Role of CXC chemokine ligand 13, CC chemokine ligand (CCL) 19, and CCL21 in the organization and function of nasal-associated lymphoid tissue

Nasal-associated lymphoid tissue (NALT) orchestrates immune responses to Ags in the upper respiratory tract. Unlike other lymphoid organs, NALT develops independently of lymphotoxin-alpha (LTalpha). However, the structure and function of NALT are impaired in Ltalpha(-/-) mice, suggesting a link between LTalpha and chemokine expression. In this study we show that the expression of CXCL13, CCL19, CCL21, and CCL20 is impaired in the NALT of Ltalpha(-/-) mice. We also show that the NALT of Cxcl13(-/-) and plt/plt mice exhibits some, but not all, of the structural and functional defects observed in the NALT of Ltalpha(-/-) mice. Like the NALT of Ltalpha(-/-) mice, the NALT in Cxcl13(-/-) mice lacks follicular dendritic cells, BP3(+) stromal cells, and ERTR7(+) lymphoreticular cells. However, unlike the NALT of Ltalpha(-/-) mice, the NALT of Cxcl13(-/-) mice has peripheral node addressin(+) high endothelial venules (HEVs). In contrast, the NALT of plt/plt mice is nearly normal, with follicular dendritic cells, BP3(+) stromal cells, ERTR7(+) lymphoreticular cells, and peripheral node addressin(+) HEVs. Functionally, germinal center formation and switching to IgA are defective in the NALT of Ltalpha(-/-) and Cxcl13(-/-) mice. In contrast, CD8 T cell responses to influenza are impaired in Ltalpha(-/-) mice and plt/plt mice. Finally, the B and T cell defects in the NALT of Ltalpha(-/-) mice lead to delayed clearance of influenza from the nasal mucosa. Thus, the B and T cell defects in the NALT of Ltalpha(-/-) mice can be attributed to the impaired expression of CXCL13 and CCL19/CCL21, respectively, whereas impaired HEV development is directly due to the loss of LTalpha.     

CC chemokine ligand 2 (CCL2) promotes prostate cancer tumorigenesis and metastasis

CCL2 is a chemokine known to recruit monocytes and macrophages to sites of inflammation. A growing body of research suggests CCL2 is progressively overexpressed in tumor beds and may play a role in the clinical progression of solid tumors. Cancer cells derived from several solid tumor types demonstrate functional receptors for CCL2, suggesting this chemokine may achieve tumorigenicity through direct effects on malignant cells; however, a variety of normal host cells that co-exist with cancer in the tumor microenvironment also respond to CCL2. These cells include macrophages, osteoclasts, endothelial cells, T-lymphocytes, and myeloid-derived immune suppressor cells (MDSCs). CCL2 mediated interactions between normal and malignant cells in the tumor microenvironment and plays a multi-faceted role in tumor progression.     

HIV type 1 glycoprotein 120 inhibits human B cell chemotaxis to CXC chemokine ligand (CXCL) 12, CC chemokine ligand (CCL)20, and CCL21

We analyzed the modulation of human B cell chemotaxis by the gp120 proteins of various HIV-1 strains. X4 and X4/R5 gp120 inhibited B cell chemotaxis toward CXCL12, CCL20, and CCL21 by 40-50%, whereas R5 gp120 decreased inhibition by 20%. This gp120-induced inhibition was strictly dependent on CXCR4 or CCR5 and lipid rafts but not on CD4 or V(H)3-expressing BCR. Inhibition did not impair the expression or ligand-induced internalization of CCR6 and CCR7. Our data suggest that gp120/CXCR4 and gp120/CCR5 interactions lead to the cross-desensitization of CCR6 and CCR7 because gp120 does not bind CCR6 and CCR7. Unlike CXCL12, gp120 did not induce the activation of phospholipase Cbeta3 and PI3K downstream from CXCR4, whereas p38 MAPK activation was observed. Similar results were obtained if gp120-treated cells were triggered by CCL21 and CCL20. Our results are consistent with a blockade restricted to signaling pathways using phosphatidylinositol-4,5-bisphosphate as a substrate. X4 and X4/R5 gp120 induced the cleavage of CD62 ligand by a mechanism dependent on matrix metalloproteinase 1 and 3, CD4, CXCR4, Galpha(i), and p38 MAPK, whereas R5 gp120 did not. X4 and X4/R5 gp120 also induced the relocalization of cytoplasmic CD95 to the membrane and a 23% increase in CD95-mediated apoptosis. No such effects were observed with R5 gp120. The gp120-induced decrease in B cell chemotaxis and CD62 ligand expression, and increase in CD95-mediated B cell apoptosis probably have major deleterious effects on B cell responsiveness during HIV infection and in vaccination trials.     

