Early local generation of C5a initiates the elicitation of contact sensitivity by leading to early T cell recruitment

We have shown previously that an early complement C5-dependent cascade is required to recruit T cells to elicit 24-h contact sensitivity (CS) responses. In this paper, we have characterized molecular events of this early required cascade by biochemically analyzing extracts of mouse ears undergoing elicitation of CS. Chemotactic activity was found after local Ag challenge, in CS ear extracts early (by 1 h), in CS ear extracts late (through 24 h), in previously immunized mice, but not in ears of vehicle-immunized or non-immune-challenged mice. The early chemotactic activity at 2 h was likely caused by C5a, because it was neutralized in vitro by anti-C5a Ab, was inactive on C5aR-deficient (C5aR-/-) macrophages, and was absent in C5-deficient mice. The activity was present in T cell-deficient mice, but elaboration was Ag-specific. This T cell-independent, Ag-specific elaboration of C5a early in CS ear responses likely led to T cell recruitment, because subsequent local IFN-gamma mRNA and protein expression, as markers of T cell arrival and activation, began by 4 h after Ag challenge. In contrast to early C5a chemotactic activity, late chemotactic activity 24 h after Ag challenge was unaffected by anti-C5, was active on C5aR-/- macrophages, was T cell-dependent, and by ELISA appeared largely due to chemokines (macrophage-inflammatory protein-1alpha and -1beta, IFN-gamma-inducible protein-10, and monocyte chemoattractant protein-1). Importantly, early generation of C5a was required for T cell recruitment because C5aR-/- mice had absent 24-h CS. Taken together, these findings indicate an important linkage of C5a as a component of early activated innate immunity that is required for later elicitation of acquired T cell immunity, probably by facilitating the initial recruitment of T cells into the Ag-challenged local site in CS responses.     

Endogenous regulatory T cells adhere in inflamed dermal vessels via ICAM-1: association with regulation of effector leukocyte adhesion

Regulatory T cells (Tregs) must express appropriate skin-homing adhesion molecules to exert suppressive effects on dermal inflammation. However, the mechanisms whereby they control local inflammation remain unclear. In this study we used confocal intravital microscopy in wild-type and Foxp3-GFP mice to examine adhesion of effector T cells and Tregs in dermal venules. These experiments examined a two-challenge model of contact sensitivity (CS) in which Treg abundance in the skin progressively increases during the course of the response. Adhesion of CD4(+) T cells increased during CS, peaking 8-24 h after an initial hapten challenge, and within 4 h of a second challenge. At these time points, 40% of adherent CD4(+) T cells were Foxp3(+) Tregs. CD4(+) T cell adhesion was highly dependent on ICAM-1, and consistent with this finding, anti-ICAM-1 prevented Treg adhesion. Skin TGF-β levels were elevated in skin during both challenges, in parallel with Treg adhesion. In the two-challenge CS model, inhibition of ICAM-1 eliminated Treg adhesion, an effect associated with a significant increase in neutrophil adhesion. Similarly, total CD4(+) T cell depletion caused an increase in adhesion of CD8(+) T cells. Because Treg adhesion was restricted by both of these treatments, these experiments suggest that adherent Tregs can control adhesion of proinflammatory leukocytes in vivo. Moreover, the critical role of ICAM-1 in Treg adhesion provides a potential explanation for the exacerbation of inflammation reported in some studies of ICAM-1-deficient mice.     

Neutrophil expression of fas ligand and perforin directs effector CD8 T cell infiltration into antigen-challenged skin

Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and perforin expression. Although DNFB sensitization of gld/perforin(-/-) mice induced hapten-specific CD8 T cells producing IFN-γ and IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS when transferred to naive gld/perforin(-/-) recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when transferred to sensitized gld/perforin(-/-) mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1, CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune responses in the skin.     

