Human leukocyte immunoglobulin-like receptors in health and disease

Human leukocyte immunoglobulin (Ig)-like receptors (LILR) are a family of 11 innate immunomodulatory receptors, primarily expressed on lymphoid and myeloid cells. LILRs are either activating (LILRA) or inhibitory (LILRB) depending on their associated signalling domains (D). With the exception of the soluble LILRA3, LILRAs mediate immune activation, while LILRB1-5 primarily inhibit immune responses and mediate tolerance. Abnormal expression and function of LILRs is associated with a range of pathologies, including immune insufficiency (infection and malignancy) and overt immune responses (autoimmunity and alloresponses), suggesting LILRs may be excellent candidates for targeted immunotherapies. This review will discuss the biology and clinical relevance of this extensive family of immune receptors and will summarise the recent developments in targeting LILRs in disease settings, such as cancer, with an update on the clinical trials investigating the therapeutic targeting of these receptors

LILRB3 (ILT5) is a myeloid cell checkpoint that elicits profound immunomodulation.

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor

A Genetic Basis of Susceptibility to Acute Pyelonephritis

For unknown reasons, urinary tract infections (UTIs) are clustered in certain individuals. Here we propose a novel, genetically determined cause of susceptibility to acute pyelonephritis, which is the most severe form of UTI. The IL-8 receptor, CXCR1, was identified as a candidate gene when mIL-8Rh mutant mice developed acute pyelonephritis (APN) with severe tissue damage.We have obtained CXCR1 sequences from two, highly selected APN prone patient groups, and detected three unique mutations and two known polymorphisms with a genotype frequency of 23% and 25% compared to 7% in controls (p<0.001 and p<0.0001, respectively). When reflux was excluded, 54% of the patients had CXCR1 sequence variants. The UTI prone children expressed less CXCR1 protein than the pediatric controls (p<0.0001) and two sequence variants were shown to impair transcription.The results identify a genetic innate immune deficiency, with a strong link to APN and renal scarring

Transepithelial neutrophil migration is CXCR1 dependent in vitro and is defective in IL-8 receptor knockout mice

Neutrophil migration across infected mucosal surfaces is chemokine dependent, but the role of chemokine receptors has not been investigated. In this study, chemokine receptors were shown to be expressed by epithelial cells lining the urinary tract, and to play an essential role for neutrophil migration across the mucosal barrier. Uroepithelial CXCR1 and CXCR2 expression was detected in human urinary tract biopsies, and in vitro infection of human uroepithelial cell lines caused a dramatic increase in both receptors. As a consequence, there was higher binding of IL-8 to the cells and the IL-8-dependent neutrophil migration across the infected epithelial cell layers was enhanced. Abs to IL-8 or to the CXCR1 receptor inhibited this increase by 60% (p<0.004), but anti-CXCR2 Abs had no effect, suggesting that CXCR1 was the more essential receptor in this process. Similar observations were made in the mouse urinary tract, where experimental infection stimulated epithelial expression of the murine IL-8 receptor, followed by a rapid flux of neutrophils into the lumen. IL-8 receptor knockout mice, in contrast, failed to express the receptor, their neutrophils were unable to cross the epithelial barrier, and accumulated in massive numbers in the tissues. These results demonstrate that epithelial cells express CXC receptors and that infection increases receptor expression. Furthermore, we show that CXCR1 is required for neutrophil migration across infected epithelial cell layers in vitro, and that the murine IL-8 receptor is needed for neutrophils to cross the infected mucosa of the urinary tract in vivo

Interleukin-8 receptor knockout mice have subepithelial neutrophil entrapment and renal scarring following acute pyelonephritis

Interleukin (IL)-8 receptor knockout (KO) mice were shown to have a dysfunctional neutrophil response to urinary tract infection and to develop renal scarring. Intravesical Escherichia coli infection stimulated epithelial chemokine secretion and IL-8 receptor expression in control mice. Neutrophils migrated through the tissues and crossed the epithelial barrier into the urinary tract lumen. In murine IL-8 receptor homologue (mIL-8Rh) KO mice, infection triggered a chemokine response, and neutrophils were recruited but failed to traverse the mucosal barrier and accumulated under the epithelium. After 7 days, control mice were healthy, and infection was cleared, but mIL-8Rh KO mice had swollen kidneys, with neutrophil abscesses and high numbers of bacteria. After 35 days, they developed kidney pathology and renal scarring. The results demonstrate that chemokine receptors drive transepithelial neutrophil migration. In their absence, the neutrophils are trapped, and the tissues are destroyed. This molecular deficiency may determine the progression from acute pyelonephritis to renal scarring

