Tonic inhibition of chemotaxis in human plasma.

We found exaggerated chemotaxis in plasma treated with EDTA and thought that the EDTA might itself be inhibiting a tonic inhibitor(s) of chemotaxis. Our plasma fractionations suggested that evidence should be sought for a lipid moiety carrying this activity, and on spectrometry (LC-MS-MS together with GC-MS analyses), the biologically active but not the inactive fraction contained oleic and arachidonic acids. Because fatty acids are largely protein bound, we flooded plasma preparations with delipidated albumin, reasoning that it would bind enough fatty acids, including inhibitory ones, to counter their tonic inhibition. Indeed, we observed dramatic increases in chemotaxis. Hence, adding delipidated albumin to plasma has a similar effect to that of adding EDTA--amplification of the chemotactic response. Oleic acid in physiologic concentrations diminishes the magnifying effects of both EDTA and of delipidated albumin, and in fact diminishes the chemotactic response even without the presence of the amplifiers of chemotaxis. In contrast, arachidonic acid amplifies further the effect of EDTA but not of delipidated albumin, and this augmentation appears to be caused by an EDTA-dependent enrichment of the chemotactic gradient with leukotriene B4 (LTB4). We conclude that oleic acid, the blood levels of which vary among individuals, is at least one tonic inhibitor of chemotaxis in plasma.

Phagocytic function of human blood polymorphonuclear leukocytes in the presence of carrageenan, a potential vaginal microbicide.

Carrageenan is currently undergoing clinical trials as the active constituent of a vaginal gel product for use as a female-controlled option to prevent the transmission of HIV during sexual intercourse. Here we show that in the presence of 0.5 mg/ml of carrageenan, human blood polymorphonuclear leukocytes (PMN) do not ingest this material, as evidenced by a lack of progressive vacuolization, but can ingest microorganisms present in the medium, excluding adjacent carrageenan. Moreover, PMN move at normal speeds, respond chemotactically, and reduce nitroblue tetrazolium (NBT) to formazan on stimulation. Hence, in the presence of carrageenan the phagocytic response appears to remain intact.

Chemotactic activity of human blood leukocytes in plasma treated with EDTA: chemoattraction of neutrophils about monocytes is mediated by the generation of NAP-2.

In slide preparations of human blood leukocytes in autologous plasma containing EDTA, many adherent monocytes are initially chemotactic for neutrophils (PMN). We have identified the chemotactic factor that they generate as neutrophil-activating peptide-2 (NAP-2), as evidenced by distraction of the gradient by authentic human NAP-2, the importance of platelets in the media, which elaborate the precursor of NAP-2, and suppression of the chemotactic response by serine protease inhibitors, which would block the monocyte-derived serine esterase that creates NAP-2 from its immediate precursor. Consistent with this conclusion is inhibition of the chemotactic response to monocytes by agents that block CXCR2, the receptor that NAP-2 uses. Later, when the monocyte moves from the center of chemoattraction, the activated PMN themselves, whose own chemotactic properties are enhanced in EDTA/plasma, appear to take over generation of the gradient, resulting in a prolonged ingress of PMN from outside the field ("second wave"). Chemoattraction by monocytes seems to be simply one way of stimulating the PMN, which, once activated, fail in EDTA/plasma to efficiently shut off their own chemoattraction for other PMN. We suggest that these exaggerated chemotactic effects are due to the loss of normal modulation by a regulatory factor(s) designed to keep the chemotactic response from getting out of hand-i.e., a tonic inhibitor of chemotaxis in plasma.

Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo.

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.

Clocking the Lyme spirochete.

In order to clear the body of infecting spirochetes, phagocytic cells must be able to get hold of them. In real-time phase-contrast videomicroscopy we were able to measure the speed of Borrelia burgdorferi (Bb), the Lyme spirochete, moving back and forth across a platelet to which it was tethered. Its mean crossing speed was 1,636 microm/min (N = 28), maximum, 2800 microm/min (N = 3). This is the fastest speed recorded for a spirochete, and upward of two orders of magnitude above the speed of a human neutrophil, the fastest cell in the body. This alacrity and its interpretation, in an organism with bidirectional motor capacity, may well contribute to difficulties in spirochete clearance by the host.

Tick saliva reduces adherence and area of human neutrophils.

During natural infection with the agent of Lyme disease, Borrelia burgdorferi, spirochetes are delivered with vector saliva, which contains anti-inflammatory and antihemostatic activities. We show here that the saliva of ixodid ticks reduces polymorphonuclear leukocyte (PMN) adhesion via downregulation of beta2-integrins and decreases the efficiency of PMN in the uptake and killing of spirochetes. Inhibition of integrin adhesion and signaling reduces anti-inflammatory functions of PMN. These effects may favor the initial survival of spirochetes in vivo.

Human phagocytic cells in the early innate immune response to Borrelia burgdorferi.

During natural infection with the agent of Lyme disease, Borrelia burgdorferi, polymorphonuclear leukocytes (PMNL) are the first cells of the innate immune system to arrive at the site of spirochete deposition in the skin. This study examined the degree of spirochete clearance likely to occur with PMNL or mononuclear cells before the development of the secondary immune response. Without specific antibody in vitro, there was very limited uptake of spirochetes by PMNL or monocytes and no intracellular colocalization of PMNL granule products with spirochetes. Most of the killing of spirochetes by PMNL was extracellular. In contrast, mature macrophages ingest and kill spirochetes avidly with or without specific antibody. Once the spirochetes are opsonized, PMNL clear them rapidly. These findings may be relevant to the initial survival of spirochetes introduced into the host.

Chemotaxis of non-compressed blood polymorphonuclear leukocytes from an adolescent with severe leukocyte adhesion deficiency.

We have defined the defect in a child with severe leukocyte adhesion deficiency-1 (LAD) as resulting from a single amino acid shift in CD18 (from a C to T mutation at position 533) that prevents heterodimerization with the CD11 antigens to produce beta(2) integrins-the first reported patient homozygous for this defect. Although beset by frequent infections, the patient has survived to adolescence despite the lack of these important adhesion molecules. Consistent with his clinical course is the ability of his PMN to respond chemotactically in slide preparations, albeit with difficulty because of their poor purchase on substrate. The operant adhesins are unknown; his polymorphonuclear leukocytes (PMN) remain chemotactically responsive in the presence of antibodies to alphavbeta(3) and beta(1) integrins and to integrin-associated protein (IAP). These findings indicate that not all patients with severe LAD are candidates for early bone marrow transplantation.