The chemokine decoy receptor M3 blocks CC chemokine ligand 2 and CXC chemokine ligand 13 function in vivo

Chemokines and their receptors play a key role in immune homeostasis regulating leukocyte migration, differentiation, and function. Viruses have acquired and optimized molecules that interact with the chemokine system. These virus-encoded molecules promote cell entry, facilitate dissemination of infected cells, and enable the virus to evade the immune response. One such molecule in the murine gammaherpesvirus 68 genome is the M3 gene, which encodes a secreted 44-kDa protein that binds with high affinity to certain murine and human chemokines and blocks chemokine signaling in vitro. To test the hypothesis that M3 directly interferes with diverse chemokines in vivo, we examined the interaction of M3 with CCL2 and CXCL13 expressed in the pancreas of transgenic mice. CCL2 expression in the pancreas promoted recruitment of monocytes and dendritic cells; CXCL13 promoted recruitment of B and T lymphocytes. Coexpression of M3 in the pancreas blocked cellular recruitment induced by both CCL2 and CXCL13. These results define M3 as multichemokine blocker and demonstrate its use as a powerful tool to analyze chemokine biology.     

Human CC chemokine CCL23, a ligand for CCR1, induces endothelial cell migration and promotes angiogenesis

A number of chemokines induce angiogenesis and endothelial cells express several chemokine receptors. To date, only a limited number of CC chemokines for CCR1 have been reported to induce angiogenic responses. We investigated the ability of CCL23 (also known as MPIF-1, MIP-3, or CKbeta8) to promote angiogenesis, which induces chemotaxis of immune cells through CCR1. CCL23 promoted the chemotactic migration and differentiation of endothelial cells, and neovascularization in the chick chorioallantoic membrane. An N-terminal truncated form of CCL23 was at least 100-fold more potent than its intact form and was comparable to that of FGF in the angiogenic activities. Treatment with either pertussis toxin or anti-CCR1 antibody completely inhibited the CCL23-induced endothelial cell migration, indicating that endothelial cell migration was mediated through CCR1. CCL23 didn't promote the migration of HT1080 human fibrosarcoma cells that did not express CCR1. Our results suggest a role of CCL23 in angiogenesis in vitro as well as in vivo.     

The CC chemokine eotaxin (CCL11) is a partial agonist of CC chemokine receptor 2b

Despite sharing considerable homology with the members of the monocyte chemoattractant protein (MCP) family, the CC chemokine eotaxin (CCL11) has previously been reported to signal exclusively via the receptor CC chemokine receptor 3 (CCR3). Using the monocyte cell line THP-1, we investigated the relative abilities of eotaxin and MCPs 1-4 to induce CCR2 signaling, employing assays of directed cell migration and intracellular calcium flux. Surprisingly, 1 microm concentrations of eotaxin were able to recruit THP-1 cells in chemotaxis assays, and this migration was sensitive to antagonism of CCR2 but not CCR3. Radiolabeled eotaxin binding assays performed on transfectants bearing CCR2b or CCR3 confirmed eotaxin binding to CCR2 with a K(d) of 7.50 +/- 3.30 nm, compared with a K(d) of 1.68 +/- 0.91 nm at CCR3. In addition, whereas 1 microm concentrations of eotaxin were able to recruit CCR2b transfectants, substimulatory concentrations of eotaxin inhibited MCP-1-induced chemotaxis of CCR2b transfectants and also inhibited MCP-1-induced intracellular calcium flux of THP-1 cells. Collectively, these findings suggest that eotaxin is a partial agonist of the CCR2b receptor. A greater understanding of the interaction of CCR2 with all of its ligands, both full and partial agonists, may aid the rational design of specific antagonists that hold great promise as future therapeutic treatments for a variety of inflammatory disorders.     

