Granulocyte/macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects.

High levels of granulocyte/macrophage-colony-stimulating factor (GM-CSF) autoantibodies are thought to cause pulmonary alveolar proteinosis (PAP), a rare syndrome characterized by myeloid dysfunction resulting in pulmonary surfactant accumulation and respiratory failure. Paradoxically, GM-CSF autoantibodies have been reported to occur rarely in healthy people and routinely in pharmaceutical intravenous immunoglobulin (IVIG) purified from serum pooled from healthy subjects. These findings suggest that either GM-CSF autoantibodies are normally present in healthy people at low levels that are difficult to detect or that serum pooled for IVIG purification may include asymptomatic persons with high levels of GM-CSF autoantibodies. Using several experimental approaches, GM-CSF autoantibodies were detected in all healthy subjects evaluated (n = 72) at low levels sufficient to rheostatically regulate multiple myeloid functions. Serum GM-CSF was more abundant than previously reported, but more than 99% was bound and neutralized by GM-CSF autoantibody. The critical threshold of GM-CSF autoantibodies associated with the development of PAP was determined. Results demonstrate that free serum GM-CSF is tightly maintained at low levels, identify a novel potential mechanism of innate immune regulation, help define the therapeutic window for potential clinical use of GM-CSF autoantibodies to treat inflammatory and autoimmune diseases, and have implications for the pathogenesis of PAP.

Activation of intracellular signaling pathways by the murine cytomegalovirus G protein-coupled receptor M33 occurs via PLC-{beta}/PKC-dependent and -independent mechanisms.

The presence of numerous G protein-coupled receptor (GPCR) homologs within the herpesvirus genomes suggests an essential role for these genes in viral replication in the infected host. Such is the case for murine cytomegalovirus (MCMV), where deletion of the M33 GPCR or replacement of M33 with a signaling defective mutant has been shown to severely attenuate replication in vivo. In the present study we utilized a genetically altered version of M33 (termed R131A) in combination with pharmacological inhibitors to further characterize the mechanisms by which M33 activates downstream signaling pathways. This R131A mutant of M33 fails to support salivary gland replication in vivo and, as such, is an important tool that can be used to examine the signaling activities of M33. We show that M33 stimulates the transcription factor CREB via heterotrimeric G(q/11) proteins and not through promiscuous coupling of M33 to the G(s) pathway. Using inhibitors of signaling molecules downstream of G(q/11), we demonstrate that M33 stimulates CREB transcriptional activity in a phospholipase C-beta and protein kinase C (PKC)-dependent manner. Finally, utilizing wild-type and R131A versions of M33, we show that M33-mediated activation of other signaling nodes, including the mitogen-activated protein kinase family member p38alpha and transcription factor NF-kappaB, occurs in the absence of G(q/11) and PKC signaling. The results from the present study indicate that M33 utilizes multiple mechanisms to modulate intracellular signaling cascades and suggest that signaling through PLC-beta and PKC plays a central role in MCMV pathogenesis in vivo.

Standardized serum GM-CSF autoantibody testing for the routine clinical diagnosis of autoimmune pulmonary alveolar proteinosis.

Autoantibodies against granulocyte/macrophage colony-stimulating factor (GMAbs) cause autoimmune pulmonary alveolar proteinosis (PAP) and measurement of the GMAb level in serum is now commonly used to identify this disease, albeit, in a clinical research setting. The present study was undertaken to optimize and standardize serum GMAb concentration testing using a GMAb enzyme-linked immunosorbent assay (GMAb ELISA) to prepare for its introduction into routine clinical use. The GMAb ELISA was evaluated using serum specimens from autoimmune PAP patients, healthy people, and GMAb-spiked serum from healthy people. After optimizing assay components and procedures, its accuracy, precision, reliability, sensitivity, specificity, and ruggedness were evaluated. The coefficient of variation in repeated measurements was acceptable (<15%) for well-to-well, plate-to-plate, day-to-day, and inter-operator variation, and was not affected by repeated freeze-thaw cycles of serum specimens or the reference standards, or by storage of serum samples at -80°C. The lower limit of quantification (LLOQ) of the PAP patient-derived polyclonal GMAb reference standard (PCRS) was 0.78ng/ml. Receiver operating characteristic curve analysis identified a serum GMAb level of 5µg/ml (based on PCRS) as the optimal cut off value for distinguishing autoimmune PAP serum from normal serum. A pharmaceutical-grade, monoclonal GMAb reference standard (MCRS) was developed as the basis of a new unit of measure for GMAb concentration: one International Unit (IU) of GMAb is equivalent to 1µg/ml of MCRS. The median [interquartile range] serum GMAb level was markedly higher in autoimmune PAP patients than in healthy people (21.54 [12.83-36.38] versus 0.08 [0.05-0.14] IU; n=56, 38; respectively; P<0.0001). Results demonstrate that serum GMAb measurement using the GMAb ELISA was accurate, precise, reliable, had an acceptable LLOQ, and could be accurately expressed in standardized units. These findings support the use of this GMAb ELISA for the routine clinical diagnosis of autoimmune PAP and introduce a new unit of measure to enable standardized reporting of serum GMAb data from different laboratories.