anti-CD11b mAb Research Articles

Pseudoginsenoside-F11 Accelerates Microglial Phagocytosis of Myelin Debris and Attenuates Cerebral Ischemic Injury Through Complement Receptor 3

After ischemic stroke, the degenerated myelin caused by ischemic injury cannot be rapidly cleared away by microglia and interferes with the recovery process. Complement receptor 3 (CR3, CD11b/CD18), belonging to β2 integrin family primarily expressed in phagocytes, is involved in the microglial phagocytosis of myelin debris. We previously found that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, exerts neuroprotective effects against ischemic stroke and neuroinflammation. In the present study, we investigated the promotion of PF11 on oxygen-glucose deprivation (OGD)-induced microglial phagocytosis of myelin debris, the neuroprotection of PF11 on permanent middle cerebral artery occlusion (pMCAO)-induced ischemic stroke, and the possible role of CR3. The results indicated that PF11 (50 μM) accelerated the OGD-induced promotion of myelin debris phagocytosis by microglia in the early stage of OGD (2 h, 4 h, 8 h), which was significantly inhibited by anti-CD11b mAb or down-regulated by CD11b-specific siRNA. Meanwhile, PF11 strengthened the OGD-activated RhoA/ROCK signaling associated with the internalization during myelin debris phagocytosis through CR3. Consistently, the anti-CD11b mAb could markedly attenuated the nrueoprotective effects of PF11 (12 mg/kg, i.v.) on infarction and brain edema, neurological functions and loss of neurons of pMCAO rats. These findings suggest that PF11 accelerates the phagocytosis of myelin debris by microglia mainly through CR3, which may likely contribute to its neuroprotection against ischemic stroke.

The Adhesion of Sickle Cell Disease Neutrophils to Endothelial Layers, In Vitro, Is Mediated by the Mac-1, LFA-1 and VLA-4 Integrins.

Leukocytes may have a propagating and, possibly, initiating role in sickle cell disease (SCD) vaso-occlusion. In vivo studies suggest that adherent leukocytes capture sickle erythrocytes in the microcirculation and in vitro studies demonstrate an increased ability of SCD neutrophils (neu) to adhere to fibronectin, endothelial cells and endothelial proteins. Previous studies suggest that the expressions of the major neu integrins, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) may only be upregulated on the surface of SCD neu following their stimulation, indicating that alterations in integrin function (affinity or avidity) contribute to alter SCD neu adhesion. The objective of this study was to identify the integrins responsible for altered SCD neu adhesion. Neus were isolated from the peripheral blood of healthy controls and SCD individuals in steady state over ficoll-paque gradients. Cell adhesion (2×106cells/ml in RPMI) to cultured human umbilical vein endothelial cells (HUVEC) at confluence was assessed using static adhesion assays (30min, 37°C, 5%CO2). Neus from SCD patients demonstrated a significantly greater adhesion to HUVEC than control neus (20.2±2.8% compared to 11.2±1.0%; n≥7; p<0.03; Mann Whitney test). Subsequently, cells were co-incubated with adhesion molecule-blocking monoclonal antibodies (mAbs) during assays. Control neu adhesion to HUVEC was significantly inhibited by the anti-CD11b mAb (6.7±1.5%;n=6; P<0.05, paired t test), but not by mAbs against CD11a, the VLA-4-integrin subunit, CD49d, or a non-specific negative control mAb (neg control) (data not shown). In contrast, the adhesion of SCD neus to HUVEC was significantly inhibited by both the anti-CD11a and the anti-CD11b mAbs (20.2±2.8% reduced to 11.4±1.2% and 9.1±1.5%; n=9; P<0.01 and P<0.001, respect.). Interestingly, a mAb against CD49d was also found to significantly decrease SCD neu adhesion to HUVEC (10.4±1.1%; n=9; P<0.01), while the neg control mAb did not significantly affect SCD neu adhesion (data not shown). Following the stimulation of HUVEC with TNF-α (10 ng/ml) (3h, 37°C, 5%CO2) to simulate an endothelial layer under inflammatory conditions, the adhesions of control and SCD neus were increased but statistically similar (38.4±2.9% and 34.4±5.0%; n≥4, respect.). Under these conditions anti-CD11a and CD11b mAbs significantly inhibited control neu adhesion to HUVEC (reduced to 28.8±2.9% and 19.6±4.6%; n=4; P<0.01 and P<0.05, respect.). In contrast, SCD neu adhesion to HUVEC was significantly inhibited by mAbs for CD11a (19.5±2.6%; n=6; p<0.01) and CD11b (15.2±2.0%; n=6; p<0.001). The anti-CD49d, but not the neg control mAb, also significantly decreased SCD neu adhesion to TNF-α-stimulated HUVEC (19.5±3.7%; n=6; p<0.05). In conclusion, data indicate that control neu adhesion to endothelial cells appears mainly to be mediated by the Mac-1 (CD11b/18) integrin with a contribution from the LFA-1 integrin (CD11a/18) under inflammatory conditions. In contrast, SCD neu adhesion to endothelium (under both basal and stimulated conditions), at least in vitro, appears to be mediated by the Mac-1 and LFA-1 integrins and, interestingly, by VLA-4 (CD49d/CD29), an integrin found expressed at low levels on neus during certain inflammatory conditions. We speculate that alterations in the affinity/ avidity of these molecules contribute to SCD neu adhesion. Approaches to inhibit the adhesion of all three integrins may be important for preventing leukocyte adhesion to the vascular endothelium and, in turn, vaso-occlusion.

