Characterization of Synthetic C3a Analog Peptides on Human Eosinophils in Comparison to the Native Complement Component C3a

Deuterostome invertebrates possess complement genes, and in limited instances complement-mediated functions have been reported in these organisms. However, the organization of the complement pathway(s), as well as the functions exerted by the cloned gene products, are largely unknown. To address the issue of the presence of an inflammatory pathway in ascidians, we expressed in Escherichia coli the fragment of Ciona intestinalis C3-1 corresponding to mammalian complement C3a (rCiC3-1a) and assessed its chemotactic activity on C. intestinalis hemocytes. We found that the migration of C. intestinalis hemocytes toward rCiC3-1a was dose dependent, peaking at 500 nM, and was specific for CiC3-1a, being inhibited by an anti-rCiC3-1a-specific Ab. As is true for mammalian C3a, the chemotactic activity of C. intestinalis C3-1a was localized to the C terminus, because a peptide representing the 18 C-terminal amino acids (CiC3-1a59–76) also promoted hemocyte chemotaxis. Furthermore, the CiC3-1a terminal Arg was not crucial for chemotactic activity, because the desArg peptide (CiC3-1a59–75) retained most of the directional hemocyte migration activity. The CiC3-1a-mediated chemotaxis was inhibited by pretreatment of cells with pertussis toxin, suggesting that the receptor molecule mediating the chemotactic effect is Gi protein coupled. Immunohistochemical analysis with anti-rCiC3-1a-specific Ab and in situ hybridization experiments with a riboprobe corresponding to the 3′-terminal sequence of CiC3-1, performed on tunic sections of LPS-injected animals, showed that a majority of the infiltrating labeled hemocytes were granular amebocytes and compartment cells. Our findings indicate that CiC3-1a mediates chemotaxis of C. intestinalis hemocytes, thus suggesting an important role for this molecule in inflammatory processes.

Section: Discussionsupporting

confidence: 49%

Section: Discussionmentioning

confidence: 99%

CiC3-1a-Mediated Chemotaxis in the Deuterostome InvertebrateCiona intestinalis(Urochordata)

Pinto

Chinnici

Kimura

et al. 2003

“…To quantify COX-1, COX-2, and GAPDH mRNA levels, quantitative RT-PCR was performed using a dsDNA-specific fluorochrome (SYBR Green-I) and the LightCycler Instrument (Roche Molecular Biochemicals) as described (22,42). Briefly, the reverse transcriptase reaction was performed as described previously (22).…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

Prostaglandin E2 Mediates IL-1β-Related Fibroblast Mitogenic Effects in Acute Lung Injury through Differential Utilization of Prostanoid Receptors

White¹,

Ding²,

Moore

et al. 2008

The fibroproliferative response to acute lung injury (ALI) results in severe, persistent respiratory dysfunction. We have reported that IL-1β is elevated in pulmonary edema fluid in those with ALI and mediates an autocrine-acting, fibroblast mitogenic pathway. In this study, we examine the role of IL-1β-mediated induction of cyclooxygenase-2 and PGE2, and evaluate the significance of individual E prostanoid (EP) receptors in mediating the fibroproliferative effects of IL-1β in ALI. Blocking studies on human lung fibroblasts indicate that IL-1β is the major cyclooxygenase-2 mRNA and PGE2-inducing factor in pulmonary edema fluid and accounts for the differential PGE2 induction noted in samples from ALI patients. Surprisingly, we found that PGE2 produced by IL-1β-stimulated fibroblasts enhances fibroblast proliferation. Further studies revealed that the effect of fibroblast proliferation is biphasic, with the promitogenic effect of PGE2 noted at concentrations close to that detected in pulmonary edema fluid from ALI patients. The suppressive effects of PGE2 were mimicked by the EP2-selective receptor agonist, butaprost, by cAMP activation, and were lost in murine lung fibroblasts that lack EP2. Conversely, the promitogenic effects of mid-range concentrations of PGE2 were mimicked by the EP3-selective agent, sulprostone, by cAMP reduction, and lost upon inhibition of Gi-mediated signaling with pertussis toxin. Taken together, these data demonstrate that PGE2 can stimulate or inhibit fibroblast proliferation at clinically relevant concentrations, via preferential signaling through EP3 or EP2 receptors, respectively. Such mechanisms may drive the fibroproliferative response to ALI.

“…To quantify mRNA levels of G-CSF, PDGF-A chain, MIP-2-␣ (gro-␤), IL-6, and GAPDH, quantitative RT-PCR was performed using a dsDNAspecific fluorochrome (SYBR Green-I) on the LightCycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany) as described (29,30). Briefly, the reverse transcriptase reaction was performed as described above.…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

Pulmonary Edema Fluid from Patients with Early Lung Injury Stimulates Fibroblast Proliferation through IL-1β-Induced IL-6 Expression

Olman

White²,

Ware

et al. 2004

121

103

Although the fibroproliferative response to lung injury occurs with a high frequency in patients with clinical acute lung injury, the mechanisms that initiate this response are largely unknown. This study was undertaken first to identify fibroblast mitogenic factors in pulmonary edema fluid, and second to examine the human lung fibroblast’s gene expression profile in response to pulmonary edema fluid. The edema fluid obtained from patients with early lung injury has an eightfold higher concentration of IL-1β and a twofold greater IL-1β-dependent mitogenic effect than does fluid obtained from control patients with hydrostatic pulmonary edema. Furthermore, fibroblasts responded to acute lung injury patient-derived edema fluid through production of soluble mediators that possess an autocrine mitogenic effect. Gene array analysis reveals that acute lung injury edema fluid induces several inflammation-modulating and proliferation-related genes in fibroblasts, whose inductions are similarly dependent on bioactive IL-1β. Most notably, the 20-fold induction of IL-6 mRNA and protein was completely blocked by IL-1 receptor antagonist. The combined addition of IL-1β and IL-6 was mitogenic, and the proliferative response to conditioned medium from IL-1β-exposed cells was blocked by antagonistically acting Abs to IL-6 or to gp130. These novel findings indicate that soluble IL-1β bioactivity and autocrine IL-1β-dependent IL-6 up-regulation are critical initiators of fibroblast activation and proliferation and that they likely play a role in the fibroproliferative response seen in human acute lung injury.