Control of leucocyte function-associated antigen-1-dependent cellular conjugation by divalent cations.

The control of integrin activation is fundamental to an understanding of the integrin-dependent cellular adhesion thought to be important for a plethora of basic cellular functions. Using a cell-cell conjugation assay the role of divalent cations in leucocyte function-associated antigen-1 (LFA-1)-dependent cellular adhesion was further investigated. The conjugation of interleukin-2 (IL-2)-activated lymphocytes to tumour cells was found to be energy dependent and required the presence of various divalent cations, removal of which decreased the level of conjugation. Increased concentrations of calcium, magnesium and manganese ions resulted in a corresponding increase in levels of conjugation. This increase in conjugation was LFA-1 dependent. Interestingly, when calcium ions were first removed from LFA-1, treatment of lymphocytes with magnesium and manganese ions gave significantly higher levels of conjugation than in the presence of calcium. Using a simple displacement study, calcium ions were shown to displace magnesium ions resulting in decreased conjugation. However, calcium ions were unable to displace manganese ions for binding to LFA-1. That manganese was exerting its effect via an LFA-1-dependent mechanism was confirmed using monoclonal antibodies to CD11a which negated the increased conjugation frequency due to manganese.     

Neuregulin-1 accelerates corneal epithelial wound healing

Corneal epithelial cells (CECs) play an important role in the function of the cornea, and are maintained by corneal epithelial stem cells (CESCs). Recent studies have shown that neuronal growth factors affect the proliferation and migration of CESCs. Neuregulin-1 (NR-1) is a neuronal growth factor that is expressed in the early stages of brain development. The aim of this study was to determine whether NR-1 activates corneal wound healing. We observed that NR-1 activated both proliferation and migration of CECs. In addition, the colony-forming efficacy of CESCs was enhanced. In mice, NR-1 treatment improved corneal wound healing. Furthermore, the expression of markers of corneal epithelium maintenance (ΔNp63) and CESC proliferation (Bmi-1 and Abcg2) was increased. These effects were mediated by intracellular signalling pathways (Stat3, Erk1/2 and p38). Taken together, our results suggest that NR-1 accelerates the recovery of corneal wounds, and may represent a novel treatment for corneal damage.     

Prolonged inhibition of an antigen-specific IgE response in vivo by monoclonal antibody against lymphocyte function-associated antigen-1

Intraperitoneal injection of ovalbumin (OVA) into BALB/c mice caused the induction of OVA-specific IgE production in vivo. However, administration of monoclonal antibody against lymphocyte function-associated antigen-1 (anti-LFA-1 mAb) at days 0 and 1 after OVA immunization resulted in an inhibition of OVA-specific primary and secondary IgE production in a dose-dependent manner. The inhibition of the antigen-specific IgE response due to anti-LFA-1 mAb was seen up to 8 weeks after anti-LFA-1 mAb administration. The OVA-specific IgG1 response was also blocked by anti-LFA-1 mAb. The spleen cells obtained from OVA-immunized mice showed enhanced proliferation against secondary stimulation with OVA in vitro. However, the spleen cells obtained from the mice treated with both OVA and anti-LFA-1 mAb revealed a markedly decreased proliferative responses to OVA, while they showed no reduced responses against keyhole limpet hemocyanin stimulation, indicating that anti-LFA-1 mAb might induce antigen-specific anergy in vivo. It was also demonstrated that treatment of the mice with anti-LFA-1 mAb significantly inhibited the interleukin-4-producing ability of OVA-immunized mouse spleen cells. These results demonstrated that LFA-1-dependent cell-cell interaction is essential for the production of IgE in vivo and may be important in IgE-dependent allergic disease.     