Differential regulation of CD11b on γδ T cells and monocytes in response to unripe apple polyphenols

Leukocyte adhesion and migration are mediated partially by CD11b/CD18 (membrane-activated complex-1, CR3). Earlier studies have demonstrated a role for green tea polyphenols in down-regulating CD11b on CD8(+) T cells and monocytes. We have shown recently a stimulatory effect of unripe apple polyphenols (APP) on gammadelta T cells. Thus, we compared the effect of APP on bovine gammadelta T cell and monocyte CD11b expression. Purified bovine monocytes and monocyte-depleted PBLs were cultured with APP. CD11b levels decreased on monocytes in response to APP. In contrast, a gammadelta T cell subset responded to APP by up-regulating CD11b. The CD11b regulation was not seen on gammadelta T cells or monocytes treated with APP fractions depleted of tannins. The APP-induced down-regulation of CD11b on monocytes was inhibited by an anti-CD11b mAb, consistent with previous studies showing that polyphenols bind CD11b. As expected, the anti-CD11b mAb had no effect on the APP response in resting gammadelta T cells, as these cells lacked CD11b. Consistent with the changes in surface CD11b expression, APP-treated gammadelta T cells showed increased adherence to plastic, whereas monocyte adhesion was reduced. APP also induced cytokine gene expression in gammadelta T cells. Some polyphenols are thought of as anti-inflammatory agents; however, these data, as well as other ongoing studies, indicate they have a proinflammatory effect on gammadelta T cells. In vivo, plant polyphenols may enhance gammadelta T cell migration and function at sites of inflammation, where they could induce rapid, immune-regulatory and innate-like immune responses.

Plant polyphenols induce differential regulation of CD11b expression and function on monocytes and γδ T cells (97.11)

Abstract Leukocyte adhesion and migration is partially mediated by CD11b/CD18 (MAC-1, CR3). Earlier studies have demonstrated a role for green tea polyphenols in down-regulating CD11b on monocytes and CD8+ T cells. We have recently shown a stimulatory effect of unripe apple polyphenols (APP) on γδ T cells. Thus, we compared the effect of APP on γδ T cell and monocyte CD11b expression. Purified bovine monocytes and monocyte-depleted peripheral blood lymphocytes were cultured with APP for 24 hours. CD11b levels decreased on monocytes in response to APP. In contrast, a subset of γδ T cells responded to APP by up-regulating CD11b. Consistant with the changes in surface CD11b expression, APP treated γδ T cells showed increased adherence to plastic, whereas monocyte adhesion was reduced. The affects of APP on monocytes could be inhibited by anti-CD11b mAb, whereas the mAb had no effect on the APP response in γδ T cells, suggesting the two cell types respond to APP via different mechanisms. Polyphenols are typically thought of as anti-inflammatory agents, however, these data, as well as other ongoing studies, indicate they have a pro-inflammatory effect on γδ T cells. In vivo, plant polyphenols may enhance γδ T cell migration to sites of inflammation, where they can enact rapid immunoregulatory and innate-like responses. Funded by: NIH –NIAID DAIT-BAA-03 -41

