Kenneth L. Bost

Production of immunoreactive growth hormone by mononuclear leukocytes

In the present study, we evaluated whether mononuclear leukocytes could synthesize and secrete growth hormone (GH) in vitro. By using RNA slot blot analysis, we detected maximum spontaneous levels of specific GH mRNA in the cytoplasm of rat leukocytes after a 4‐h incubation. Northern gel analysis demonstrated that the specific leukocyte GH RNA was polyadenylated and had a molecular mass of 1.0 kb. Further studies using immunofluorescence, antibody affinity chromatography, and Sephacryl gel filtration indicate that leukocytes secrete a high molecular weight ( > 300,000) and a low molecular weight (~ 22,000) immunoreactive GH (irGH). A substantial amount of the high molecular weight irGH can be converted to the lower molecular weight form after reduction with mercaptoethanol. The irGH appeared to be de novo synthesized because it could be radiolabeled with tritiated amino acids and its production could be blocked by previous incubation of leukocytes with cycloheximide. The replication of Nb2 rat node lymphoma cells was stimulated by affinity‐purified human lymphocyte‐derived irGH. The growth stimulation was blocked by specific antibodies to hGH. We conclude that lymphocytes produce an irGH that is similar to if not identical to pituitary GH in terms of bioactivity, antigenicity, and molecular weight. The findings demonstrate a potential regulatory loop between the immune and neuroendocrine tissues.—Weigent, D. A.; Baxter, J. B.; Wear, W. E.; Smith, L. R.; Bost, K. L.; Blalock, J. E. Production of immunoreactive growth hormone by mononuclear leukocytes. FASEB J. 2: 2812‐2818; 1988.

Salmonella efficiently enter and survive within cultured CD11c+ dendritic cells initiating cytokine expression.

While Salmonella infects macrophages, this cell population may not be the only one important for disseminating intracellular bacteria from mucosal sites. Dendritic cells (DC) are present in the Peyers patches and are mobilized following stimulation. Such characteristics would seem to be ideal for the dissemination of an intracellular, mucosal pathogen. However, it has been difficult to obtain sufficient numbers of DC to assess their ability to harbor Salmonella or to monitor DC in vivo. In the present study, this problem has been addressed by expanding DC in vivo using flt3 ligand, followed by the purification of CD11c+ cells using antibody‐coated magnetic beads or by fluorescence‐activated cell sorting. Salmonella dublin were found to be efficiently internalized, and to survive and replicate within purified CD11c+ DC, and also in CD11c+, CD8α+ or CD11c+, CD11b+ DC subpopulations. The ability of Salmonella to enter DC is of similar magnitude to that reported for macrophages, suggesting that this cell population could be an important host cell for dissemination of this pathogen from mucosal sites. Furthermore, infected DC responded to Salmonella by secretion of IL‐1, IL‐6 and IL‐12. As such, these cells may be important sources of these cytokines during the host response against Salmonella infection.

Neuroendocrine peptide hormones and their receptors in the immune system: production, processing and action

Recent findings indicate that the immune and neuroendocrine systems interact and modulate one another functionally. The mechanism for this seems to be that the 2 systems share a set of receptors and ligands (hormones). Cells of the immune system are able to synthesize neuroendocrine peptide hormones which are biologically active and produced in physiologically significant quantities. Furthermore, leukocytes possess functional receptors for these same neuroendocrine hormones which will specifically modulate immune responses. The structural and functional evidence for these interactions is reviewed and discussed in the context of a bidirectional regulatory circuit between the immune and neuroendocrine systems.

Staphylococcus aureus infection of mouse or human osteoblasts induces high levels of interleukin-6 and interleukin-12 production

Staphylococcus aureus is the principal causative agent of the inflammatory bone disease osteomyelitis. Unfortunately, the pathogenesis of this often chronic infection is poorly understood and is complicated by the recent observation that bone-forming osteoblasts can harbor S. aureus. Such an infection presents a significant challenge for the host immune response, because osteoblasts are not known to initiate protective cell-mediated immune responses. Cultured mouse and human osteoblasts infected with S. aureus were found to express high levels of interleukin (IL)-6 and IL-12p75, on the basis of complementary investigations demonstrating both S. aureus-induced up-regulation of expression of IL-6 and IL-12p40 mRNA and secretion of IL-6 and IL-12p75 by these cells. Additionally, a quantitative bioassay demonstrated that IL-12p75 secreted after infection was biologically active. These studies are the first to demonstrate induced IL-12p75 expression by osteoblasts and suggest a previously unrecognized role for osteoblasts in initiating immune responses after S. aureus infection.

Dormant Microbes in Interstitial Cystitis

Interstitial cystitis (IC) is an inflammatory disease of the urinary bladder that has no known etiology. A microbial association with this disease has not been supported since routine cultures of urine from IC patients are usually negative. However, we have demonstrated the presence of bacterial 16S rRNA genes in bladder biopsies from 29% of patients with IC, but not from control patients with other urological diseases. The ability to identify the presence of bacterial DNA in these patients was accomplished using a sensitive and specific nested PCR method capable of amplifying 16S rRNA genes from a wide variety of bacterial genera. Cloning and sequencing of 16S rRNA gene fragments amplified from bladder tissue of IC patients showed that these genes were derived from genera representing Gram-negative bacteria. In addition to the molecular data, a novel finding of 0.22 micron. filterable forms has been isolated in culture from the biopsy tissue of 14 of 14 IC patients and from 1 of 15 controls. The forms contain nucleic acids and resemble cell wall-deficient bacteria in gross morphology; however, their swirled myelin-like ultrastructure is unusual and suggests a heretofore unclassified microbe. These results demonstrate for the first time an association of Gram-negative bacterial DNA and filterable forms with affected bladder tissue from patients with IC.