Inhibition of very late antigen-4 and leukocyte function-associated antigen-1 in experimental autoimmune uveoretinitis

B10.RIII mice were immunized with interphotoreceptor retinoid binding protein peptide to induce uveitis. Mice were injected intraperitoneally with anti-very late antigen-4 (VLA-4), anti-leukocyte function-associated antigen-1 (LFA-1), or a control Ab every other day from Day 5 to Day 13 post-immunization. The eyes and spleens were harvested on Day 14 or 28. The eyes were used for histologic/cytokine mRNA expression analyses. The spleens were used for Ag-recall cytokine production assays and intracellular cytokine assays. Treatment with both Abs led to a profoundly significant reduction in severity of uveitis and cytokine mRNA expression in the eye. However, cytokine production by splenocytes was significantly upregulated. Discontinuation of Ab treatment led to an increase in uveitis severity and cytokine mRNA expression in the eye, but led to a decrease in cytokine production and intracellular IFN-γ⁺ and IL-17A⁺cytokine profile by splenocytes. Thus, blockade of these molecules using specific Abs may be a therapeutic option for patients with uveitis; however, such treatment must be continued.     

Role of T-cell-associated lymphocyte function-associated antigen-1 in the pathogenesis of experimental colitis

The ß₂ integrin lymphocyte function-associated antigen-1(LFA-1; CD11a/CD18) is important for lymphocyte traffickingand activation as well as recruitment to sites of tissue inflammation.The objective of this study was to assess the role of ‘T-cell-associated’LFA-1 in the pathogenesis of chronic colitis in vivo. Transferof CD4⁺CD25^– T cells isolated from wild-type (wt) miceinto immunodeficient recipients [recombinase-activating gene-1-deficient(RAG-1^–/–)] produced moderate to severe colitis,whereas RAG-1^–/– mice injected with CD11a-deficient(CD11a^–/–; LFA-1^–/–) donor T cells displayedminimal macroscopic and histological evidence of colitis. Surfaceexpression of L-selectin, ₄, ₄ß₇ and chemokine receptor-7were similar for wt and CD11a^–/– donor T cells.Attenuated disease in the CD11a^–/– RAG-1^–/–animals was associated with decreased numbers of CD4⁺ T cellsin the mesenteric lymph nodes (MLNs), spleen and intestinallamina propria (LP). In addition, significant reductions inT_h1 cytokines were observed following ex vivo stimulation ofmononuclear cells obtained from the MLNs and colonic LP. Interestingly,mononuclear cells obtained from the spleens of CD11a^–/– RAG-1^–/– exhibited enhanced pro-inflammatory cytokineproduction compared with splenocytes obtained from wt RAG-1^–/–colitic mice. Taken together, our data suggest that T-cell-associatedCD11a (LFA-1) expression plays a dual role in the initiationof chronic gut inflammation by facilitating naive T-cell priming/activationand expansion within MLNs and by augmenting pro-inflammatorycytokine production following secondary stimulation by antigen-presentingcells in the colonic interstitium.     

胶原润眼液能够加快眼角膜上皮损伤的修复*

背景：Ⅰ型胶原不仅是角膜的主要组成成分,还能对受伤角膜起到营养修复作用,具有很好的药物缓释功能。 目的：了解胶原润眼液对兔损伤角膜的修复作用,并评价其生物安全性。 方法：采用角膜环钻制备兔双眼角膜损伤模型,抗感染处理后,右眼滴胶原润眼液作为实验组,左眼滴生理盐水作为对照组,以结膜分泌物及水肿度、2%荧光素角膜染色评分、损伤愈合率等指标评价胶原润眼液对损伤角膜的修复作用。 结果与结论：对照组兔术后结膜分泌物增多,水肿严重;实验组动物术后结膜分泌物较少,无严重水肿和充血。实验组术后第11天,13天角膜损伤愈合率明显高于对照组(P < 0.05)。体外细胞毒性试验不大于2级反应,且无致敏和刺激反应,结果显示胶原润眼液无生物学危害,能够加快兔损伤角膜的愈合。 关键词：胶原润眼液;角膜;修复;安全性;组织工程 doi:10.3969/j.issn.1673-8225.2012.03.018      

