Mammalian ␤-defensins are small cationic peptides possessing broad antimicrobial and physiological activities. Because dogs are particularly resilient to sexually transmitted diseases, it has been proposed that their antimicrobial peptide repertoire might provide insight into novel antimicrobial therapeutics and treatment regimens. To investigate this proposal, we cloned the full-length cDNA of three canine ␤-defensin isoforms (cBD-1, -2, and -3) from canine testicular tissues. Their predicted peptides share identical N-terminal 65-amino-acid residues, including the ␤-defensin consensus six-cysteine motif. The two longer isoforms, cBD-2 and -3, possess 4 and 34 additional amino acids, respectively, at the C terminus. To evaluate the antimicrobial activity of cBD, a 34-amino-acid peptide derived from the shared mature peptide region was synthesized. Canine ␤-defensin displayed broad antimicrobial activity against gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus; MICs of 6 and 100 g/ml, respectively), gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Neisseria gonorrhoeae; MICs of 20 to 50, 20, and 50 g/ml, respectively), and yeast (Candida albicans; MIC of 5 to 50 g/ml) and lower activity against Ureaplasma urealyticum and U. canigenitalium (MIC of 200 g/ml). Antimicrobial potency was significantly reduced at salt concentrations higher than 140 mM. All three canine ␤-defensins were highly expressed in testis. In situ hybridization indicated that cBD-1 was expressed primarily in Sertoli cells within the seminiferous tubules. In contrast, cBD-2 was located primarily within Leydig cells. The longest isoform, cBD-3, was detected in Sertoli cells and to a lesser extent in the interstitium. The tissue-specific expression and broad antimicrobial activity suggest that canine ␤-defensins play an important role in host defense and other physiological functions of the male reproductive system. The mucosal physical barrier is an important component protecting luminal surfaces from bacteria. Discovery of epithelium-derived antimicrobial peptides with activity at mucosal surfaces extends luminal protection from the traditional barrier effect to innate immune capabilities. These endogenous peptides are natural antibiotics that are widely distributed in nature and represent an important mechanism of innate defenses against microbial infection (1, 5). Among these naturally occurring antimicrobial peptides, defensins form a unique family of cysteine-rich, small cationic peptides (17).The defensin family is large and has been documented in mammals, plants, insects, and mollusks (6,27,29,39,47). In mammals, defensins are subdivided into ␣-, ␤-, and -defensins based on the organization of conserved six-cysteine motifs (43,50). With the exception of bovine neutrophils, ␤-defensins are synthesized primarily in epithelial tissues (18,27). Because epithelia of the male reproductive tract are essentially isolated from adaptive immune capabilities and because the luminal contents of the testis and...

No abstract

Obesity is associated with a proinflammatory state, with macrophage infiltration into adipose tissue. We tested the hypothesis that communication between macrophages and adipocytes affects insulin resistance by disrupting insulin-stimulated glucose transport, adipocyte differentiation, and macrophage function. To test this hypothesis, we cocultured 3T3-L1 adipocytes with C2D macrophages or primary peritoneal mouse macrophages and examined the impacts of macrophages and adipocytes on each other. Adipocytes and preadipocytes did not affect C2D macrophage TNF-␣, IL-6, or IL-1␤ transcript concentrations relative to those obtained when C2D macrophages were incubated alone. However, preadipocytes and adipocytes increased PEC-C2D macrophage IL-6 transcript levels, while preadipocytes inhibited IL-1␤ transcript levels compared to those obtained when PEC-C2D macrophages were incubated in medium alone. We found that adipocyte coculture increased macrophage consumption of tumor necrosis factor alpha (TNF-␣), interleukin 1␤ (IL-1␤), and, in some cases, IL-6. C2D macrophages increasingly downregulated GLUT4 transcript levels in differentiated adipocytes. Recombinant TNF-␣, IL-1␤, and IL-6 also downregulated GLUT4 transcript levels relative to those for the control. However, only IL-6 was inhibitory at concentrations detected in macrophage-adipocyte cocultures. IL-6 and TNF-␣, but not IL-1␤, inhibited Akt phosphorylation within 15 min of insulin stimulation, but only IL-6 was inhibitory 30 min after stimulation. Lastly, we found that adipocyte differentiation was inhibited by macrophages or by recombinant TNF-␣, IL-6, and IL-1␤, with IL-6 having the most impact. These data suggest that the interaction between macrophages and adipocytes is a complex process, and they support the hypothesis that the macrophage-adipocyte interaction affects insulin resistance by disrupting insulin-stimulated glucose transport, adipocyte differentiation, and macrophage function.

No abstract