The Immunomodulatory Activity of C3 Binding Glycoprotein (C3bgp) is Mediated by the Complement Receptor Type III and Mitogen-Activated Protein Kinase Signal Transduction Pathways

C3 binding glycoprotein (C3bgp) is immunomodulating molecule isolated from the plant Cuscuta europea. When neutrophils were incubated with C3bgp the subsequent binding of anti-CD11b mAb became significantly higher. C3bgp induced moderate TNF-α production in human PBMC and primary monocytes. This production was significantly inhibited by the specific inhibitors of JNK and p38 MAPKs. The inhibition of JNK reduced PBMC viability. We concluded that: (i) C3bgp utilized CD11b polypeptide chain of CR3 and mediated a part of its immunomodulatory properties by activation of JNK and p38 and (ii) PBMC viability at in vitro conditions depends of JNK signal transduction pathway activation.

Mechanism of Immune System Activation by (1,4)-α-D-glucan Isolated from Tinospora cordifolia in Macrophages.

We have characterized and reported the immunostimulating properties of a novel polysaccharide - (1,4)-α-D-glucan (RR1)- isolated from the medicinal plant Tinospora cordifolia [23]. This novel glucan is water soluble and having (1, 4)-α-D-glycosidic linkages in the main chain and (1, 6)-α-D-glycosidic linked side chains at an interval of 6, 7 glucose units. The signaling mechanism of the novel (1,4)-a-D-glucan (RR1) was investigated in macrophages to evaluate its immunostimulating properties. When RAW264.7 macrophages were incubated with RR1 at 4°C, the novel glucan inhibited the phagocytosis of unopsonized zymosan A bioparticles in a dose-dependent manner. RR1 also inhibited the binding and internalization of opsonized zymosan A bioparticles, although at a lower level than laminarin. Incubation of macrophages with anti-CD11b mAb followed by RR1 failed to show any inhibitory effect on RR1-induced TNF-α synthesis confirming that complement receptor 3 (CR3) is not involved in the opsonic binding and internalization of RR1 in macrophages unlike zymosan A. The anti-CD11b mAb has significant inhibitory effect on the zymosan A-induced tumor necrosis factor (TNF)-α synthesis. RR1 induced TNF-α synthesis in macrophages in a dose-dependent manner which can be completely inhibited by the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or curcumin. RR1 activated NF-κB in a time- and dose-dependent manner and this modulation of nuclear NF-κB activity is associated with the degradation of I-κB a thus facilitating the translocation of NF-κB into the nucleus. RR1-induced NF-κB activity peaks at 8 h of RR1 stimulation while I-κB a degradation occurred within 1 h of stimulation. RR1-induced NF-κB activation occurred through TLR6 signaling as evidenced by the synthesis of IL-8 in TLR6-transfected HEK293 cells. These results show that the novel (1,4)-α-D glucan from Tinospora cordifolia activates the immune system through the activation of macrophages that occurs through TLR6 signaling, NF-κB translocation and cytokine production. (Supported by Miami Children's Hospital Foundation research funds).

Mechanism of macrophage activation by (1,4)-α- d-glucan isolated from Tinospora cordifolia

The signaling mechanism of the novel (1,4)-α- d-glucan (RR1) isolated from the medicinal plant Tinospora cordifolia was investigated in macrophages to evaluate its immunostimulating properties. When RAW264.7 macrophages were incubated with RR1 at 4 °C, the novel glucan inhibited the phagocytosis of unopsonized zymosan A bioparticles in a dose-dependent manner. RR1 also inhibited the binding and internalization of opsonized zymosan A bioparticles, although at a lower level than laminarin. Incubation of macrophages with anti-CD11b mAb followed by RR1 failed to show any inhibitory effect on RR1-induced TNF-α synthesis confirming that complement receptor 3 (CR3) is not involved in the opsonic binding and internalization of RR1 in macrophages unlike zymosan A. The anti-CD11b mAb has significant inhibitory effect on the zymosan A-induced tumor necrosis factor (TNF)-α synthesis. RR1 induced TNF-α synthesis in macrophages in a dose-dependent manner which can be completely inhibited by the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or curcumin. RR1 activated NF-κB in a time- and dose-dependent manner and this modulation of nuclear NF-κB activity is associated with the degradation of I-κB α thus facilitating the translocation of NF-κB into the nucleus. RR1-induced NF-κB activity peaks at 8 h of RR1 stimulation while I-κB α degradation occurred within 1 h of stimulation. RR1-induced NF-κB activation occurred through TLR6 signaling as evidenced by the synthesis of IL-8 in TLR6-transfected HEK293 cells. These results show that the novel (1,4)-α- d-glucan from Tinospora cordifolia activates the immune system through the activation of macrophages that occurs through TLR6 signaling, NF-κB translocation and cytokine production.

