TLR5 Activation Exacerbates Airway Inflammation in Asthma.

INTRODUCTION: Innate immune activation through exposure to indoor and outdoor pollutants is emerging as an important determinant of asthma severity. For example, household levels of the bacterial product lipopolysaccharide (LPS) are associated with increased asthma severity. We hypothesized that activation of the innate immune receptor TLR5 by its bacterial ligand flagellin will exacerbate airway inflammation and asthma symptoms. METHODS: We determined the effect of flagellin co-exposure with ovalbumin in a murine model of allergic asthma. We evaluated the presence of flagellin activity in house dust of asthma patients. Finally, we analyzed the association of a dominant-negative polymorphism in TLR5 (rs5744168) with asthma symptoms in patients with asthma. RESULTS: We showed that bacterial flagellin can be found in the house dust of patients with asthma and that this bacterial product exacerbates allergic airway inflammation in an allergen-specific mouse model of asthma. Furthermore, a dominant-negative genetic polymorphism in TLR5, the receptor for flagellin, is associated with decreased symptoms in patients with asthma. CONCLUSION: Together, our results reveal a novel genetic protective factor (TLR5 deficiency) and a novel environmental pollutant (microbial flagellin) that influence asthma severity. (Clinical trials NCT01688986 and NCT01087307).

MyD88-dependent dendritic and epithelial cell crosstalk orchestrates immune responses to allergens.

Sensitization to inhaled allergens is dependent on activation of conventional dendritic cells (cDCs) and on the adaptor molecule, MyD88. However, many cell types in the lung express Myd88, and it is unclear how signaling in these different cell types reprograms cDCs and leads to allergic inflammation of the airway. By combining ATAC-seq with RNA profiling, we found that MyD88 signaling in cDCs maintained open chromatin at select loci even at steady state, allowing genes to be rapidly induced during allergic sensitization. A distinct set of genes related to metabolism was indirectly controlled in cDCs through MyD88 signaling in airway epithelial cells (ECs). In mouse models of asthma, Myd88 expression in ECs was critical for eosinophilic inflammation, whereas Myd88 expression in cDCs was required for Th17 cell differentiation and consequent airway neutrophilia. Thus, both cell-intrinsic and cell-extrinsic MyD88 signaling controls gene expression in cDCs and orchestrates immune responses to inhaled allergens.

Distinct Tlr4-expressing cell compartments control neutrophilic and eosinophilic airway inflammation.

Allergic asthma is a chronic, inflammatory lung disease. Some forms of allergic asthma are characterized by T helper type 2 (Th2)-driven eosinophilia, whereas others are distinguished by Th17-driven neutrophilia. Stimulation of Toll-like receptor 4 (TLR4) on hematopoietic and airway epithelial cells (AECs) contributes to the inflammatory response to lipopolysaccharide (LPS) and allergens, but the specific contribution of TLR4 in these cell compartments to airway inflammatory responses remains poorly understood. We used novel, conditionally mutant Tlr4(fl/fl) mice to define the relative contributions of AEC and hematopoietic cell Tlr4 expression to LPS- and allergen-induced airway inflammation. We found that Tlr4 expression by hematopoietic cells is critical for neutrophilic airway inflammation following LPS exposure and for Th17-driven neutrophilic responses to the house dust mite (HDM) lysates and ovalbumin (OVA). Conversely, Tlr4 expression by AECs was found to be important for robust eosinophilic airway inflammation following sensitization and challenge with these same allergens. Thus, Tlr4 expression by hematopoietic and airway epithelial cells controls distinct arms of the immune response to inhaled allergens.

Trif-dependent induction of Th17 immunity by lung dendritic cells.

Allergic asthma is thought to stem largely from maladaptive T helper 2 (Th2) responses to inhaled allergens, which in turn lead to airway eosinophilia and airway hyperresponsiveness (AHR). However, many individuals with asthma have airway inflammation that is predominantly neutrophilic and resistant to treatment with inhaled glucocorticoids. An improved understanding of the molecular basis of this form of asthma might lead to improved strategies for its treatment. Here, we identify novel roles of the adaptor protein, TRIF (TIR-domain-containing adapter-inducing interferon-ß), in neutrophilic responses to inhaled allergens. In different mouse models of asthma, Trif-deficient animals had marked reductions in interleukin (IL)-17, airway neutrophils, and AHR compared with wild-type (WT) mice, whereas airway eosinophils were generally similar in these two strains. Compared with lung dendritic cells (DCs) from WT mice, lung DCs from Trif-deficient mice displayed impaired lipopolysaccharide (LPS)-induced migration to regional lymph nodes, lower levels of the costimulatory molecule, CD40, and produced smaller amounts of the T helper 17 (Th17)-promoting cytokines, IL-6, and IL-1ß. When cultured with allergen-specific, naive T cells, Trif-deficient lung DCs stimulated robust Th2 cell differentiation but very weak Th1 and Th17 cell differentiation. Together, these findings reveal a TRIF-CD40-Th17 axis in the development of IL-17-associated neutrophilic asthma.