Comparative biochemical and morphometric studies on corneal wound healing

Comparative biochemical and morphological studies were carried out on wounded corneas in order to correlate biochemical findings with morphometric observations during the healing process. Experimental production of corneal wounds, biochemical determinations and quantitative morphometric studies are described in an attempt to correlate corneal matrix macromolecules biosynthesis during the healing process with the morphological modifications of the tissue. The central part and the peripheral part of the corneas were examined separately and compared to the central and peripheral parts of the controlateral corneas. The DNA content of the central portion of the wounded corneas progressively increased and reached after 60 days the same level as the corresponding portion of the controlateral corneas. The DNA contents of the peripheral portions of wounded and controlateral corneas are persistently higher than the DNA contents in the central portion. In peripheral portions of wounded corneas the DNA content is higher than in controlateral corneas and continues to increase steadily during the 60 days of experimentation. Cell density, as determined by morphometry, increased also during that period. The differences between the evolution of the DNA contents and cell density curves may be attributed to variations in cell sizes. The collagen content, estimated from hydroxyproline determinations remained lower in the wounded corneas as compared to the controlateral corneas, even 60 days after operation. This was true for the central as well as for the peripheral portions, suggesting a collagenolytic process as a result of wounding. This is confirmed by morphometric evaluation of fiber density.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of natural killer cells by the mAb YTA-1 that recognizes leukocyte function-associated antigen-1

The mAb YTA-1, which brightly stains CD3^–CD16⁺ large granularlymphocytes (LGL)/natural killer (NK) cells and CD8⁺ T cellsby immunofluorescence, is specific for leukocyte function-associatedantigen (LFA)-1. Some mAbs recognizing the LFA-1 chain (CD11a)or LFA-1ß chain (CD18) inhibited the binding of YTA-1to peripheral blood mononuclear cells. YTA-1 mAb could be chemicallycross-linked to 170 and 96 kDa molecules, whose molecular weightscorrespond to those of LFA-1 and ß respectively.YTA-1bound to COS-7 cells co-transfected with CD11a and CD18 cDNAs,but not to untransfected cells. Reactivities of YTA-1 to K562cells transfected with LFA-1 and ß(CD11a/CD18) cDNAsand to CHO cells transfected with Mac-1 (CD11b/CD18) or p150,95 (CD11c/CD18) cDNAs strongly suggest that YTA-1 recognizeseither LFA-1 or an epitope formed by a combination of LFA-1and ß. Treatment of fresh CD3^–CD16⁺ LGL withYTA-1 augmented cytolytic activity and induced proliferation.F(ab')₂ fragments of YTA-1 augmented NK cytotoxicity, indicatingthat the NK activating signal was transmitted through LFA-1without involvement of Fc receptor III. In contrast, the othermAbs against LFA-1 could not activate NK cells. These resultscollectively indicate that YTA-1 recognizes a unique epitopeof LFA-1, which is involved in activation of fresh NK cells.     

Leukocyte function-associated antigen-1 expression on peripheral blood mononuclear cell subsets in HIV-1 seropositive patients

In order to further investigate immune dysfunctions in HIV-1 infection, we studied the intensity of leukocyte function-associated antigen-1 (LFA-1) expression using a novel application of immunofluorescence analysis in 14 adults and 5 children seropositive for HIV-1 and in 14 healthy adults and 5 healthy children seronegative for HIV-1. While almost all lymphocytes in human peripheral blood expressed LFA-1 and while the percentage of the LFA-1 positive cells was not modified during the course of the HIV-1 infection in both adults and children, our results showed an increase of the LFA-1 expression on selected peripheral blood mononuclear cell subsets. Some LFA-1-labeled functional peripheral blood mononuclear cell subsets such as the CD16, CD14, CD3, and CD8 lymphocyte subpopulations expressed higher levels of the LFA-1 molecule during the HIV-1 infection. The LFA-1 dim cell subsets (CD4 cells) and the LFA-1 low cell subpopulation (CD19 lymphocytes) were not affected by the HIV-1 infection. Moreover, in the CD8 and CD3 cell subsets displaying a heterogeneous LFA-1 expression (dim and bright), we also observed a decrease of the LFA-1 dim/LFA-1 bright cell ratio.     

Anti-leucocyte function-associated antigen-1 antibodies inhibit T-cell activation following low-avidity and adhesion-independent interactions.