Ameliorating effect of saporin‐conjugated anti‐CD11b monoclonal antibody in a murine T‐cell‐mediated chronic colitis

Crohn's disease (CD) is an inflammatory bowel disease that is associated with several changes in the immune system, including an increased number of infiltrating macrophages. These macrophages release a variety of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) which are critically involved in the onset and the development of CD. The present study was performed to explore the initial involvement of macrophages in the development of T-cell-mediated chronic colitis. The effects were evaluated of saporin-conjugated anti-CD11b monoclonal antibody (mAb) on the development of chronic colitis in severe combined immunodeficiency (SCID) mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells as an animal model of CD. Significantly increased CD11b-expressing macrophages as well as CD4(+) T cells were found in inflamed colon from colitic mice. Administration of saporin-conjugated anti-CD11b mAb markedly ameliorated the clinical and histopathological disease. In vivo treatment with saporin-conjugated anti-CD11b mAb decreased CD4(+) T-cell infiltration in the colon and suppressed interferon-gamma (IFN-gamma) and TNF-alpha production by lamina propria CD4(+) T cells. Collectively, the present results suggest an initial role of macrophages in the pathogenesis of T-cell-mediated chronic colitis. Furthermore, the macrophage-specific targeting may be a promising strategy for therapeutic intervention in CD.

Macrophage Colony-Stimulating Factor Induces Vascular Endothelial Growth Factor Production in Skeletal Muscle and Promotes Tumor Angiogenesis

Although M-CSF has been used for myelosuppression due to chemotherapy in patients with solid tumors, the effect of exogenous M-CSF on tumor angiogenesis has not been studied. In this study we showed that M-CSF has the ability to accelerate solid tumor growth by enhancing angiogenesis with a novel mechanism. M-CSF accelerated intratumoral vessel density in tumors inoculated into mice, although it did not accelerate the proliferation of malignant cells and cultured endothelial cells in vitro. In both the absence and the presence of tumors, M-CSF significantly increased the circulating cells that displayed phenotypic characteristics of endothelial progenitor cells in mice. Moreover, M-CSF treatment induced the systemic elevation of vascular endothelial growth factor (VEGF). VEGFR-2 kinase inhibitor significantly impaired the effect of M-CSF on tumor growth. In vivo, M-CSF increased VEGF mRNA expression in skeletal muscles. Even after treatment with carageenan and anti-CD11b mAb in mice, M-CSF increased VEGF production in skeletal muscles, suggesting that systemic VEGF elevation was attributed to skeletal muscle VEGF production. In vitro, M-CSF increased VEGF production and activated the Akt signaling pathway in C2C12 myotubes. These results suggest that M-CSF promotes tumor growth by increasing endothelial progenitor cells and activating angiogenesis, and the effects of M-CSF are largely based on the induction of systemic VEGF from skeletal muscles.

CD11b-mediated migratory property of peripheral blood B cells

CD11b belongs to the integrin family and is expressed on neutrophils, monocytes, natural killer cells, and a subset of lymphocytes. Although CD11b expressed on neutrophils and monocytes has been extensively investigated and has been reported to play an important role in the migration of these subsets of leukocytes, the function of CD11b expressed on a subset of B cells has not yet been clarified. To elucidate the functional activity of CD11b expressed on B cells, we characterized the CD11b-expressing cells among the B-cell population and investigated their migratory ability. Isolated peripheral blood CD19 + B cells were analyzed by flow cytometry. The migratory ability of B cells was evaluated by the transwell assay, and the contribution of CD11b to this ability was investigated by using an anti-CD11b blocking mAb. The majority of CD27 - IgD + naive B cells were CD11b - , whereas most CD27 + memory cells were CD11b +. Among the CD27 + memory cells, expression of CD11b was stronger on the IgD - cells than on the IgD + cells. In the transwell assay, the migrating cells were predominantly CD27 + IgD - cells, most of which expressed CD11b. The addition of an anti-CD11b blocking mAb resulted in the significant reduction of the number of migrating B cells. Memory B cells express CD11b and, in contrast with naive B cells, have high migratory ability. CD11b plays an essential role in the homing process of memory cells.