Migratory properties of pulmonary dendritic cells are determined by their developmental lineage.

The chemokine receptor, CCR7, directs the migration of dendritic cells (DCs) from peripheral tissue to draining lymph nodes (LNs). However, it is unknown whether all pulmonary DCs possess migratory potential. Using novel Ccr7(gfp) reporter mice, we found that Ccr7 is expressed in CD103⁺ and a CD14(med/lo) subset of CD11b(hi) classical (c)DCs but not in monocyte-derived (mo)DCs, including Ly-6C(hi)CD11b(hi) inflammatory DCs and CD14(hi)CD11b(hi) DCs. Consequently, cDCs migrated to lung-draining LNs but moDCs did not. Mice lacking the chemokine receptor, CCR2, also lacked inflammatory DCs in the lung after lipopolysaccharide inhalation but retained normal levels of migratory DCs. Conversely, the lungs of fms-like tyrosine kinase 3 ligand (Flt3L)-deficient mice lacked cDCs but retained moDCs, which were functionally mature but did not express Ccr7 and were uniformly non-migratory. Thus, the migratory properties of pulmonary DCs are determined by their developmental lineage.

Pulmonary CD103(+) dendritic cells prime Th2 responses to inhaled allergens.

Allergic asthma stems largely from the actions of T helper 2 (Th2) cells, but the pathways that initiate Th2 responses to inhaled allergens are not fully understood. In the lung, there are two major subsets of dendritic cells (DCs), displaying CD11b or CD103. We found that after taking up inhaled ovalbumin in vivo, purified CD103(+) DCs from the lung or lung-draining lymph nodes primed Th2 differentiation ex vivo. Th2 induction by CD103(+) DCs was also seen when cockroach or house dust mite allergens were used. In contrast, CD11b(hi) DCs primed Th1 differentiation. Moreover, mice lacking CD103(+) DCs displayed diminished Th2 priming to various inhaled allergens and did not develop asthma-like responses following subsequent allergen challenge. Low-level antigen presentation by CD103(+) DCs was necessary, but not sufficient for Th2 priming. Together, these findings show that CD103(+) DCs have a significant role in priming Th2 responses to inhaled allergens.

Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity.

We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

Tachykinin NK3-receptor deficiency does not inhibit pulmonary eosinophilia in allergic mice.

Tachykinins are important in the development of pulmonary inflammation in mice but the tachykinin receptor subtype mediating this response has not been defined. To elucidate the role of tachykinin NK3-receptors on allergen-induced pulmonary inflammation, studies were performed on ovalbumin (OVA) sensitized and challenged mice with genetic disruption of the tachykinin NK3-receptor (NK3-/-). Aerosol OVA (0.5%) challenge produced eosinophil influx into the bronchoalveolar lavage fluid and lung tissue, goblet cell hyperplasia and damage to the airway epithelium of both NK3-/- mice and in wild type control mice (NK3+/+). There was no difference in the magnitude of these allergic inflammatory pulmonary responses between NK3-/- and NK3+/+ mice. These results find no role for tachykinin NK3-receptors on the pulmonary eosinophilia and lung damage after antigen challenge in mice.

Macrophage inflammatory protein-1alpha uses a novel receptor for primitive hemopoietic cell inhibition.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of the chemokine family of proinflammatory mediators. In addition to its inflammatory roles, MIP-1alpha has been shown to be active as an inhibitor of primitive hemopoietic cell proliferation. Indeed, a dysfunction in this inhibitory process has been postulated to contribute to leukemogenesis. Research has been aimed at characterizing the receptor involved in cellular inhibition by MIP-1alpha. This study demonstrates that of all the beta-chemokines tested, only MIP-1alpha is capable of inhibiting primitive hemopoietic cell proliferation. Because no MIP-1alpha-specific receptors have been identified, this suggests that inhibition is mediated by an uncharacterized receptor. Further evidence for the involvement of a novel receptor in this process is the equivalent potencies of MIP-1alphaS and MIP-1alphaP variants of human MIP-1alpha and the fact that primitive cells from bone marrow derived from individual MIP-1alpha receptor null mice display a full response to MIP-1alpha inhibition.

