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Leukotoxin (LtxA) (trade name, Leukothera) is a protein secreted by the oral bacterium Aggregatibacter actinomycetemcomitans A. actinomycetemcomitans is an oral pathogen strongly associated with development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans by binding to the β 2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs) and causing cell death. In addition, because of its specificity for malignant and activated WBCs, LtxA is being investigated as a therapeutic agent for treatment of hematological malignancies and autoimmune diseases. Here, we report the successful generation and characterization of Jurkat T lymphocytes with deletions in CD18, CD11a, and Fas that were engineered using CRISPR/Cas9 gene editing. Using these clones, we demonstrate the specificity of LtxA for cells expressing LFA-1. We also demonstrate the requirement of the cell death receptor Fas for LtxA-mediated cell death in T lymphocytes. We show that LFA-1 and Fas are early events in the LtxA-mediated cell death cascade as caspase activation and mitochondrial perturbation do not occur in the absence of either receptor. To our knowledge, LtxA is the first molecule, other than FasL, known to require the Fas death receptor to initiate cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the understanding of LtxA as a bacterial virulence factor and development of it as a potential therapeutic agent., (Copyright © 2019 American Society for Microbiology.)

Adhesion molecules are known to play major roles in the initiation and stabilization of cell-to-cell contacts during the immunological response. Human immunodeficiency virus type 1 (HIV-1) exploits those interactions to facilitate infection and propagation processes. The primary objective of the present study was to investigate the ability of antagonists specific for lymphocyte function-associated antigen 1 (LFA-1) to diminish HIV-1 infection and transmission. We demonstrate here that LFA-1 antagonists can significantly reduce HIV-1 replication in primary human cells and virus propagation by affecting cell-to-cell interactions. Moreover, the inhibition of LFA-1-mediated adhesion events also potentiates the antiviral efficacy of the peptide fusion inhibitor T-20. Altogether, our data suggest that LFA-1 antagonists represent promising antiviral agents. Antiadhesion therapy could be considered a complementary strategy targeting cellular functions essential for HIV-1 spreading and against which the combined therapy currently used displays a limited efficacy.

Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here, we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1), we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4(+) T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together, our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore, the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures.

Although there is now convincing evidence that the infectivity of human immunodeficiency virus type 1 (HIV-1) is increased by incorporation of host intercellular adhesion molecule 1 (ICAM-1) in budding virions, the exact mechanism(s) through which ICAM-1 can so significantly affect HIV-1 biology remains obscure. To address this question, we focused our attention on the most proximal events in the virus life cycle. We made comparative analyses to estimate attachment and internalization of isogenic HIV-1 particles either lacking or bearing host-derived ICAM-1. Using attachment-and-entry assays and confocal fluorescence microscopy, we found that virus binding and uptake were both markedly enhanced by insertion of ICAM-1 within the virus envelope when PM1 lymphoid cells and primary human cells (i.e., peripheral blood lymphocytes and purified CD4(+) T cells) were used as targets. Moreover, ICAM-1-bearing virions entered cells with faster uptake kinetics than viruses devoid of ICAM-1. Experiments conducted with fully competent viruses further confirmed the positive effect of virion-anchored host ICAM-1 on HIV-1 replication. Interestingly, subcellular-fractionation assays revealed that ICAM-1 incorporation modifies the HIV-1 entry route by increasing the level of viral material released in the cytosol, a process of internalization known to be mediated mainly by pH-independent membrane fusion and to result in productive infection. A virion-based fusion assay confirmed that the acquisition of ICAM-1 increases the efficiency of productive HIV-1 entry in primary CD4(+) T lymphocytes. These observations provide new insights into how interactions other than those with gp120 and CD4-coreceptor complex can modulate the process of productive HIV-1 infection in CD4(+) T lymphocytes, a cell target highly relevant to HIV-1 pathogenesis.