Modulation of immune cell proliferation and chemotaxis towards CC chemokine ligand (CCL)-21 and CXC chemokine ligand (CXCL)-12 in undenatured whey protein-treated mice

Whey protein concentrates (WPCs) enhance innate mucosal immunity during early life and have a protective role in some immune disorders. To further elucidate the potential benefits of this protein, the present study investigated the effect of dietary supplementation with WPCs on blood parameters, plasma cytokine profiles, and immune cell proliferation and chemotaxis. A total of 45 male mice were equally distributed into three experimental groups and treated daily for 21 days as follows: group I was a control group that was orally supplemented with distilled water, group II was orally supplemented with undenatured WP (100 mg/kg body weight), and group III was orally supplemented with bovine serum albumin (100 mg/kg body weight). We found that the plasma cytokine levels of interleukin (IL)-1α, IL-1β, IL-10 and tumor necrosis factor-α and the levels of reactive oxygen species, cholesterol, triglycerides and the lipid profile were significantly decreased in the WP-treated group compared to the control group. In contrast, the levels of IL-2, IL-4, IL-7, IL-8 and glutathione were significantly elevated, and consequently, the ability of peripheral blood mononuclear cells to proliferate in response to stimulation with different antigens was significantly increased in the WP-treated group. Moreover, the in vitro chemotaxis of B, T and bone-marrow-derived dendritic cells toward CC chemokine ligand-21 and CXC chemokine ligand-12 was significantly increased, by twofold, in WP-treated mice compared to the control group. Taken together, our data reveal the benefits of WP supplementation in enhancing immune cell proliferation and migration to the secondary lymphoid organs.     

Kinetics of virus-specific CD8+ -T-cell expansion and trafficking following central nervous system infection

CD8+ T cells control acute infection of the central nervous system (CNS) by neurotropic mouse hepatitis virus but do not suffice to achieve sterile immunity. To determine the lag between T-cell priming and optimal activity within the CNS, the accumulation of virus-specific CD8+ T cells in the CNS relative to that in peripheral lymphoid organs was assessed by using gamma interferon-specific ELISPOT assays and class I tetramer staining. Virus-specific CD8+ T cells were first detected in the cervical lymph nodes. Expansion in the spleen was delayed and less pronounced but also preceded accumulation in the CNS. The data further suggest peripheral acquisition of cytolytic function, thus enhancing CD8+ -T-cell effector function upon cognate antigen recognition in the CNS.     

Murine CD8+ recent thymic emigrants are alphaE integrin-positive and CC chemokine ligand 25 responsive

Recent thymic emigrants (RTE) are an important subpopulation of naive CD8+ T cells because of their ability to reconstitute a diverse immune system after periods of T cell depletion. In neonatal mice, the majority of peripheral T lymphocytes are RTE, cells that have recently left the thymus to populate the periphery. Postulating that these cells could have unique trafficking mechanisms, we compared adhesion molecule and chemokine receptor expression of neonatal RTE with mature adult lymphocytes. Neonatal CD8+ splenocytes uniformly express alpha(E) integrin and exhibit a high responsiveness to CC chemokine ligand (CCL25) (as compared with adult CD8+ splenocytes). Mature CD8+ thymocytes have a similar alpha(E) integrin(+) CCL25 responsive phenotype, as do adult CD8+ RTE identified by intrathymic FITC injection. With increasing age, the frequency of CD8+ alpha(E) integrin(+) splenocytes decreases, roughly correlating with thymic involution. Moreover, halting thymic output by thymectomy accelerates the age-dependent decline in peripheral CD8+ alpha(E) integrin(+) RTE phenotype cells. Low expression of CD44 distinguishes these CD8+ RTE from a population of memory phenotype alpha(E) integrin(+) CD8+ cells that are CD44(high). We conclude that CD8+ RTE have unique adhesive and chemotactic properties that distinguish them from naive CD8+ T cells. These properties may enable specialized microenvironmental and cell-cell interactions contributing to the fate of RTE in the periphery during the early post-thymic period. This phenotype will also facilitate the identification and isolation of RTE for further studies.     

Molecular cloning of a novel human CC chemokine (Eotaxin-3) that is a functional ligand of CC chemokine receptor 3.