Regulatory effects of macrophage inflammatory protein 1alpha/CCL3 on the development of immunity to Cryptococcus neoformans depend on expression of early inflammatory cytokines.

Macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3 prevents the development of eosinophilic pneumonia (EP) driven by a nonprotective T2-type immunity during infection with a highly virulent strain of Cryptococcus neoformans. The present study evaluated the interaction of MIP-1alpha with other innate immune system cytokines by comparing the immune responses that followed pulmonary infections with high- (C. neoformans 145A) and low (C. neoformans 52D)-virulence strains. In contrast to what was found for C. neoformans 145A infection, lack of MIP-1alpha in C. neoformans 52D infection did not cause the development of EP. C. neoformans 52D induced tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), and MCP-1 in the lungs of infected wild-type (WT) and MIP-1alpha knockout (KO) mice by day 7 postinfection. Both WT and MIP-1alpha KO mice subsequently cleared this infection. Thus, the robust expression of early inflammatory cytokines in C. neoformans 52D-infected mice promoted the development of protective immunity even in the absence of MIP-1alpha. Alternatively, C. neoformans 145A-infected WT and MIP-1alpha KO mice had diminished TNF-alpha, IFN-gamma, and macrophage chemoattractant protein 1 (MCP-1) responses, indicating that virulent C. neoformans 145A evaded early innate host defenses. However C. neoformans 145A-infected WT mice had an early induction of MIP-1alpha and subsequently did not develop EP. In contrast, C. neoformans 145A-infected MIP-1alpha KO mice developed EP and had increased C. neoformans dissemination into the brain by day 35. We conclude that, in the absence of other innate immune response effector molecules, MIP-1alpha is crucial to prevent the development of EP and to control C. neoformans dissemination to the brain.

Requirement for the chemokine receptor CCR6 in allergic pulmonary inflammation.

Allergic asthmatic responses in the airway are associated with airway hyperreactivity, eosinophil accumulation in the lung, and cytokine production by allergen-specific, T helper cell type 2 (Th2) lymphocytes. Here, we show that in a cockroach antigen (CA) model of allergic pulmonary inflammation, the chemokine macrophage inflammatory protein (MIP)-3alpha is expressed in the lung within hours of allergen challenge. To determine the biologic relevance of this expression, mice lacking CCR6, the only known receptor for MIP-3alpha, were studied for their response to CA. CCR6-deficient mice were immunized to the same extent as their wild-type counterparts, as judged by cytokine production in antigen-challenged lymphocytes. However, compared with CA-challenged wild-type mice, challenged CCR6-deficient mice had reduced airway resistance, fewer eosinophils around the airway, lower levels of interleukin 5 in the lung, and reduced serum levels of immunoglobulin E. Together, these data demonstrate that MIP-3alpha and CCR6 function in allergic pulmonary responses and suggest that these molecules might represent novel therapeutic targets for treatment of asthma.

Wound healing in MIP-1alpha(-/-) and MCP-1(-/-) mice.

A salient feature of normal wound healing is the development and resolution of an acute inflammatory response. Although much is known about the function of inflammatory cells within wounds, little is known about the chemotactic and activation signals that influence this response. As the CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1) are abundant in acute wounds, wound repair was examined in MIP-1alpha(-/-) and MCP-1(-/-) mice. Surprisingly, wound re-epithelialization, angiogenesis, and collagen synthesis in MIP-1alpha(-/-) mice was nearly identical to wild-type controls. In contrast, MCP-1(-/-) mice displayed significantly delayed wound re-epithelialization, with the greatest delay at day 3 after injury (28 +/- 5% versus 79 +/- 14% re-epithelialization, P < 0.005). Wound angiogenesis was also delayed in MCP-1(-/-) mice, with a 48% reduction in capillary density at day 5 after injury. Collagen synthesis was impeded as well, with the wounds of MCP-1(-/-) mice containing significantly less hydroxyproline than those of control mice (25 +/- 3 versus 50 +/- 8 microg/wound at day 5, P < 0.0001). No change in the number of wound macrophages was observed in MCP-1(-/-) mice, suggesting that monocyte recruitment into wounds is independent of this chemokine. The data suggest that MCP-1 plays a critical role in healing wounds, most likely by influencing the effector state of macrophages and other cell types.

Absence of macrophage-inflammatory protein-1alpha delays central nervous system demyelination in the presence of an intact blood-brain barrier.