Actinobacillus actinomycetemcomitans, an oral bacterium implicated in the etiology of periodontal diseases, produces a leukotoxin that selectively lyses primate neutrophils and monocytes, the major populations of defense cells in the periodontium. Though lysis requires expression of the receptor lymphocyte function-associated molecule 1 (LFA-1) on the cell surface, not all LFA-1-expressing leukocyte populations are equally susceptible to the toxin. In this study, the susceptibility of human leukocytes to leukotoxin-induced lysis is compared to their expression of LFA-1 and the activity of caspase 1. Cytolysis was determined by the activity of lactate dehydrogenase released from peripheral human leukocytes after 1-h exposure to leukotoxin. Monocytes were lysed at leukotoxin concentrations of > or = 5 ng/ml, while the corresponding values for neutrophils and lymphocytes were approximately 10 times greater. Similar LFA-1 expression was found in all susceptible cell populations irrespective of their degree of sensitivity to the toxin. Exposure of monocytes to leukotoxin increased their caspase 1 activity about fivefold within 10 to 20 min. Presence of the caspase 1 inhibitor Ac-YVAD-CMK significantly blocked the leukotoxin-induced lysis of monocytes only. At sublytic concentrations, leukotoxin induced no apoptotic activity in monocytes, as revealed by the lack of caspase 3 activation and DNA fragmentation. Monocytes are the most lysis-sensitive leukocytes for A. actinomycetemcomitans leukotoxin. Their lysis by this toxin depends on caspase 1 activation and proceeds through a process that differs from classical apoptosis.

The ability to activate macrophages in vitro for nitric oxide production and killing of Leishmania major parasites is dependent on tumor necrosis factor, although L. major-infected mice lacking the TNF receptor p55 (TNFRp55(-/-) mice) or both the TNFRp55 and TNFRp75 (TNFRp55p75(-/-) mice) are able to produce NO in vivo and eliminate the parasites. Here we report that activated T cells cocultured with macrophages results in TNFR-independent activation sufficient to control parasites and that both CD40/CD40L and LFA-1 contribute to T-cell-mediated macrophage activation. Thus, anti-CD3-stimulated T cells activated TNFR-deficient macrophages, while T cells from CD40L(-/-) mice were partially defective in triggering NO production by TNFRp55p75(-/-) macrophages. Moreover, in the presence of gamma interferon, anti-CD40 monoclonal antibody (MAb) activated TNFR-deficient macrophages. Finally, MAb blockade of LFA-1 completely inhibited macrophage NO production. Our data indicate that T cells can activate macrophages in the absence of TNF, thus providing a mechanism for how TNFR-deficient mice can control intracellular pathogens.

Mouse cytotoxic T lymphocytes (CTL) reactive with a H-2Db-presented 9-mer peptide of the human papillomavirus type 16 protein E7(49-57) (RAHYNIVTF) were generated from the spleen cells of wild-type C57BL/6 (B6) or B6 perforin-deficient (B6.P0) mice. CD8(+) B6 CTL displayed peptide-specific perforin- and Fas-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7), while CD8(+) CTL from B6.P0 mice lysed RMA-E7 cells via Fas ligand (FasL) exclusively. Rapid and efficient lysis of syngeneic bystander B6 blasts or RMA cells by either B6 or B6.P0 Ag-activated CTL was mediated by a FasL-Fas mechanism. Fas-resistant bystanders were not lysed, nor were allogeneic Fas-sensitive C3H/HeJ (H-2(k)) or BALB/c (H-2(d)) bystander blasts. Interestingly, however, phorbol myristate acetate-ionomycin preactivation of B6.P0 effectors enabled lysis of allogeneic H-2(k) and H-2(d) bystanders even in the absence of antigenic stimulation. Lysis of syngeneic bystander cells was always FasL-Fas dependent and required effector-bystander contact and, in particular, an interaction between CTL LFA-1 and bystander ICAM-1. Thus, in the context of major histocompatibility complex class I molecule-peptide ligation of the T-cell receptors of CD8(+) CTL, neighboring bystander cells that are syngeneic and Fas sensitive and express the adhesion molecule ICAM-1 are potential targets of CTL attack.