Previously, we mapped the novel CC chemokine myeloid progenitor inhibitory factor 2 (MPIF-2)/eotaxin-2 to chromosome 7q11.23 (Nomiyama, H., Osborne, L. R., Imai, T., Kusuda, J., Miura, R., Tsui, L.-C., and Yoshie, O. (1998) Genomics 49, 339-340). Since chemokine genes tend to be clustered, unknown chemokines may be present in the vicinity of those mapped to new chromosomal loci. Prompted by this hypothesis, we analyzed the genomic region containing the gene for MPIF-2/eotaxin-2 (SCYA24) and have identified a novel CC chemokine termed eotaxin-3. The genes for MPIF-2/eotaxin-2 (SCYA24) and eotaxin-3 (SCYA26) are localized within a region of approximately 40 kilobases. By Northern blot analysis, eotaxin-3 mRNA was constitutively expressed in the heart and ovary. We have generated recombinant eotaxin-3 in a baculovirus expression system. Eotaxin-3 induced transient calcium mobilization specifically in CC chemokine receptor 3 (CCR3)-expressing L1.2 cells with an EC(50) of 3 nM. Eotaxin-3 competed the binding of (125)I-eotaxin to CCR3-expressing L1.2 cells with an IC(50) of 13 nM. Eotaxin-3 was chemotactic for normal peripheral blood eosinophils and basophils at high concentrations. Collectively, eotaxin-3 is yet another functional ligand for CCR3. The potency of eotaxin-3 as a CCR3 ligand seems, however, to be approximately 10-fold less than that of eotaxin. Identification of eotaxin-3 will further promote our understanding of the control of eosinophil trafficking and other CCR3-mediated biological phenomena. The strategy used in this study may also be applicable to identification of other unknown chemokine genes.     

Lung-specific overexpression of CC chemokine ligand (CCL) 2 enhances the host defense to Streptococcus pneumoniae infection in mice: role of the CCL2-CCR2 axis

Mononuclear phagocytes are critical components of the innate host defense of the lung to inhaled bacterial pathogens. The monocyte chemotactic protein CCL2 plays a pivotal role in inflammatory mononuclear phagocyte recruitment. In this study, we tested the hypothesis that increased CCL2-dependent mononuclear phagocyte recruitment would improve lung innate host defense to infection with Streptococcus pneumoniae. CCL2 transgenic mice that overexpress human CCL2 protein in type II alveolar epithelial cells and secrete it into the alveolar air space showed a similar proinflammatory mediator response and neutrophilic alveolitis to challenge with S. pneumoniae as wild-type mice. However, CCL2 overexpressing mice showed an improved pneumococcal clearance and survival compared with wild-type mice that was associated with substantially increased lung mononuclear phagocyte subset accumulations upon pneumococcal challenge. Surprisingly, CCL2 overexpressing mice developed bronchiolitis obliterans upon pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block the CCL2-CCR2 axis in CCL2 overexpressing mice, though completely abrogating bronchiolitis obliterans, led to progressive pneumococcal pneumonia. Collectively, these findings demonstrate the importance of the CCL2-CCR2 axis in the regulation of both the resolution/repair and remodelling processes after bacterial challenge and suggest that overwhelming innate immune responses may trigger bronchiolitis obliterans formation in bacterial lung infections.     

Immediate early effector functions of virus-specific CD8+CCR7+ memory cells in humans defined by HLA and CC chemokine ligand 19 tetramers

Memory T cells exhibit a high degree of heterogeneity in terms of their phenotype and functional characteristics. It has been proposed that the CCR7 chemokine receptor divides memory T cell populations into central memory T cells and effector memory T cells with distinct functions in secondary immune responses. We were interested whether this hypothesis holds true in experiments performed on Ag-specific CD8(+) T cells. To identify CCR7(+) cells, we engineered a fluorescent ligand for CCR7; results with the new CC chemokine ligand 19 chemotetramer were verified by staining with a CCR7 mAb. Staining with the CC chemokine ligand 19 chemotetramer reveals two subsets within CCR7(+) cells: a CCR7(int) population containing memory cells and a CCR7(high) population containing naive T cells. Phenotypic analysis of MHC class I/peptide tetramer-positive cells revealed that HLA-A2-restricted CMV-specific CD8 T cells exhibit the lowest percentage of CCR7(+) cells (0.5-5%), while HLA-A2-restricted flu- and HLA-B8-restricted EBV-specific CD8 T cells showed the highest (45-70%). Intracellular staining of unstimulated cells revealed that both CCR7(int)- and CCR7(-)-specific CD8 T cells exhibit a detectable level of perforin. Both CCR7(int) and CCR7(-) Ag-specific CD8(+) T cells produced IFN-gamma and TNF-alpha following short-term peptide stimulation. Therefore, our finding that CCR7(+)CD8(+) T cells are able to exert immediate effector functions requires a substantial revision to the central and effector memory hypothesis.     