Substance P activates NF-κB independent of elevations in intracellular calcium in murine macrophages and dendritic cells

Professional antigen presenting cells, such as macrophages, can be activated by intracellular calcium-dependent as well as calcium-independent mechanisms, depending upon the stimulus used. In this report, we addressed the mechanism of substance P-induced intracellular signalling in murine macrophages and dendritic cells. While no increases in intracellular calcium concentration were detected in macrophages or dendritic cells using sensitive fluorimetric techniques, substance P did induce rapid enhanced activation of NF-kappaB, a transcriptional activator known to regulate pro-inflammatory cytokines. These data provide an important mechanism by which substance P may augment the production of pro-inflammatory molecules.

IL-4 and IFN-γ Up-Regulate Substance P Receptor Expression in Murine Peritoneal Macrophages

While the ability of macrophages to express authentic substance P receptors (i.e., NK-1 receptors) has been inferred from radioreceptor binding assays and functional assays and, most recently, by identification of NK-1 receptor mRNA expression, we know little about NK-1 expression at the protein level or what host factors might up-regulate expression of this receptor. In the present study we demonstrate that the cytokines IL-4 and IFN-γ can increase the expression of NK-1 receptors on murine peritoneal macrophages. Specifically, we show that IL-4 and IFN-γ can elicit increases in the level of mRNA encoding the NK-1 receptor by up to 12- and 13-fold, respectively. Furthermore, these cytokines can significantly increase the expression of the NK-1 receptor protein as measured by Western blot and FACS analysis using specific Abs developed in our laboratory. In addition, we have demonstrated the ability of both IL-4 and IFN-γ to enhance the ability of macrophages to bind substance P as measured by radiolabeled binding assay. The observation that the level of expression of this receptor protein can be enhanced by cytokines that promote either cell-mediated (Th1) or humoral (Th2) immune responses supports the idea that this receptor can be induced during either type of immune response. As such, these results may point to a more ubiquitous role for substance P in the generation of optimal immune responses than previously appreciated.

Expression of functional NK‐1 receptors in murine microglia

Cells of myeloid origin such as microglia have the potential to contribute significantly to the development of inflammatory responses in the CNS. The ability of the neuropeptide substance P to augment proinflammatory responses by other myeloid cell types such as macrophages and dendritic cells is well recognized. In the present study, we demonstrate the presence of mRNA encoding NK‐1 (substance P) receptors in murine microglia cell lines. Importantly, we have utilized specific antibodies developed by our laboratory to detect the expression of the NK‐1 receptor protein in murine microglia cell lines by Western blot analysis and flow cytometry. Furthermore, we have investigated the presence of this receptor on primary murine microglia and report the presence of authentic NK‐1 receptors as determined by Western blot analysis and flow cytometry. In addition, we demonstrate that NK‐1 receptors expressed on microglia are functional as demonstrated by the ability of nanomolar concentrations of substance P to initiate activation of the transcriptional activator, NF‐κB. Given the weight of evidence supporting the role of substance P–substance P receptor interactions in the initiation of optimal proinflammatory responses by myeloid cells, the demonstration of authentic and functional NK‐1 receptors in microglia identifies this neuropeptide as a potentially important contributor to CNS inflammatory responses during disease states. GLIA 37:258–267, 2002.

Expression of authentic substance P receptors in murine and human dendritic cells

Recent studies from our laboratory have shown that substance P can elicit transcription factor activation in dendritic cells. In the present study, we extend these findings by demonstrating the presence of authentic substance P (NK-1) receptors on both normal murine and human dendritic cells. Specifically, we demonstrate the presence of mRNA encoding NK-1 tachykinin receptors and have utilized specific antibodies to detect the expression of NK-1 receptor protein in dendritic cells by Western blot analysis and flow cytometry. These data provide a crucial first step in determining the potential of substance P to modulate dendritic cell function.

Osteoblasts express the inflammatory cytokine interleukin-6 in a murine model of Staphylococcus aureus osteomyelitis and infected human bone tissue

Staphylococcus aureus is the single most common cause of osteomyelitis in humans. Incidences of osteomyelitis caused by S. aureus have increased dramatically in recent years, in part due to the appearance of community-acquired antibiotic resistant strains. Therefore, understanding the pathogenesis of this organism has become imperative. Recently, we have described the surprising ability of bone-forming osteoblasts to secrete a number of important immune mediators when exposed to S. aureus in vitro. In the present study, we provide the first evidence for the in vivo production of such molecules by osteoblasts during bacterial infection of bone. These studies demonstrate the expression of the key inflammatory cytokine interleukin-6 by osteoblasts in organ cultures of neonatal mouse calvaria, and in vivo using a mouse model that closely resembles the pathology of trauma-induced staphylococcal osteomyelitis, as determined by confocal microscopic analysis. Importantly, we have established the clinical relevancy of these findings in infected human bone tissue from patients with S. aureus-associated osteomyelitis. As such, these studies demonstrate that bacterial challenge of osteoblasts during bone diseases, such as osteomyelitis, induces cells to produce inflammatory molecules that can direct appropriate host responses or contribute to progressive inflammatory damage.