Leukocyte adherence to endothelial cells has been implicated in the pathogenesis of microvascular injury as well as in host defense against various infectious microorganisms. Administration of monoclonal antibodies directed against the beta chain of the leukocyte integrins inhibits leukocyte-endothelial-cell adherence and has been reported to modulate ischemia-reperfusion and inflammatory injury. However, such inhibition of adhesion molecule function adversely affects resistance to infection. The following studies were carried out to determine whether monoclonal antibodies to other adhesion molecules, including L-selectin (CD62L), and CD11a (the alpha chain of LFA-1), also increase susceptibility to infection. New Zealand White rabbits were shaved and given subcutaneous injections on their dorsa with 10(9) CFU of Staphylococcus aureus ATCC 25923 at two sites and with 10(8) CFU at two sites. A second set of rabbits were given subcutaneous injections with 10(8) CFU of P. aeruginosa ATCC 27853 at two sites and with 10(7) CFUs at two sites. The animals were monitored for 1 week. There were three blinded experimental groups: controls given saline and two groups given blocking monoclonal antibodies to either L-selectin (Dreg-200) or CD11a (R7.1). In contrast to monoclonal antibodies to CD18, none of the monoclonal antibodies significantly increased the risk of abscess formation by S. aureus, although inhibition of CD11a increased the rate of abscess formation by P. aeruginosa.

Granuloma formation in murine schistosomiasis is dependent on CD4+ Th lymphocytes and requires recruitment and accumulation of inflammatory cells at the site of egg deposition. The present study examined the role of three adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1), and very late antigen-4 (VLA-4), that participate in cellular recruitment, interaction, and lymphocyte activation during in vitro activation of acutely and chronically infected spleen and liver granuloma lymphocytes. Blockade of ICAM-1, LFA-1, or VLA-4 by rat monoclonal antibody inhibited spleen and granuloma lymphocyte interleukin-2 (IL-2) and IL-4 production as well as lymphoproliferative responses at similar levels (66 to 87%). The down-modulated cytokine and proliferative responses of chronically infected lymphocytes were inhibited to the same extent as their acutely infected counterparts. Cell sorting analysis demonstrated that acutely and chronically infected splenic and granuloma lymphocytes expressed similar levels of LFA-1, ICAM-1, and VLA-4 and that more ICAM-1 was expressed on infected than on uninfected mouse lymphocytes. By exposure of cells to paired monoclonal antibodies at suboptimal doses, it was determined that whereas all three adhesion molecules may participate, only ICAM-1 and LFA-1 showed synergistic interactions in determining lymphocyte responsiveness. These data suggest that spleen and liver granuloma lymphocytes are equally well armed with functional adhesion receptors. Thus, ICAM-1, LFA-1, and VLA-4 play an important accessory role in inflammatory cytokine production and lymphocyte proliferation, and therefore these adhesion molecules may participate in the initiation and maintenance of the granulomatous inflammation.

Infection of CBA mice with Plasmodium berghei ANKA results in severe malaria, which is characterized by mortality 6 to 10 days after infection and is associated with alterations of the brain microcirculation. These alterations consist of (i) intravascular sequestration of monocytes, (ii) an increase in vascular permeability as documented by Evans blue diffusion, and (iii) microhemorrhages. This syndrome may be due to an increase of production of tumor necrosis factor alpha which upregulates the endothelial expression of ICAM-1 and thus leads to adhesion of CD11a/CD18 (LFA-1)-bearing cells. During severe malaria, we found an important sequestration of the CD11a-bearing polymorphonuclear neutrophil leukocytes (PMN) in the lung but not in the brain. Treatment with a monoclonal antibody (MAb) against PMN, which induces profound neutropenia, prevented mortality and Evans blue diffusion in the brain and the lung, while it unexpectedly increased the occurrence of microhemorrhages. The anti-PMN MAb abolished PMN sequestration in the lung and also partially decreased monocyte sequestration in the brain and the lung. Treatment with an anti-CD11a MAb also prevented mortality, Evans blue diffusion, and PMN and monocyte sequestration. This study shows that PMN contribute to the mortality and the microvascular lesions resulting from severe malaria. This may be due to their CD11a-dependent sequestration in the lung and also to their indirect influence on vascular permeability and the sequestration of monocytes.