Anti-leucocyte function-associated antigen-1 (LFA-1) antibodies can provide either stimulatory or inhibitory signals to T cells, depending on the epitope they recognize, type and stage of activation of the T cells, and nature of the activation stimulus. Because of the low affinity of interaction between the T-cell receptor (TcR) and the antigen/major histocompatibility complex (MHC), it was proposed that the LFA-1 molecule strengthens the adhesion between the interacting cells, thus contributing in an additive manner to TcR-specific interactions. To check if high-avidity, TcR-specific interactions still require the accessory function of the adhesion molecule, we studied the effect of anti-LFA-1 antibodies on T-cell triggering mediated through chimeric receptors composed of an Fv of an antibody and a constant region of the TcR. Such chimeric TcR (cTcR) confer on T cells antibody-type specificity and affinity. We made use of transfected T-cell hybridomas expressing various amounts of either one cTcR chain (composed of VH linked to C beta) or double-chain cTcR (VHC beta + VLC alpha). When such transfectants were stimulated with hapten-modified cells, anti-LFA-1 antibodies inhibited activation predominantly mediated through cTcR composed of a single chimeric chain and did not inhibit stimulation of the double-chain transfectants. Moreover, these anti-LFA-1 antibodies blocked antigen-specific T-cell activation regardless of whether the stimulus was adhesion dependent or not, such as in the case of stimulation by immobilized hapten-protein conjugates. These studies show that the 'off-signal' provided by anti-LFA-1 antibodies is adhesion independent and affects mainly low-avidity TcR-antigen interactions.     

Higher level expression of lymphocyte function-associated antigen-1 (LFA-1) on in vivo natural killer cells

Normal mouse spleen cells express low levels of lymphocyte function-associated antigen-1 (LFA-1) as well as other lymphoid cells. However, fractionation of spleen cells with Percoll discontinuous gradients resulted in the appearance of lymphocytes expressing high levels of LFA-1 molecule (LFA-1 high lymphocytes) in parallel with the enrichment of natural killer (NK) activity. Lower density spleen cells (fractions 1 and 2) expressed higher level of LFA-1 antigen than unfractionated spleen cells and showed a higher NK activity. In contrast, higher density spleen cells (fractions 3 and 4) expressed lower levels of LFA-1 antigen and revealed lower NK activity. LFA-1 high lymphocytes possessed a high level of asialo GM1, which was the cell surface marker for NK cells. Moreover, sorting of LFA-1 high lymphocytes from spleen cells caused a great enrichment of NK cells. These results demonstrated that in vivo NK cells expressed higher levels of LFA-1 molecule, which was an important adhesion molecule for NK cell-mediated cytotoxicity.     

Cloning and characterisation of the primary structure of the sheep lymphocyte function-associated antigen-1 alpha subunit

The leukocyte integrins play a critical role in a number of cellular adhesive interactions during the immune response. The ovine cDNA encoding CD11a, the predominant alpha subunit of the beta2-integrin family, was sequenced and compared with the human, bovine and murine sequences. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues. Along with the ovine CD18-encoding cDNA, which is available for a few months, the sequence data provided here will allow the Ovis aries beta2-integrin CD11a/CD18 (LFA-1, alpha(L)beta2) expression in vitro as a tool to examine the specificities of inflammation in the ovine species.     

Acidic fibroblast growth factor overexpression in corneal epithelial wound healing.

aFGF expression was studied in normal and regenerating cornea of adult rats. aFGF mRNA and proteins were expressed mainly in corneal epithelium but not in stroma. After burning of the epithelium by iodine vapours, the intact epithelial cells migrated to cover the wounded area during the first 4 days and then divided to reconstitute a normal multilayered epithelium 6 days after injury. aFGF mRNA localized by in situ hybridization on regenerating epithelium showed a peak between 6 hr and 2 days after denudation, decreasing to basal levels 6 days later. This induction of aFGF mRNA preceded the increased amount of aFGF peptides, as assessed by indirect immunofluorescence staining. Thus aFGF overexpression is clearly correlated with active migration in epithelial wound healing.     