C5a-mediated leukotriene B4-amplified neutrophil chemotaxis is essential in tumor immunotherapy facilitated by anti-tumor monoclonal antibody and beta-glucan

Intravenous and orally administered beta-glucans promote tumor regression and survival by priming granulocyte and macrophage C receptor 3 (CR3, iC3bR and CD11b/CD18) to trigger the cytotoxicity of tumor cells opsonized with iC3b via anti-tumor Abs. Despite evidence for priming of macrophage CR3 by oral beta-glucan in vivo, the current study in C57BL/6 and BALB/c mice showed that granulocytes were the essential killer cells in mAb- and oral beta-glucan-mediated tumor regression, because responses were absent in granulocyte-depleted mice. Among granulocytes, neutrophils were the major effector cells, because tumor regression did not occur when C5a-dependent chemotaxis was blocked with a C5aR antagonist, whereas tumor regression was normal in C3aR(-/-) mice. Neutrophil recruitment by C5a in vivo required amplification via leukotriene B(4), because both C5a-mediated leukocyte recruitment into the peritoneal cavity and tumor regression were suppressed in leukotriene B(4)R-deficient (BLT-1(-/-)) mice.     

The leukotriene B4 receptor (BLT1) is required for effector CD8+ T cell-mediated, mast cell-dependent airway hyperresponsiveness

Studies in both humans and rodents have suggested that CD8+ T cells contribute to the development of airway hyperresponsiveness (AHR) and that leukotriene B4 (LTB4) is involved in the chemotaxis of effector CD8+ T cells (T(EFF)) to the lung by virtue of their expression of BLT1, the receptor for LTB4. In the present study, we used a mast cell-CD8-dependent model of AHR to further define the role of BLT1 in CD8+ T cell-mediated AHR. C57BL/6+/+ and CD8-deficient (CD8-/-) mice were passively sensitized with anti-OVA IgE and exposed to OVA via the airways. Following passive sensitization and allergen exposure, C57BL/6+/+ mice developed altered airway function, whereas passively sensitized and allergen-exposed CD8-/- mice failed to do so. CD8-/- mice reconstituted with CD8+ T(EFF) developed AHR in response to challenge. In contrast, CD8-/- mice reconstituted with BLT1-deficient effector CD8+ T cells did not develop AHR. The induction of increased airway responsiveness following transfer of CD8+ T(EFF) or in wild-type mice could be blocked by administration of an LTB4 receptor antagonist confirming the role of BLT1 in CD8+ T cell-mediated AHR. Together, these data define the important role for mast cells and the LTB4-BLT1 pathway in the development of CD8+ T cell-mediated allergic responses in the lung.     

Divergent functions of CD4+ T lymphocytes in acute liver inflammation and injury after ischemia-reperfusion

Hepatic ischemia-reperfusion results in an acute inflammatory response culminating in the recruitment of activated neutrophils that directly injure hepatocytes. Recent evidence suggests that CD4+ lymphocytes may regulate this neutrophil-dependent injury, but the mechanisms by which this occurs remain to be elucidated. In the present study, we sought to determine the type of CD4+ lymphocytes recruited to the liver after ischemia-reperfusion and the manner in which these cells regulated neutrophil recruitment and tissue injury. Wild-type and CD4 knockout (CD4-/-) mice were subjected to hepatic ischemia-reperfusion. CD4+ lymphocytes were recruited in the liver within 1 h of reperfusion and remained for at least 4 h. These cells were comprised of conventional (alphabetaTCR-expressing), unconventional (gammadeltaTCR-expressing), and natural killer T cells. CD4-/- mice were then used to determine the functional role of CD4+ lymphocytes in hepatic ischemia-reperfusion injury. Compared with wild-type mice, CD4-/- mice had significantly greater liver injury, yet far less neutrophil accumulation. Adoptive transfer of CD4+ lymphocytes to CD4-/- mice recapitulated the wild-type response. In wild-type mice, neutralization of interleukin (IL)-17, a cytokine released by activated CD4+ lymphocytes, significantly reduced neutrophil recruitment in association with suppression of MIP-2 expression. Finally, oxidative burst activity of liver-recruited neutrophils was higher in CD4-/- mice compared with those from wild-type mice. These data suggest that CD4+ lymphocytes are rapidly recruited to the liver after ischemia-reperfusion and facilitate subsequent neutrophil recruitment via an IL-17-dependent mechanism. However, these cells also appear to attenuate neutrophil activation. Thus the data suggest that CD4+ lymphocytes have dual, opposing roles in the hepatic inflammatory response to ischemia-reperfusion.     