The role of platelets was investigated in two models of lipopolysaccharide (LPS)-induced toxicity in mice: the systemic reaction, provoked by intravenous LPS injection in D-galactosamine-sensitized recipients, which results in host death, and the local reaction, elicited in the skin by sequential injections of LPS and tumor necrosis factor alpha at 24-h intervals, which results in hemorrhagic necrosis. In both models, the depletion of platelets with a rabbit polyclonal or a mouse monoclonal antiplatelet immunoglobulin G afforded significant protection. In the local reaction, studies of the distribution of 111In-labelled platelets as well as optical and electron microscopy showed that platelets are localized in the dermal venules before hemorrhage occurs. Anti-CD11a (LFA-1) and anti-CD54 (ICAM-1) monoclonal antibodies prevented both platelet localization and hemorrhagic necrosis, and these determinants were detected on mouse platelets by immunofluorescence. The antiplatelet monoclonal antibody did not reduce the localization of polymorphonuclear leukocytes in the dermal venules, as shown by histological sections. Thus, in the local reaction, the stimulation with LPS and tumor necrosis factor alpha leads to a binding of platelets to the endothelium of venules by their beta 2 integrins, which seems necessary for the development of the hemorrhagic necrosis.

We previously demonstrated that the induction by staphylococcal toxic shock syndrome toxin 1 (TSST-1) of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion by human monocytes requires direct T cell-monocyte contact. In the present study, a role for the adhesion molecule lymphocyte function associated antigen 1 (LFA-1) in TSST-1-induced cytokine secretion by human monocytes among 12 normal healthy donors was investigated. Monoclonal antibodies to the alpha chain (anti-CD11a) and to the beta chain (anti-CD18) of LFA-1 significantly inhibited TSST-1-induced TNF-alpha and IL-1 beta secretion (P < 0.025; Wilcoxon signed-rank test, two tailed), while a control monoclonal antibody directed against the monocyte CD14 antigen had no effect. These results suggest that LFA-1 may play an important role in the secretion of TNF-alpha and IL-1 beta by TSST-1-stimulated human monocytes, likely by promoting cell-cell adhesion between monocytes and lymphocytes.

The role of complement receptor type 3 (CR3) in nonopsonic recognition of group B streptococci (GBS) by macrophages was investigated. Monoclonal anti-CR3 (anti-Mac-1) inhibited phagocytosis of GBS strains by as much as 50% in serum-free cultures of both mouse peritoneal macrophages and the macrophage cell line PU5-1.8. GBS uptake was unaffected by the presence of anti-C3 or salicylhydroxamate, an inhibitor of the covalent binding reaction of C3. Soluble antibodies to LFA-1 or to the common beta-chain (CD18) of the LFA-1/CR3/p150,95 family of cell adhesion molecules did not inhibit GBS uptake. Down-modulation of surface Mac-1 on macrophages following adherence to anti-Mac-1- or anti-CD18-coated surfaces also inhibited uptake of GBS. Further evidence for GBS interaction with CR3 was demonstrated by reduction of EC3bi rosette formation in macrophages adherent to GBS-coated plates. These studies suggest that GBS can interact with macrophage CR3, promoting phagocytosis in a C3-independent fashion. In the absence of specific immunity in neonates, this recognition mechanism may be a significant virulence determinant for GBS which poorly activate the alternate complement pathway.