The lectin KM+ induces corneal epithelial wound healing in rabbits

Neutrophil influx is essential for corneal regeneration ( Gan et al. 1999 ). KM+, a lectin from Artocarpus integrifolia , induces neutrophil migration ( Santos-de-Oliveira et al. 1994 ). This study aims at investigating a possible effect of KM+ on corneal regeneration in rabbits. A 6.0-mm diameter area of debridement was created on the cornea of both eyes by mechanical scraping. The experimental eyes received drops of KM+ (2.5 μg/ml) every 2 h. The control eyes received buffer. The epithelial wounded areas of the lectin-treated and untreated eyes were stained with fluorescein, photographed and measured. The animals were killed 12 h (group 1, n  = 5), 24 h (group 2, n  = 10) and 48 h (group 3, n  = 5) after the scraping. The corneas were analysed histologically (haematoxylin and eosin and immunostaining for proliferation cell nuclear antigen, p63, vascular endothelial growth factor, c-Met and laminin). No significant differences were found at the epithelial gap between treated and control eyes in the group 1. However, the number of neutrophils in the wounded area was significantly higher in treated eyes in this group. Three control and seven treated eyes were healed completely and only rare neutrophils persisted in the corneal stroma in group 2. No morphological distinction was observed between treated and control eyes in group 3. In treated corneas of group 2, there was an increase in immunostaining of factors involved in corneal healing compared to controls. Thus, topical application of KM+ may facilitate corneal epithelial wound healing in rabbits by means of a mechanism that involves increased influx of neutrophils into the wounded area induced by the lectin.     

Bioactive interpenetrating polymer network hydrogels that support corneal epithelial wound healing

The development and characterization of collagen-coupled poly(ethylene glycol)/poly(acrylic acid) (PEG/PAA) interpenetrating polymer network hydrogels is described. Quantitative amino acid analysis and FITC-labeling of collagen were used to determine the amount and distribution of collagen on the surface of the hydrogels. The bioactivity of the coupled collagen was detected by a conformation-specific antibody and was found to vary with the concentration of collagen reacted to the photochemically functionalized hydrogel surfaces. A wound healing assay based on an organ culture model demonstrated that this bioactive surface supports epithelial wound closure over the hydrogel but at a decreased rate relative to sham wounds. Implantation of the hydrogel into the corneas of live rabbits demonstrated that epithelial cell migration is supported by the material, although the rate of migration and morphology of the epithelium were not normal. The results from the study will be used as a guide toward the optimization of bioactive hydrogels with promise in corneal implant applications such as a corneal onlay and an artificial cornea.     

The outcome of T-cell costimulation through intercellular adhesion molecule-1 differs from costimulation through leucocyte function-associated antigen-1

Optimal T-cell activation requires both an antigen-specific and a costimulatory signal. The outcome of T-cell activation can be influenced by the nature of the costimulatory signal the T cell receives. We recently demonstrated the ability of stimulation through intercellular adhesion molecule-1 (ICAM-1), resident on the T-cell surface, to provide a second signal for T-cell activation, and have extended that work here to begin an examination of the functional outcome of this set of signals. Costimulation through ICAM-1 resulted in a greater percentage of cells having undergone more than three divisions when compared to costimulation through leucocyte function-associated antigen-1 (LFA-1). Costimulation through ICAM-1 also had an effect similar to costimulation through CD28 in its ability to down-regulate the cyclin dependent kinase inhibitor p27kip1. Costimulation through ICAM-1 provided greater protection from apoptosis than costimulation through LFA-1, especially in cells having divided more than three times. This was supported by the ability of costimulation through ICAM-1 to up-regulate the anti-apoptotic protein Bcl-2. Finally, costimulation through ICAM-1 or CD28 produced a greater number of T cells with a memory phenotype than costimulation through LFA-1.     