454. Evaluation of Polyethylene Glycol (PEG) Modification of Adenoviral Vectors To Reduce Innate Immune Response and Increase Vector Persistence

Gene therapy is a therapeutic strategy that may potentially treat a multitude of clinically important conditions. To these ends, adenovirus vectors (Ad-vectors) are a promising means to deliver specific genes of interest to target cells. The efficacy of Ad-vectors is limited by the innate immune response against these infectious particles. Following transduction in vivo, Ad-vectors activate the innate immune system resulting in rapid vector clearance and acute inflammation of transduced tissues. The infiltration of effector leukocytes, such as neutrophils, is a hallmark of the innate immune response and may play a major role in Ad-vector clearance. The aims of this work were to explore neutrophil-Ad-vector interactions and to identify the essential molecules mediating this interaction. C57/B6 mice were administered a Cy2-labeled first generation Ad-vector, AdLuc, intravenously and sacrificed at 3 hours. Neutrophil populations were isolated from the liver and analyzed by flow-cytometry for fluorescence. Compared to vehicle, neutrophils from Cy2-AdLuc treated mice demonstrated significant vector uptake confirming an important role for these innate cells in the host response to Ad-vectors. To further study Ad-vector neutrophil interactions, purified populations of human peripheral blood neutrophils were incubated with Cy2-AdLuc and analyzed using fluorescence microscopy and flow cytometry. Ad-vector uptake by neutrophils increased significantly over 20-60 minutes. Neutrophil uptake of Ad-vectors did not require viral capsid RGD motifs since internalization of the RGD-deleted Ad-vector, AdL.PB over 20-60 minutes was similar to AdLuc. Neutrophil pre-incubation with a blocking anti-CD11b mAb (ICRF44), but not control IgG, inhibited Ad-vector uptake demonstrating that this β2-integrin is required for Ad-vector internalization. To determine activation, neutrophil L-selectin expression was determined by flow cytometry following incubation with AdLuc. L-selectin expression, decreased significantly over 60 minutes following incubation with AdLuc consistent with neutrophil activation. These data confirm that neutrophils interact significantly with and are activated by Ad-vectors. Furthermore, neutrophil-expressed CD11b, but not viral capsid RGD motifs, is essential for adenoviral uptake. Future studies will focus on elucidating the mechanism of Ad-vector-mediated neutrophil activation and its role in vector clearance in vivo.

Anti-CD11 b monoclonal antibody suppresses brain dissemination of Cryptococcus neoformans in mice.

To elucidate the role of the beta2 integrin family of adhesion molecules in the disseminated infection of Cryptococcus neoformans from the lung to the central nervous system, we examined the effects of monoclonal antibodies (mAbs) against CD11a, CD11b, CD11c and CD18 on the number of live microorganisms in both the lung and brain of mice three weeks after intratracheal infection. Administration of anti-CD11b mAb partially, but reproducibly, reduced the fungal loads in the brain in three independent experiments, while the lung loads were not affected. In addition, the same treatment significantly decreased the number of live microorganisms in the blood. In sharp contrast, the brain loads one week after intravenous injection with C. neoformans were not affected by treatment with anti-CD11b mAb. Finally, administration of mAb against other adhesion molecules (CD11a, CD11c or CD18) failed to affect the fungal loads in the brain as well as in the lung three weeks after intratracheal instillation, except for anti-CD18 mAb which rather increased the brain loads. Our results suggested that CD11b might be involved at least in part in the process of fungal dissemination from lung to brain, although the significance of other beta2 integrin family adhesion molecules remains to be substantiated.