Tumor necrosis factor-alpha (TNF-alpha) induces integrin CD11b/CD18 (Mac-1) up-regulation and migration to the CC chemokine CCL3 (MIP-1alpha) on human neutrophils through defined signalling pathways

Strong evidence suggests that neutrophils may play an active role in acute and chronic inflammatory disorders, such as rheumatoid arthritis and atherosclerosis. Given the role of pro-inflammatory cytokine TNF-alpha in these inflammatory processes, we planned the present study to investigate the effect of short term incubation with TNF-alpha on neutrophil migration to CCL3, a chemokine produced in inflammatory sites and normally devoid of neutrophil chemotactic properties. We found that TNF-alpha primed neutrophils for migration to CCL3 via CCR5. TNF-alpha-induced migration was a consequence of the TNF-alpha-induced up-regulation of integrin CD11b/CD18 (Mac-1) on neutrophil surface. Furthermore, TNF-alpha activity was found to be strictly dependent on the activation of ERK 1/2 p44, cooperating with the intracellular pathways involving Src kinases, PI3K/Akt, p38 MAPK, well known as activated in response to classical chemoattractants (CXCL8) or priming agents (GM-CSF). On the contrary, the effect of TNF-alpha on neutrophil migration to CCL3 was not dependent on JNK 1/2. In conclusion, the present report shows that TNF-alpha unveils a previously unknown capacity of neutrophils to migrate to CCL3 through the intervention of Mac-1. TNF-alpha regulates Mac-1 up-regulation through signalling pathways, involving various kinases, but not JNK 1/2. Although highly speculative, ERK 1/2 p44 may represent a selective target for the pharmacologic manipulation of neutrophil-mediated adverse activities in TNF-alpha-mediated inflammatory states.     

The host environment regulates the function of CD8+ graft-versus-host-reactive effector cells

We have examined how the host environment influences the graft-vs-leukemia (GVL) response following transfer of donor T cells to allogeneic chimeras. Donor T cells induce significant GVL when administered in large numbers to established mixed chimeras (MC). However, when using limiting numbers of T cells, we found that late transfer to MC induced less GVL than did early transfer to freshly irradiated allogeneic recipients. Late donor T cell transfer to MC was associated with marked accumulation of anti-host CD8 cells within the spleen, but delayed kinetics of differentiation, reduced expression of effector molecules including IFN-gamma, impaired cytotoxicity, and higher rates of sustained apoptosis. Furthermore, in contrast to the spleen, we observed a significant delay in donor CD8 cell recruitment to the bone marrow, a key location for hematopoietic tumors. Increasing the numbers of T cells transferred to MC led to the enhancement of CTL activity and detectable increases in absolute numbers of IFN-gamma(+) cells without inducing graft-vs-host disease (GVHD). TLR-induced systemic inflammation accelerated differentiation of functional CTL in MC but was associated with severe GVHD. In the absence of inflammation, both recipient T and non-T cell populations impeded the full development of GVHD-inducing effector function. We conclude that per-cell deficits in the function of donor CD8 cells activated in MC may be overcome by transferring larger numbers of T cells without inducing GVHD.     

Neonates mount robust and protective adult-like CD8(+)-T-cell responses to DNA vaccines

Neonates are thought to mount less vigorous adaptive immune responses than adults to antigens and infectious agents. This concept has led to a delay in the administration of many currently available vaccines until late infancy or early childhood. It has recently been shown that vaccines composed of plasmid DNA can induce both humoral and cell-mediated antimicrobial immunity when administered within hours of birth. In most of these studies, immune responses were measured weeks or months after the initial vaccination, and it is therefore questionable whether the observed responses were actually the result of priming of splenocytes within the neonatal period. Here we show that DNA vaccination at birth results in the rapid induction of antigen-specific CD8(+) T cells within neonatal life. Analyses of T-cell effector functions critical for the resolution of many viral infections revealed that neonatal and adult CD8(+) T cells produce similar arrays of cytokines. Furthermore, the avidities of neonatal and adult CD8(+) T cells for peptide and the rapidity with which they upregulate cytokine production after recall encounters with antigen are similar. Protective immunity against the arenavirus lymphocytic choriomeningitis virus, which is mediated by CD8(+) cytotoxic T cells, is also rapidly acquired within the neonatal period. Collectively these data imply that, at least in the case of CD8(+) T cells, neonates are not as immunodeficient as previously supposed and that DNA vaccines may be an effective and safe means of providing critical cell-mediated antiviral immunity extremely early in life.     