Leukotriene B4 receptor-1 is essential for allergen-mediated recruitment of CD8+ T cells and airway hyperresponsiveness

Recent studies in both human and rodents have indicated that in addition to CD4+ T cells, CD8+ T cells play an important role in allergic inflammation. We previously demonstrated that allergen-sensitized and -challenged CD8-deficient (CD8-/-) mice develop significantly lower airway hyperresponsiveness (AHR), eosinophilic inflammation, and IL-13 levels in bronchoalveolar lavage fluid compared with wild-type mice, and that all these responses were restored by adoptive transfer of in vivo-primed CD8+ T cells or in vitro-generated effector CD8+ T cells (T(EFF)). Recently, leukotriene B4 and its high affinity receptor, BLT1, have been shown to mediate in vitro-generated T(EFF) recruitment into inflamed tissues. In this study we investigated whether BLT1 is essential for the development of CD8+ T cell-mediated allergic AHR and inflammation. Adoptive transfer of in vivo-primed BLT1+/+, but not BLT1-/-, CD8+ T cells into sensitized and challenged CD8-/- mice restored AHR, eosinophilic inflammation, and IL-13 levels. Moreover, when adoptively transferred into sensitized CD8-/- mice, in vitro-generated BLT1+/+, but not BLT1-/-, T(EFF) accumulated in the lung and mediated these altered airway responses to allergen challenge. These data are the first to show both a functional and an essential role for BLT1 in allergen-mediated CD8+ T(EFF) recruitment into the lung and development of AHR and airway inflammation.     

Oral administration of hapten inhibits in vivo induction of specific cytotoxic CD8+ T cells mediating tissue inflammation: a role for regulatory CD4+ T cells

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2, 4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-gamma-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.     

Hapten application to the skin induces an inflammatory program directing hapten-primed effector CD8 T cell interaction with hapten-presenting endothelial cells

Contact hypersensitivity is a CD8 T cell-mediated response to hapten sensitization and challenge of the skin. Effector CD8 T cell recruitment into the skin parenchyma to elicit the response to hapten challenge requires prior CXCL1/KC-directed neutrophil infiltration within 3-6 h after challenge and is dependent on IFN-γ and IL-17 produced by the hapten-primed CD8 T cells. Mechanisms directing hapten-primed CD8 T cell localization and activation in the Ag challenge site to induce this early CXCL1 production in response to 2,4-dinitrofluorobenzene were investigated. Both TNF-α and IL-17, but not IFN-γ, mRNA was detectable within 1 h of hapten challenge of sensitized mice and increased thereafter. Expression of ICAM-1 was observed by 1 h after challenge of sensitized and nonsensitized mice and was dependent on TNF-α. The induction of IL-17, IFN-γ, and CXCL1 in the challenge site was not observed when ICAM-1 was absent or neutralized by specific Ab. During the elicitation of the contact hypersensitivity response, endothelial cells expressed ICAM-1 and produced CXCL1 suggesting this as the site of CD8 T cell localization and activation. Endothelial cells isolated from challenged skin of naive and sensitized mice had acquired the hapten and the ability to activate hapten-primed CD8 T cell cytokine production. These results indicate that hapten application to the skin of sensitized animals initiates an inflammatory response promoting hapten-primed CD8 T cell localization to the challenge site through TNF-α-induced ICAM-1 expression and CD8 T cell activation to produce IFN-γ and IL-17 through endothelial cell presentation of hapten.     

Leukotriene B4 mediates neutrophil migration induced by heme

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.     

Leukotriene B4 is essential for selective eosinophil recruitment following allergen challenge of CD4+ cells in a model of chronic eosinophilic inflammation

Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class II expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4+ (but not CD4-) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4+ cells ex vivo. MK886 blocked induction of CCL17. Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalbumin-restimulated CD4+ cells initiate eosinophil recruitment which is strictly dependent on LTB4 production.     

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2 in mice

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor α (TNFα), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ET(A)/ET(B) receptor antagonist bosentan, and selective ET(A) or ET(B) receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFα and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c(+) markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ET(A)- and ET(B)-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2-dependent mechanism.     

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5(+), CD19(+), CD22(+), B220(+), Thy1(+), and Mac1(+), suggesting that they are B-1 B cells. DTH initiation is absent in micro MT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.     