IFN-β-induced IL-1Ra synthesis in human monocytes involves PI 3-kinase-STAT1 signaling pathway

IFN-β displays an anti-inflammatory property by inducing IL-1Ra without triggering synthesis of IL-1β in human monocytes (Mo). IFN-β initiates JAK-STAT pathway that may cross-talk with components of MAP- and PI 3-kinase pathways. Since maximal activation of transcription by several STATs requires both Tyr and Ser phosphorylation, we investigated the role of MAP- (ERK1/2) and PI 3-kinases in IFN-β-induced IL-1Ra production in Mo. The PI 3-kinase inhibitor Ly294002 but not the MAP kinase inhibitor PD98052 suppresses, in a dose-dependent way, IL-1Ra production in Mo at a protein level correlating with the reduction of steady state levels of IL-1Ra mRNA. IFN-β treatment of Mo leads to rapid Ser-phosphorylation and nuclear translocation of STAT1 that is inhibited by Ly294002. Interestingly, suppression of PI 3-kinase activity in Mo stimulated by IFN-β and anti-CD11b mAb results in inhibition of IL-1Ra and upregulation of IL-1β production, suggesting that PI 3-kinase might be a check-point signaling molecule favoring IL-1Ra synthesis. Involvement of PI 3-kinase pathway in IL-1Ra synthesis seems to be independent of the differentiation state of Mo: M-CSF differentiated Mo requires activation of PI 3-kinase to synthesize IL-1Ra following IFN-β treatment. Thus, IFN-β induced IL-1Ra production in Mo by simultaneously activating components of JAK-STAT and PI 3-kinase signaling pathways.

Monoclonal Lym-1 antibody-targeted lysis of B lymphoma cells by neutrophils. Evidence for two mechanisms of FcγRII-dependent cytolysis

Human neutrophils incubated with the anti-HLA-DR mAb Lym-1, plus PMA, induced significant cytolysis of B lymphoma cells compared with Lym-1 and PMA alone. The effect of PMA was independent of the ability of the compound to stimulate neutrophil-respiratory burst. In fact, first, neutrophils from a patient with chronic granulomatous disease were cytolytically effective in spite of their inability to produce oxidants. Second, various kinase inhibitors exerted different effects on the PMA-stimulated cytolytic system and neutrophil-oxidative burst. Previous studies have shown the involvement of the FcgammaRII, CD11b-CD18 integrins, and CD66b glycoproteins in the Lym-1 mAb-dependent cytolysis by GM-CSF-stimulated neutrophils. The present PMA-stimulated system was inhibited by the anti-FcgammaRII mAb IV.3, the anti-CD18 mAb MEM 48, and the anti-CD11b mAb 2LPM19c but not by the anti-CD66b mAb 80H3 and N-acetyl-D-glucosamine. Furthermore, the PMA- and GM-CSF-stimulated cytolysis was insensitive and sensitive to inhibition by pertussis toxin, respectively. Thus, the use of PMA and GMCSF as neutrophil stimulants uncovers the existence of distinct mechanisms of Lym-1 mAb-mediated cytolysis.

Heparin attenuates TNF-α induced inflammatory response through a CD11b dependent mechanism

BACKGROUNDIn addition to its anticoagulant properties, heparin has anti-inflammatory effects, the molecular and mechanistic bases of which are incompletely defined.AIMSThe current studies were designed to test the hypothesis that heparin...

The β-Glucan-Binding Lectin Site of Mouse CR3 (CD11b/CD18) and Its Function in Generating a Primed State of the Receptor That Mediates Cytotoxic Activation in Response to iC3b-Opsonized Target Cells