Chemokines are small chemotactic cytokines that modulate leukocyte recruitment and activation during inflammation. Here, we describe the role of macrophage inflammatory protein-1alpha (MIP-1alpha) during cuprizone intoxication, a model where demyelination of the CNS features a large accumulation of microglia/macrophage without T cell involvement or blood-brain barrier disruption. RNase protection assays showed that mRNA for numerous chemokines were up-regulated during cuprizone treatment in wild-type, C57BL/6 mice. RANTES, inflammatory protein-10, and monocyte chemoattractant protein-1 showed greatest expression with initiation of insult at 1-2 wk of treatment, whereas MIP-1alpha and beta increased later at 4-5 wk, coincident with peak demyelination and cellular accumulation. The function of MIP-1alpha during demyelination was tested in vivo by exposing MIP-1alpha knockout mice (MIP-1alpha(-/-)) to cuprizone and comparing pathology to wild-type mice. Demyelination at 3.5 wk of treatment was significantly decreased in MIP-1alpha(-/-) mice ( approximately 36% reduction), a result confirmed by morphology at the electron microscopic level. The delay in demyelination was correlated to apparent decreases in microglia/macrophage and astrocyte accumulation and in TNF-alpha protein levels. It was possible that larger effects of the MIP-1alpha deficiency were being masked by other redundant chemokines. Indeed, RNase protection assays revealed increased expression of several chemokine transcripts in both untreated and cuprizone-treated MIP-1alpha(-/-) mice. Nonetheless, despite this possible compensation, our studies show the importance of MIP-1alpha in demyelination in the CNS and highlight its effect, particularly on cellular recruitment and cytokine regulation.

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs.

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.

Generation and analysis of mice lacking the chemokine fractalkine.

Fractalkine (CX(3)CL1) is the first described chemokine that can exist either as a soluble protein or as a membrane-bound molecule. Both forms of fractalkine can mediate adhesion of cells expressing its receptor, CX(3)CR1. This activity, together with its expression on endothelial cells, suggests that fractalkine might mediate adhesion of leukocytes to the endothelium during inflammation. Fractalkine is also highly expressed in neurons, and its receptor, CX(3)CR1, is expressed on glial cells. To determine the biologic role of fractalkine, we used targeted gene disruption to generate fractalkine-deficient mice. These mice did not exhibit overt behavioral abnormalities, and histologic analysis of their brains did not reveal any gross changes compared to wild-type mice. In addition, these mice had normal hematologic profiles except for a decrease in the number of blood leukocytes expressing the cell surface marker F4/80. The cellular composition of their lymph nodes did not differ significantly from that of wild-type mice. Similarly, the responses of fractalkine(-/-) mice to a variety of inflammatory stimuli were indistinguishable from those of wild-type mice.

Aberrant in vivo T helper type 2 cell response and impaired eosinophil recruitment in CC chemokine receptor 8 knockout mice.

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (-/-) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8(-/)- mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.

Impaired pulmonary host defense in mice lacking expression of the CXC chemokine lungkine.

Lungkine (CXCL15) is a novel CXC chemokine that is highly expressed in the adult mouse lung. To determine the biologic function of Lungkine, we generated Lungkine null mice by targeted gene disruption. These mice did not differ from wild-type mice in their hematocrits or in the relative number of cells in leukocyte populations of peripheral blood or other tissues, including lung and bone marrow. However, Lungkine null mice were more susceptible to Klebsiella pneumonia infection, with a decreased survival and increased lung bacterial burden compared with infected wild-type mice. Histologic analysis of the lung and assessment of leukocytes in the bronchioalveolar lavage revealed that neutrophil numbers were normal in the lung parenchyma, but reduced in the airspace. The production of other neutrophil chemoattractants in the Lungkine null mice did not differ from that in wild-type mice, and neutrophil migration into other tissues was normal. Taken together, these findings demonstrate that Lungkine is an important mediator of neutrophil migration from the lung parenchyma into the airspace.

Leukocytes expressing green fluorescent protein as novel reagents for adoptive cell transfer and bone marrow transplantation studies.