Design of recombinant antibody microarrays for cell surface membrane proteomics

Generating proteomic maps of membrane proteins, common targets for therapeutic interventions and disease diagnostics, has turned out to be a major challenge. Antibody-based microarrays are among the novel rapidly evolving proteomic technologies that may enable global proteome analysis to be performed. Here, we have designed the first generation of a scaleable human recombinant scFv antibody microarray technology platform for cell surface membrane proteomics as well as glycomics targeting intact cells. The results showed that rapid and multiplexed profiling of the cell surface proteome (and glycome) could be performed in a highly specific and sensitive manner and that differential expression patterns due to external stimuli could be monitored

Type 1 fimbriae deliver an LPS- and TLR4-dependent activation signal to CD14-negative cells

Fimbriae target bacteria to different mucosal surfaces and enhance the inflammatory response at these sites. Inflammation may be triggered by the fimbriae themselves or by fimbriae-dependent delivery of other host activating molecules such as lipopolysaccharide (LPS). Although LPS activates systemic inflammation through the CD14 and Toll-like receptor 4 (TLR4) pathways, mechanisms of epithelial cell activation by LPS are not well understood. These cells lack CD14 receptors and are unresponsive to pure LPS, but fimbriated Escherichia coli overcome this refractoriness and trigger epithelial cytokine responses. We now show that type 1 fimbriae can present an LPS- and TLR4-dependent signal to the CD14-negative epithelial cells. Human uroepithelial cells were shown to express TLR4, and type 1 fimbriated E. coli strains triggered an LPS-dependent response in those cells. A similar LPS- and fimbriae-dependent response was observed in the urinary tract of TLR4-proficient mice, but not in TLR4-defective mice. The moderate inflammatory response in the TLR4-defective mice was fimbriae dependent but LPS independent. The results demonstrate that type 1 fimbriae present LPS to CD14-negative cells and that the TLR4 genotype determines this response despite the absence of CD14 on the target cells. The results illustrate how the host "sees" LPS and other microbial products not as purified molecules but as complexes, and that fimbriae determine the molecular context in which LPS is presented to host cells

Neutrophil recruitment and resistance to urinary tract infection

This study examined the role of neutrophil leukocytes for the antibacterial defense at mucosal infection sites. Urinary tract infection (UTI) was established by injection into the bladder lumen of Escherichia coli 1177, a fully virulent clinical isolate. Infection of C3H/HeN (lpsn, lpsn) mice recruited neutrophils into the urinary tract, and bacteria were cleared from kidneys and bladders. The neutrophil response was absent in C3H/HeJ (lpsd, lpsd) mice, and bacteria persisted in the tissues. Peripheral neutrophil depletion of C3H/HeN mice was subsequently achieved by pretreatment with the granulocyte-specific antibody RB6-8C5. The E. coli-induced neutrophil recruitment was inhibited, as shown by immunohistochemistry and tissue myeloperoxidase quantitation. As a consequence, bacterial clearance from kidneys and bladders was drastically impaired. Antibody treatment of C3H/HeJ mice had only a marginal effect. The results show that neutrophils are essential for bacterial clearance from the urinary tract and that the neutrophil recruitment deficiency in C3H/HeJ mice explains their susceptibility to gram-negative UTI

Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation

Fimbriae mediate bacterial attachment to host cells and provide a mechanism for tissue attack. They activate a host response by delivery of microbial products such as lipopolysaccharide (LPS) or through direct fimbriae-dependent signalling mechanisms. By coupling to glycosphingolipid (GSL) receptors, P fimbriae trigger cytokine responses in CD14 negative host cells. Here we show that P fimbriae utilize the Toll-like receptor 4 (TLR4)-dependent pathway to trigger mucosal inflammation. Escherichia coli strains expressing P fimbriae as their only virulence factor stimulated chemokine and neutrophil responses in the urinary tract of TLR4 proficient mice, but TLR4 defective mice failed to respond to infection. Mucosal cells were CD14 negative but expressed several TLR species including TLR4, and TLR4 protein was detected. Infection with P fimbriated bacteria stimulated an increase in TLR4 mRNA levels. The activation signal did not involve the LPS-CD14 pathway and was independent of lipid A myristoylation, as shown by mutational inactivation of the msbB gene. Co-staining experiments revealed that TLR4 and the GSL receptors for P fimbriae co-localized in the cell membrane. The results demonstrate that P fimbriae activate epithelial cells by means of a TLR4-dependent signalling pathway, and suggest that GSL receptors for P fimbriae can recruit TLR4 as co-receptors