AbstractMouse leukocyte CR3 (Mac-1, αMβ2 integrin) was shown to function as a receptor for β-glucans in the same way as human CR3. Soluble zymosan polysaccharide (SZP) or pure β-glucans labeled with FITC or 125I bound in a saturable and reversible manner to neutrophils, macrophages, and NK cells. This lectin activity was blocked by anti-CD11b mAb M1/70 or 5C6 and did not occur with leukocytes from CR3−/− (CD11b-deficient) mice. SZP preparations containing primarily mannose or glucose bound to CR3, and the binding of 125I-labeled β-glucan to CR3 was competitively inhibited by β-glucans from barley or seaweed, but not by yeast α-mannan. Also, as with human CR3, the lectin site of mouse CR3 was inhibited by α- or β-methylglucoside (but not d-glucose), α- or β-methylmannoside, and N-acetyl-d-glucosamine. Phagocytosis of zymosan and serum-opsonized zymosan was partially inhibited by anti-CR3 and was reduced to &amp;lt;40% of normal with leukocytes from CR3−/− mice. As with neutrophils from patients with CD18 deficiency, neutrophils from CR3−/− mice exhibited no phagocytosis of particulate β-glucan. SZP or β-glucans primed CR3 of neutrophils, macrophages, and NK cells for cytotoxicity of iC3b-opsonized tumor cells that otherwise did not trigger killing. β-Glucan priming for cytotoxicity was inhibited by anti-CR3 and did not occur with leukocytes from CR3−/− mice. The primed state of macrophage and NK cell CR3 remained detectable for 18 to 24 h after pulsing with β-glucans. The similarity of mouse and human CR3 in response to β-glucans highlights the utility of mouse tumor models for development of therapeutic β-glucans.

A mAb to the beta2-leukocyte integrin Mac-1 (CD11b/CD18) reduces intimal thickening after angioplasty or stent implantation in rabbits.

Leukocytes are recruited early and abundantly to experimentally injured vessels, in direct proportion to cell proliferation and intimal growth. Activated circulating leukocytes and Mac-1 (CD11b/CD18, alphaMbeta2) expression are markers of restenosis risk in patients undergoing angioplasty. As angioplastied vessels lack endothelium but have extensive fibrin(ogen) and platelet deposition, we hypothesized that Mac-1-dependent adhesion to fibrin(ogen) is an important determinant of leukocyte recruitment and function, which may in turn promote intimal growth. To study this hypothesis we administered M1/70, an anti-CD11b blocking mAb, to rabbits (1 mg/kg i.v.) immediately before, and every 48 hr for 3, 6, or 14 days after, iliac artery balloon denudation or deeper stent-induced injury. M1/70, which bound to isolated rabbit monocytes and dose-dependently inhibited Mac-1-mediated fibrinogen binding in vitro, reduced leukocyte recruitment more than 2-fold 3, 6, and 14 days after injury. Neointimal growth 14 days after injury was markedly attenuated by treatment with M1/70 (intimal area after balloon injury, 0.12 +/- 0.09 mm2, compared with 0.32 +/- 0.08 mm2 in vehicle-treated controls, P < 0.01, and 0.38 +/- 0.08 mm2 in IgG-treated controls, P < 0.005; intimal area after stent injury, 0. 56 +/- 0.16 mm2, compared with 0.84 +/- 0.13 mm2 in vehicle-treated controls, P < 0.05, and 0.90 +/- 0.15 mm2 in IgG-treated controls, P < 0.02). Mac-1 blockade reduces experimental neointimal thickening, suggesting that leukocyte recruitment to and infiltration of injured arteries may be a valid target for preventing intimal hyperplasia.

Ligation of the beta2 integrin triggers activation and degranulation of human eosinophils.

Evidence suggests that cellular adhesion is critical for eosinophil effector functions. Here, we tested the hypothesis that an adhesion molecule, specifically beta2 integrin, participates in intracellular signaling events of eosinophils. Eosinophils stimulated with interleukin (IL)-5 and adherent to protein-coated tissue culture plates via beta2 integrin (CD18) showed tyrosine phosphorylation of a number of proteins. Among these proteins, tyrosine phosphorylation of the 105 kD and 115 kD proteins and the product of the c-cbl protooncogene, Cbl, was specifically inhibited using soluble anti-CD18 monoclonal antibody (mAb) to block eosinophil cell adhesion. Furthermore, phosphoinositide turnover of IL-5-stimulated adherent eosinophils was also inhibited by anti-CD18 mAb, suggesting that cellular adhesion plays important roles in eosinophil signal transduction. alphaM beta2 (Mac-1, CD11b/18) was one of the beta2 integrins involved in eosinophil adhesion to protein-coated plates. We found that direct ligation of eosinophil alphaM beta2 with anti-CD11b mAb coupled to polystyrene microbeads induced tyrosine phosphorylation of a 115 kD protein and Cbl. Furthermore, anti-CD11b mAb microbeads induced increases in both phosphoinositide hydrolysis and the eosinophil degranulation response. Control antibodies, such as mouse myeloma IgG1 and anti-HLA class I antigen mAb, did not induce these cellular responses. These results suggest that engagement of beta2 integrin either by cell adhesion or by anti-CD11b mAb triggers activation of an intracellular signaling cascade, including protein tyrosine phosphorylation and phosphoinositide turnover, and subsequent cellular degranulation in human eosinophils. Tyrosine phosphorylation of a 115 kD protein and Cbl may play important roles in adhesion-dependent cellular functions of eosinophils.