Transgenic mice expressing green fluorescent protein (GFP) were generated to provide a source of labeled leukocytes for cell transfer studies. The transgene comprises the GFP coding region under the transcriptional control of the chicken ss-actin promoter and human cytomegalovirus enhancer. Mice expressing this GFP transgene were generated in the B6D2 and in the 129SvEv backgrounds. Flow cytometric analysis of cells from the blood, spleen, and bone marrow of these transgenic mice revealed that most leukocytes, including dendritic cells and memory T cells, express GFP. In allogeneic cell transfers, donor GFP+ splenocytes were detected in the spleen and mesenteric lymph nodes of recipient mice within 2 hours after transfer and for at least 9 days thereafter. In syngeneic experiments using 129-derived GFP+ donor splenocytes, donor cells were detected in multiple tissues of 129 recipients from 2 hours to 3 weeks after transfer. In bone-marrow transplantation experiments using irradiated allogeneic recipients, the percent of GFP+ donor cells in recipients at 3 weeks was comparable to that seen in similar tissues of GFP+ donor mice. These data demonstrate that GFP+ transgenic mice provide a ready source of GFP-expressing primary cells that can be easily monitored after their transfer to recipient animals.

The role of macrophage inflammatory protein-1 alpha/CCL3 in regulation of T cell-mediated immunity to Cryptococcus neoformans infection.

Macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) is a CC chemokine required for optimal recruitment of leukocytes in response to cryptococcal Ags. MIP-1alpha is expressed in the lungs by day 6 post Cryptococcus neoformans infection and could play a role in the development of cell-mediated immunity. To address this possibility, wild-type (MIP-1alpha(+/+)) mice and MIP-1alpha knockout (MIP-1alpha(-/-)) mice were infected intratracheally with a highly virulent strain of C. neoformans (145A). MIP-1alpha message was detected in the lungs on days 3, 7, and 14 in MIP-1alpha(+/+) mice, but it was undetectable in MIP-1alpha(-/-) mice. On day 16, MIP-1alpha(-/-) mice had a 7-fold increase in C. neoformans burden in the lungs, but no decrease in pulmonary leukocyte recruitment. MIP-1alpha(+/+) and MIP-1alpha(-/-) mice had similar numbers of recruited lymphocytes and monocytes/macrophages. Notably, MIP-1alpha(-/-) mice had a significantly greater number of eosinophils. MIP-1alpha(-/-) mice had extremely high levels of serum IgE. This switch of immune response to a T(2) phenotype was associated with enhanced IL-4 and IL-13 expression in the lungs of MIP-1alpha(-/-) mice compared with MIP-1alpha (+/+) mice. Progression of pulmonary cryptococcosis in the presence of nonprotective T(2) immunity resulted in profound lung damage in MIP-1alpha(-/-) mice (eosinophilic crystal deposition, destruction of lung parenchyma, and pulmonary hemorrhage). Twelve-week survival was dramatically decreased in MIP-1alpha(-/-) mice. These studies, together with our previous studies, demonstrate that MIP-1alpha plays a role in both the afferent (T(1)/T(2) development) and efferent (T(1)-mediated leukocyte recruitment) phases of cell-mediated immunity to C. neoformans.

T-lymphocyte production of macrophage inflammatory protein-1alpha is critical to the recruitment of CD8(+) T cells to the liver, lung, and spleen during graft-versus-host disease.

To investigate the mechanism by which macrophage inflammatory protein-1alpha (MIP-1alpha) affects graft-versus-host disease (GVHD), the expression and function of MIP-1alpha in 2 murine models of GVHD were evaluated. In irradiated class I and class II disparate recipients, the expression of messenger RNA (mRNA) and protein for MIP-1alpha was significantly increased in GVHD target organs after transfer of allogeneic lymphocytes compared to syngeneic lymphocytes. When lymphocytes unable to make MIP-1alpha were transferred, there was a decrease in the production of MIP-1alpha in the liver, lung, and spleen of bm1 (B6.C-H2(bm1)/By) and bm12 (B6.C-H2(bm12)/KhEg) recipients compared to the transfer of wild-type splenocytes. At day 6 there was a 4-fold decrease in the number of transferred CD8(+) T cells in the lung and approximately a 2-fold decrease in the number of CD8(+) T cells in the liver and spleen in bm1 recipients after transfer of MIP-1alpha-deficient (MIP-1alpha(-/-)) splenocytes compared to wild-type (MIP-1alpha(+/+)) splenocytes. These differences persisted for 13 days after splenocyte transfer. In contrast, the number of donor CD4(+) T cells found in the liver and lung was significantly increased after the transfer of MIP-1alpha(-/-) compared to wild-type splenocytes in bm12 recipients from day 6 through day 10. Thus, the transfer of allogeneic T cells was associated with the enhanced expression of MIP-1alpha in both a class I and class II mismatch setting. However, the increased expression only led to enhanced recruitment of CD8(+), but not CD4(+), donor T cells. Production of MIP-1alpha by donor T cells is important in the occurrence of GVHD and functions in a tissue-dependent fashion.