A study of the proliferative properties of corneal tissues during wound healing

Summary The process of healing of the injured cornea was investigated in rabbits. This process was followed up for the period ranging from 1 hr to 30 days after infliction of the injury. Biphasic course of regeneration of the epithelium and migration of cells from the epithelial layer to the border zone was not revealed. It was demonstrated that the structure of the epithelial wedge depended on the conditions of the experiment. Connective tissue possesses a lower reactivity than epithelium and it regenerates with its own cells. When the wound is inflicted near the limbus the elements of the latter may take part in its regeneration.Presented by Active Member Acad. Med. Sci. USSR N. G. Khlopin     

Acceleration of cutaneous wound healing by transient p53 inhibition

The increase of cell proliferation during early wound healing is thought to be regulated by a decrease of apoptosis. In contrast, the reduction of cellularity during final wound maturation may be controlled by an increase of apoptotic cell death. Herein we studied whether p53 is involved in wound healing-associated apoptosis and whether transient inhibition of p53 is effective to improve the early healing process of cutaneous wounds. Using intravital microscopic and immunohistochemical techniques in hairless mice, we demonstrated that in vivo inhibition of p53 by pifithrin-alpha (PFT-alpha; 2.2 mg/kg ip) accelerates early epithelialization and neovascularization of cutaneous wounds by (i) promoting leukocyte recruitment, (ii) increasing cell proliferation, and (iii) reducing apoptotic cell death. We further show that final wound closure with down-regulation of cell proliferation is not inhibited by PFT-alpha treatment, indicating that transient blockade of p53 function does not affect the process of wound maturation. Western blot analysis revealed that PFT-alpha lowered nuclear but not cytoplasmic p53, implying that cytoplasmic retention of p53 mediates the antiapoptotic effects of PFT-alpha. Furthermore, PFT-alpha significantly increased expression of proliferating cell nuclear antigen protein in whole extracts of cutaneous tissue and caused a rise in proliferation of wild-type, but not mutant, p53-expressing keratinocytes. From our study we conclude that transient inhibition of p53 supports the early cell proliferation required for rapid tissue repair and that this may represent an attractive approach in the treatment of delayed wound healing.     

Ex vivo multiphoton analysis of rabbit corneal wound healing following conductive keratoplasty

Ex vivo multiphoton imaging is used to characterize rabbit corneal wound healing after conductive keratoplasty (CK) procedures. CK is performed on the right eyes from eight New Zealand albino rabbits while the left eyes are punctured by a keratoplast tip without energy application. Rabbits are humanely sacrificed 1 day, 1, 2, and 4 weeks after the CK procedure. Eye balls are enucleated and placed on the microscope for multiphoton imaging. Multiphoton imaging reveals damage of corneal epithelium and stroma caused by the CK procedure and the subsequent wound healing process can be followed without histological procedures. Multiphoton excited autofluorescence images demonstrate that re-epithelilialization is accomplished within 1 week in both CK and control groups. However, epithelial hyperplasia is observed in CK corneas. In addition, stromal wounds in the control group become inconspicuous within 1 week while obvious wounds still exist in CK corneas for at least 4 weeks. Postconductive keratoplasty corneal damage and wound healing can be characterized by multiphoton microscopy without histological procedures. Our results suggest that multiphoton microscopy has potential in the clinical evaluation of corneal damage due to refractive surgery, and can be used to study and reduce the unwanted side effects of these procedures.     

Monoclonal antibodies to lymphocyte function-associated antigen-1 inhibit invasion of human lymphoma and metastasis of murine lymphoma

The leukocyte integrins are cell adhesion molecules which play pivotal roles in the development of a variety of immune responses including T-cell-mediated cytotoxicity, lymphocyte proliferation, macrophage presentation of antigen, and adhesion of leukocytes to vascular endothelium. The relevance of lymphocyte function-associated antigen-1 (LFA-1) to leukocyte malignancies is currently under examination in a number of laboratories. Here, we present evidence demonstrating that LFA-1 plays a role during the in vitro invasion of human endothelium by JY lymphoma cells and during in vivo metastasis of two distinct models of murine leukemia: P815 mastocytoma and EL4 lymphoma. When assayed in vitro, a murine anti-human LFA-1 ( subunit) monoclonal antibody (mAb) inhibits up to 80% of JY lymphoma cell invasion. When assayed in vivo, a rat anti-LFA-1 (a subunit) mAb significantly inhibited the development of experimental metastases, when administered concomitantly with either P815 or EL4 tumor cells. The leukocyte integrins, particularly LFA-1, may represent useful targets for the therapeutic modulation of metastasis.