It has been known for many years that neutrophils and platelets participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. We report several cellular events that led to enhanced trapping of bacteria in blood vessels: platelet TLR4 detected TLR4 ligands in blood and induced platelet binding to adherent neutrophils. This led to robust neutrophil activation and formation of neutrophil extracellular traps (NETs). Plasma from severely septic humans also induced TLR4-dependent platelet-neutrophil interactions, leading to the production of NETs. The NETs retained their integrity under flow conditions and ensnared bacteria within the vasculature. The entire event occurred primarily in the liver sinusoids and pulmonary capillaries, where NETs have the greatest capacity for bacterial trapping. We propose that platelet TLR4 is a threshold switch for this new bacterial trapping mechanism in severe sepsis.

We have cloned and sequenced a human mRNA specific for mammalian T-lymphoid cells. The message was found to be expressed in human and murine T lymphoblasts, thymocytes and phytohaemagglutinin-stimulated T lymphocytes. The protein deduced from the cDNA sequence has a molecular weight of 34,938 and shows extensive similarity to the entire length of the variable, joining and constant regions of mammalian immunoglobulin light chains. In addition, the relative positions of the cysteine residues are similar to those of the light chains of murine and human immunoglobulin molecules. These properties suggest that the cDNA clone may correspond to a message that specifies part of the human T-cell receptor.

Multiple DNA and protein sequence alignments have been constructed for the human T-cell receptor cVS, [3, and y (TCRA/D, B, and G) variable (V) gene segments. The traditional classification into subfamilies was confirmed using a much larger pool of sequences. For each sequence, a name was derived which complies with the standard nomenclature. The traditional numbering of V gene segments in the order of their discovery was continued and changed when in conflict with names of other segments_ By discriminating between alleles at the same locus versus genes from different loci, we were able to reduce the number of more than 150 different TCRBV sequences in the database to a repertoire of only 47 functional TCRBV gene segments. An extension of this analysis to the over 1 O0 TCRAV sequences results in a predicted repertoire of 42 functional TCRAV gene segments. Our alignment revealed two residues that distinguish between the highly homologous V8 and Vo~ one at a site that in VH contacts the constant region, the other at the interface between immunoglobulin Vn and VL. This site may be responsible for restricted pairing between certain V8 and Vy chains. On the other hand, V[3 and V 7 appear to be related by the fact that their CDR2 length is increased by four residues as compared with that of VcV8 peptides.The alignment data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the alignment number DS23485. The data are available by the EBI FTP server and file server

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-α production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-α signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.