Z-100, a polysaccharide-rich preparation extracted from the human type Mycobacterium tuberculosis, improves the resistance of Meth-A tumor-bearing mice to endogenous septic infection.

The effect of Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, on cecal ligation and puncture (CLP)-induced sepsis in mice bearing Meth-A fibrosarcoma was investigated. When normal BALB/c mice were subjected to the CLP procedure, their mortality rate was 17%. On the other hand, an increased mortality was observed in tumor-bearing mice subjected to CLP 10 days after tumor inoculation, and then all mice died when tumor-bearing mice were subjected to CLP 20 days after tumor inoculation. However, the increased percent mortality was decreased by 50% when these mice were injected intraperitoneally with a 10 mg/kg dose of Z-100. When splenocytes (5 x 10(7) cells), obtained from Meth-A tumor-bearing mice 20 days after tumor inoculation, were transferred intravenously to normal mice (recipient mice), mortality of these recipient mice were increased by 62% as compared with that of the control (22%). However, no increased mortality (25%) was observed in recipient mice which were transferred with splenocytes from tumor-bearing mice injected intraperitoneally with Z-100 (10 mg/kg). In addition, suppressor cell activity was demonstrated in splenocytes from Meth-A tumor-bearing mice at 20 days after tumor inoculation using one-way mixed lymphocyte reaction. However, the suppressor cell activity was significantly decreased by the intraperitoneal administration of a 10 mg/kg dose of Z-100 (p < 0.01). The increase of mortality in recipient mice by adoptive transfer of mononuclear cells (MNCs) from tumor-bearing mice was not detected when these MNCs were treated with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb, but an increase was seen with anti-Lyt 1.2 mAb or anti-immunoglobulin antiserum treated MNCs. These results suggest that the suppressor cells affect the mortality of CLP-induced sepsis and Z-100 may have a therapeutic activity against opportunistic infections in immunocompromised hosts through the regulation of suppressor T-cells.

Reversal of immunosuppression inducible through ultraviolet-exposed skin by in vivo anti-CD11b treatment.

In both human in vitro models and murine in vivo adoptive transfer studies, UV-induced class II MHC+ CD11b+ leukocytes that infiltrate the epidermis appear to mediate UV-induced immunosuppression. In the present study, their role is further probed using an anti-CD11b mAb (clone 5C6), which is effective in vivo in blocking CD11b+ monocyte/macrophage diapedesis into inflammatory lesions. A single exposure, low dose UV protocol (72 mJ/cm2) that resulted in tolerance only when dinitroflurobenzene was applied 48 h later through the UV-irradiated skin, but not through a distant non-UV-irradiated site, was used. In vivo anti-CD11b treatment in non-UV-irradiated mice did not block contact sensitivity responses. However, the ability to induce a primary contact sensitivity response was completely restored in UV-irradiated mice receiving anti-CD11b. This restoration was associated with partial restoration of papillary dermal class II MHC+ NLDC-145- cells. In vivo anti-CD11b treatment also blocked tolerance induction, which was associated with a 50% reduction in the infiltration of class II MHC+ CD11b+ Gr-1+ monocyte/macrophages into UV-irradiated skin. In addition, anti-CD11b treatment partially protected against epidermal UV injury, in that the epidermal structure was better preserved and the keratinocytes were less severely damaged. CD11b+ leukocytes may thus affect UV-irradiated skin through at least two mechanisms: 1) a class II MHC+ CD11b+ Gr-1+ monocyte/macrophage population inducing a state of tolerance to Ag(s) acquired in UV-irradiated skin, and 2) CD11b+ leukocytes capable of inflicting additional injury to both keratinocytes and constitutive APC damaged by UV photons.