Bacterial superantigen exposure after resolution of influenza virus infection perturbs the virus-specific memory CD8(+)-T-cell repertoire

Heterologous viral infections have been shown to impact the preexisting memory CD8(+)-T-cell repertoire. Bacterial superantigens are products of common human pathogenic bacteria, including staphylococci and streptococci, that are potent T-cell-stimulatory molecules. In this report, we show that exposure to staphylococcal enterotoxin B, a bacterial superantigen, causes a selective functional deletion of cross-reactive influenza virus-specific CD8(+) memory T cells. This perturbation of the memory repertoire can have a significant impact on viral clearance after secondary challenge.     

Distinct cycling CD4(+)- and CD8(+)-T-cell profiles during the asymptomatic phase of simian immunodeficiency virus SIVmac251 infection in rhesus macaques

Elevated CD4 T-cell turnover may lead to the exhaustion of the immune system during human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) infections. However, this hypothesis remains controversial. Most studies of this subject have concerned the blood, and information about the lymph nodes is rare and controversial. We used Ki67 expression to measure cycling T cells in the blood and lymph nodes of uninfected macaques and of macaques infected with a pathogenic SIVmac251 strain or with a nonpathogenic SIVmac251Deltanef clone. During the asymptomatic phase of infection, the number of cycling CD8(+) T cells progressively increased (two- to eightfold) both in the blood and in the lymph nodes of macaques infected with SIVmac251. This increase was correlated with viral replication and the progression to AIDS. In contrast, no increases in the numbers of cycling CD4(+) T cells were found in the blood or lymph nodes of macaques infected with the pathogenic SIVmac251 strain in comparison with SIVmac251Deltanef-infected or healthy macaques during this chronic phase. However, the lymph nodes of pre-AIDS stage SIVmac251-infected macaques contained more cycling CD4(+) T cells (low baseline CD4(+)-T-cell counts in the blood). Taken together, these results show that the profiles of CD4(+)- and CD8(+)-T-cell dynamics are distinct both in the lymph nodes and blood and suggest that higher CD4(+)-T-cell proliferation at the onset of AIDS may lead to the exhaustion of the immune system.     

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.     

PSGL-1 regulates the migration and proliferation of CD8(+) T cells under homeostatic conditions

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1(null) mice. Naive CD4(+) and CD8(+) T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1(null) T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1(+/+) T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1(null) CD8(+) T cell proliferation was observed under steady-state conditions and PSGL-1(null) CD8(+) T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1(null) mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1(null) mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1(null) mice.     

Differences in potency of CXC chemokine ligand 8-, CC chemokine ligand 11-, and C5a-induced modulation of integrin function on human eosinophils

The hypothesis was tested that different chemoattractants have different effects on the activity of integrins expressed by the human eosinophil. Three chemoattractants, CXCL8 (IL-8), CCL11 (eotaxin-1), and C5a were tested with respect to their ability to induce migration and the transition of eosinophils from a rolling interaction to a firm arrest on activated endothelial cells under flow conditions. CCL11 and C5a induced a firm arrest of eosinophils rolling on an endothelial surface, whereas CXCL8 induced only a transient arrest of the cells. The CXCL8- and CCL11-induced arrest was inhibited by simultaneously blocking alpha4 integrins (HP2/1) and beta2 integrins (IB4). In contrast, the C5a-induced arrest was only inhibited by 30% under these conditions. The potency differences of C5a>CCL11>CXCL8 to induce firm adhesion under flow condition was also observed in migration assays and for the activation of the small GTPase Rap-1, which is an important signaling molecule in the inside-out regulation of integrins. Interestingly, only C5a was able to induce the high activation epitope of alphaMbeta2 integrin recognized by MoAb CBRM1/5. The C5a-induced appearance of this epitope and Rap activation was controlled by phospholipase C (PLC), as was shown with the PLC inhibitor U73122. These data show that different chemoattractants are able to induce distinct activation states of integrins on eosinophils and that optimal chemotaxis is associated with the high activation epitope of the alphaMbeta2 integrin. Furthermore, PLC plays an important role in the inside-out signaling and, thus, the activation status of integrins on eosinophils.