Rescue of tumor-infiltrating lymphocytes from activation-induced cell death enhances the antitumor CTL response in CD5-deficient mice

The CD5 coreceptor is expressed on all T cells and on the B1a B cell subset. It is associated with TCR and BCR, and modulates intracellular signals initiated by both Ag receptor complexes. Human CD5 contributes to regulation of the antitumor immune response and susceptibility of specific CTL to activation-induced cell death (AICD) triggered by the tumor. In this study, we compared the T cell response to the B16F10 melanoma engrafted into CD5-deficient and wild-type C57BL/6 mice. Compared with wild-type mice, CD5 knockout animals displayed delayed tumor growth, associated with tumor infiltration by T cell populations exhibiting a more activated phenotype and enhanced antitumor effector functions. However, control of tumor progression in CD5(-/-) mice was transient due to increased AICD of CD8(+) tumor-infiltrating T lymphocytes. Remarkably, in vivo protection of T cells from TCR-mediated apoptosis by an adenovirus engineered to produce soluble Fas resulted in a dramatic reduction in tumor growth. Our data suggest that recruitment of tumor-specific T cells in the tumor microenvironment occurs at early stages of cancer development and that tumor-mediated AICD of tumor-infiltrating T lymphocytes is most likely involved in tumor escape from the immune system.     

Type I IFN modulates host defense and late hyperinflammation in septic peritonitis

TLRs are considered important for the control of immune responses during endotoxic shock or polymicrobial sepsis. Signaling by TLRs may proceed through the adapter proteins MyD88 or TIR domain-containing adaptor inducinng IFN-beta. Both pathways can lead to the production of type I IFNs (IFN-alphabeta). In the present study, the role of the type I IFN pathway for host defense and immune pathology in sepsis was investigated using a model of mixed bacterial peritonitis. Systemic levels of IFN-alphabeta protein were markedly elevated during septic peritonitis. More detailed analyses revealed production of IFN-beta, but not IFN-alpha subtypes, and identified CD11b+ CD11c- macrophage-like cells as major producers of IFN-beta. The results further demonstrate that in IFN-alphabeta receptor I chain (IFNARI)-deficient mice, the early recruitment of neutrophils to the infected peritoneal cavity was augmented, most likely due to an increased local production of MCP-1 and leukotriene B4. In the absence of IFNARI, peritoneal neutrophils also exhibited enhanced production of reactive oxygen intermediates and elevated expression of Mac-1. Conversely, administration of recombinant IFN-beta resulted in reduced leukotriene B4 levels and decreased peritoneal neutrophil recruitment and activation. Analysis of the cytokine response to septic peritonitis revealed that IFNARI deficiency strongly attenuated late, but not early, hyperinflammation. In accordance with these findings, bacterial clearance and overall survival of IFNARI(-/-) mice were improved. Therefore, the present study reveals critical functions of the type I IFN pathway during severe mixed bacterial infections leading to sepsis. The results suggest that type I IFN exerts predominantly adverse effects under these conditions.     

Rapid up-regulation of CXC chemokines in the airways after Ag-specific CD4+ T cell activation

Ag-specific activation of CD4(+) T cells is known to be causative for the cytokine production associated with lung allergy. Chemokine-induced leukocyte recruitment potentially represents a critical early event in Ag-induced lung inflammation. Whether Ag-specific, lung CD4(+) T cell activation is important in lung chemokine production is currently not clear. Using alphabeta-TCR transgenic BALB/c DO11.10 mice, we investigated the ability of Ag-specific CD4(+) T cell activation to induce lung chemokine production and leukocyte recruitment. Within 1 h of exposure of DO11. 10 mice to OVA aerosol, lung mRNA and protein for the neutrophil chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly increased. Accordingly, neutrophils in the airways increased by >50-fold, and KC and MIP-2 proved to be functional because their neutralization significantly reduced airway neutrophilia. CD4(+) T cell activation was critical because CD4(+) but not CD8(+) T cell depletion reduced KC production, which correlated well with the previously observed inhibition of neutrophil influx after CD4(+) T cell depletion. In vitro studies confirmed that OVA-induced KC and MIP-2 production was conditional upon the interaction of CD4(+) T cells with APCs. A likely secondary mediator was TNF-alpha, and a probable source of these chemokines in the lung was alveolar macrophages. Thus, Ag-specific CD4(+) T cell activation in the lung leads to rapid up-regulation of neutrophil chemokines and the recruitment of neutrophils to the site of Ag exposure. This may be a key early event in the pathogenesis of Ag-induced lung inflammation.     

