcinoma cells, where KBF1/NF-kappa B activity cannot be detected, both types of stimuli are ineffective.

NF-kappa B-dependent induction of the NF-kappa B p50 subunit gene promoter underlies self-perpetuation of human immunodeficiency virus transcription in monocytic cells.

The molecular mechanisms underlying the sustained nuclear translocation of NF-kappa B observed in U937 monocytic cells chronically infected with human immunodeficiency virus (HIV) were studied. The activity of the promoter regulating the synthesis of the p105 precursor of the NF-kappa B p50 subunit was enhanced in these cells. Deletions in this promoter indicated that this upregulation was mediated through the NF-kappa B- but not the AP-1-binding motif, by bona fide p50/p65 heterodimers. Analysis of cytosolic extracts indicated that NF-kappa B levels were increased in HIV-infected cells. In contrast to the transient NF-kappa B activation induced by phorbol ester, the permanent NF-kappa B translocation induced by HIV infection was not dependent on PKC isoenzymes alpha and beta as shown by the use of a specific inhibitor (GF 109203X). These observations indicate that during chronic HIV infection of U937 cells, continuous NF-kappa B (p50/p65) translocation results in p105 promoter upregulation with subsequent cytosolic NF-kappa B accumulation, ready for further translocation. This HIV-mediated mechanism results in a self-perpetuating loop of NF-kappa B production.

The characterization of the promoter of the gene encoding the p50 subunit of NF-kappa B indicates that it participates in its own regulation.

In order to characterize the regulation of the gene encoding the p50 subunit of the transcription factor NF-kappa B, we have isolated a human genomic clone, and sequenced the promoter of this gene. By in situ hybridization we have mapped the gene encoding the p50 subunit of NF-kappa B to the 4q23-4q25 region of the human genome and the H1-H3 region of the murine chromosome 3. The p50 promoter lacks TATA and CAAT elements, but contains NF-kappa B, AP-1 and HIP-1 binding sequence. The kappa B motif binds NF-kappa B, KBF1, and heterodimers of p50 and c-rel, suggesting that the gene is regulated by members of this family. Co-transfection experiments demonstrate that the p50 promoter can be activated by either of the two subunits of NF-kappa B (p50 and p65), and more strongly by the combination of both. The promoter of p50 can be activated by phorbol esters and tumor necrosis factor alpha but not by forskolin and these responses are mediated through the NF-kappa B binding sequence. The involvement of NF-kappa B in the regulation of the p50 gene is also supported by the inhibition of the PMA activation of the promoter by an NF-kappa B transdominant negative mutant, as well as the product of the v-rel oncogene.

Eosinophil differentiation of human umbilical cord mononuclear cells and prolonged survival of mature eosinophils by murine EL-4 thymoma cell conditioned medium.

Umbilical cord mononuclear cells, HL-60 cells, HL-60 clones selected for eosinophil differentiation, and the eosinophil leukemia cell line EoL were tested for their ability to produce eosinophil peroxidase. HL-60 clones selected for eosinophil differentiation produced eosinophil peroxidase, as judged by staining of cells for cyanide-resistant peroxidase activity; however, these cells lost their ability to produce eosinophil peroxidase in long-term culture. In contrast, eosinophil precursors from human umbilical cord blood mononuclear cells stimulated with murine EL-4 conditioned medium (EL-4 CM) were regularly induced to eosinophil protein synthesis, including eosinophil peroxidase, major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin, as assessed by cyanide-resistant peroxidase and immunofluorescence staining. This induction by EL-4 CM is either at the level of gene transcription or mRNA stabilization, as shown by the increase of total mRNA for eosinophil peroxidase, major basic protein, and eosinophil-derived neurotoxin by Northern blot analyses. Purified peripheral blood eosinophils incubated for 4 days with EL-4 CM had increased survival over control eosinophils. Moreover, this enhanced survival was specifically blocked by antiserum to interleukin 5. Our results suggest that the effects of EL-4 CM on human umbilical cord mononuclear cells and mature eosinophils are due to the presence of interleukin 5.

Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant.

The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family.

The molecular biology of eosinophil granule proteins.

Here, we briefly review the molecular biology of the human eosinophil granule proteins, major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). The nucleotide sequence of MBP cDNA indicates that MBP is translated as a 25.2-kilodalton preproprotein; the mpb gene consists of 6 exons and 5 introns spanning 3.3 kilobases (kb). The approximately 2.1-kb nucleotide sequence of EPO cDNA corresponds to a prosequence, light chain and heavy chain in that order; similarities to other peroxidases suggest the existence of a multigene family. EDN and ECP cDNAs and genes are remarkably similar throughout, suggesting a relatively recent divergence. Promoter regions of the 4 genes show interesting differences and similarities which may be related to differential gene regulation.

Participation of eosinophils in the toxic oil syndrome.

The participation of eosinophils in the Spanish toxic oil syndrome (TOS) was investigated. Eosinophil infiltration and degranulation in tissues from 52 patients with the TOS were examined by immunofluorescence staining for the eosinophil granule major basic protein (MBP). Serum MBP levels were determined in sera from 323 patients. Eosinophil infiltration and degranulation were found in several tissues, especially during the acute phase of the TOS, and serum MBP was significantly elevated during all phases of the disease, suggesting that eosinophils play a role in the pathogenesis of the TOS.

Structure and chromosome localization of the human eosinophil-derived neurotoxin and eosinophil cationic protein genes: evidence for intronless coding sequences in the ribonuclease gene superfamily.

Human genomic DNAs for the eosinophil granule proteins, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), were isolated from genomic libraries. Alignment of EDN (RNS2) and ECP (RNS3) gene sequences demonstrated remarkable nucleotide similarities in noncoding sequences, introns, and flanking regions, as well as in the previously known coding regions. Detailed examination of the 5'-noncoding regions yielded putative TATA and CAAT boxes, as well as similarities to promoter motifs from unrelated genes. A single intron of 230 bases was found in the 5' untranslated region and we suggest that a single intron in this region and an intronless coding region are features common to many members of the RNase gene superfamily. The RNS2 and RNS3 genes were localized to the q24-q31 region of human chromosome 14. It is likely that these two genes arose as a consequence of a gene duplication event that took place approximately 25-40 million years ago and that a subset of anthropoid primates possess both of these genes or closely related genes.