Subunits of IgM reconstitute defective contact sensitivity in B-1 cell-deficient xid mice: kappa light chains recruit T cells independent of complement

The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP kappa L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while kappa L chains act via another C-independent but mast cell-dependent pathway.     

Overlapping roles for L-selectin and P-selectin in antigen-induced immune responses in the microvasculature

Although L-selectin mediates lymphocyte attachment to endothelial venules of peripheral lymph nodes, its role in leukocyte recruitment into tissues following Ag challenge is less well established. The objective of this study was to systematically examine the role of L-selectin in leukocyte rolling in the peripheral microvasculature during the first 24 h of an immune response. A type I hypersensitivity response was elicited in wild-type (C57BL/6) and L-selectin-deficient mice by systemic (i.p.) sensitization and intrascrotal challenge with chicken egg OVA. The cremaster microcirculation was observed in untreated and sensitized mice 4, 8, and 24 h post-Ag challenge by intravital microscopy. Leukocyte recruitment in L-selectin-deficient mice and wild-type mice treated with an L-selectin function-blocking mAb was examined at each time point. Ag challenge induced a significant increase in leukocyte rolling (60 cells/min/venule to approximately 300 cells/min/venule) in wild-type mice at 4-24 h. This response was reduced by approximately 60-70% in L-selectin-deficient mice and in wild-type mice treated with an L-selectin-blocking mAb. P-selectin blockade by Ab completely inhibited leukocyte rolling at 4-24 h in wild-type animals and also blocked the residual rolling seen in L-selectin-deficient mice. Blocking E-selectin function had no effect on leukocyte rolling flux at any time point in wild-type or L-selectin-deficient mice. Despite reduced rolling, leukocyte adhesion and emigration were not measurably reduced in the L-selectin-deficient mice in this vascular bed. In conclusion, leukocyte rolling is L-selectin-dependent post-Ag challenge with L-selectin and P-selectin sharing overlapping functions.     

Subset-specific reductions in lung lymphocyte accumulation following intratracheal antigen challenge in endothelial selectin-deficient mice

We previously demonstrated induction and expression of CD62E and CD62P in the lungs of mice primed and then challenged with intratracheal (i.t.) SRBC. The current study examined accumulation of endogenous lymphocytes in the lungs of endothelial E- and P-selectin-deficient (E(-)P(-)) mice after i.t. SRBC challenge. Compared with syngeneic wild-type (wt) mice, E(-)P(-) mice showed an 85-95% decrease in CD8(+) T cells and B cells in the lungs at both early and late time points. In contrast, CD4(+) T cell accumulation was reduced by approximately 60% early, but equivalent to wt levels later. Surprisingly, many gammadelta T cells were found in lungs and blood of E(-)P(-) mice but were undetectable in the lungs and blood of wt mice. Absolute numbers of peripheral blood CD4, CD8, and B lymphocytes in E(-)P(-) mice equaled or exceeded the levels in wt mice, particularly after challenge. Trafficking studies using alphabeta T lymphoblasts confirmed that the recruitment of circulating cells after challenge was markedly reduced in E(-)P(-) mice. Furthermore, Ag priming occurred normally in both the selectin-deficient and wt mice, because primed lymphocytes from both groups transferred Ag sensitivity into naive wt mice. Lung production of mRNA for six CC and two CXC chemokines after challenge was equivalent by RT-PCR analysis in wt and E(-)P(-) mice. Therefore, reduced lung accumulation of alphabeta T cells and B cells in E(-)P(-) mice did not result from reduced delivery of circulating lymphocytes to the lungs, unsuccessful Ag priming, or defective pulmonary chemokine production. Selectin-dependent lymphocyte recruitment into the lungs following i.t.-SRBC challenge is subset specific and time dependent.