Macrophage inflammatory protein-1alpha expression plasmid enhances DNA vaccine-induced immune responses against HSV-2

In this study, we examined the effectiveness of macrophage inflammatory protein (MIP)-1alpha cDNA as a HSV-2 DNA vaccine adjuvant. pcDNA3-gD (pgD) and pcDNA3-MIP-1alpha (pMIP-1alpha) were co-injected to examine the modulatory effects of MIP-1alpha on immune phenotype and protection against lethal challenge with HSV-2. We found that Th-cell proliferative responses were dramatically enhanced by co-injection of pgD and pMIP-1alpha compared with injection of pgD alone. The secretion of IL-2 and IFN-gamma was also significantly increased by pgD and pMIP-1alpha co-injection; however, the production of cytokines IL-4 and IL-10 was not affected by co-injection. pgD and pMIP-1alpha co-injection resulted in a moderate enhancement of systemic gD-specific antibody level, but mucosal secretory IgA was markedly enhanced. When BALB/c mice were challenged intravaginally with 100 LD50 of HSV-2 strain Sav, pMIP-1alpha co-injection with pgD improved their survival rate and significantly reduced both the number of mice with lesions and the lesion severity. Therefore, MIP-1alpha cDNA as a HSV-2 DNA vaccine adjuvant drives antigen-specific Th1-type responses, reducing HSV-2-derived morbidity and mortality.     

CCL3 (MIP-1alpha) induces in vitro migration of GM-CSF-primed human neutrophils via CCR5-dependent activation of ERK 1/2

CCL3 (MIP-1alpha), a prototype of CC chemokines, is a potent chemoattractant toward human neutrophils pre-treated with GM-CSF for 15 min. GM-CSF-treated neutrophils migrate also to the selective CCR5 agonist CCL4 (MIP-1beta). CCL3- and CCL4-triggered migration of GM-CSF-primed neutrophils was inhibited by the CCR5 antagonist TAK-779. Accordingly, freshly isolated neutrophils express CCR5. Extracellular signal-regulated kinases (ERK)-1/2 and p38 mitogen-activated protein kinase (MAPK) inhibitors blocked CCL3-induced migration of GM-CSF-primed neutrophils. When the activation of ERK-1/2 and p38 MAPK by CCL3 and the classical neutrophilic chemokine CXCL8 (IL-8) were compared, both the chemokines were capable of activating p38 MAPK. On the contrary, whereas both ERK-1 and ERK-2 were activated by CXCL8, no ERK-1 band was detectable after CCL3 triggering. Finally, neutrophil pre-treatment with GM-CSF activated both ERK-1 and ERK-2. This suggests that by activating ERK-1, GM-CSF renders neutrophils rapidly responsive to CCL3 stimulation throughout CCR5 which is constitutively expressed on the cell surface.     

Antigen receptor engagement selectively induces macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta chemokine production in human B cells

We show herein that B cell Ag receptor (BCR) triggering, but not stimulation by CD40 mAb and/or IL-4, rapidly induced the coordinated expression of two closely related T cell chemoattractants, macrophage inflammatory protein-1 beta (MIP-1 beta) and MIP-1 alpha, by human B cells. Naive, memory, and germinal center B cells all produced MIP-1 alpha/beta in response to BCR triggering. In contrast to MIP-1 alpha/beta, IL-8, which is spontaneously produced by germinal center B cells but not by naive and memory B cells, was not regulated by BCR triggering. Culturing follicular dendritic cell-like HK cells with activated B cells did not regulate MIP-1 alpha/beta production, but it did induce production of IL-8 by HK cells. Microchemotaxis assays showed that CD4+CD45RO+ T cells of the effector/helper phenotype actively migrated along a chemotactic gradient formed by BCR-stimulated B cells. This effect was partially blocked by anti-MIP-1 beta and anti-CC chemokine receptor 5 Ab, but not by anti-MIP-1 alpha Ab suggesting that MIP-1 beta plays a major role in this chemoattraction. Since maturation of the B cell response to a peptide Ag is mostly dependent on the availability of T cell help, the ability of Ag-stimulated B cells to recruit T cells via MIP-1 alpha/beta, may represent one possible mechanism enabling cognate interactions between rare in vivo Ag-specific T and B cells.     

The role of MIP-1 alpha in the development of systemic inflammatory response and organ injury following trauma hemorrhage

Although MIP-1alpha is an important chemokine in the recruitment of inflammatory cells, it remains unknown whether MIP-1alpha plays any role in the development of systemic inflammatory response following trauma-hemorrhage (T-H). C57BL/6J wild type (WT) and MIP-1alpha-deficient (KO) mice were used either as control, subjected to sham operation (cannulation or laparotomy only or cannulation plus laparotomy) or T-H (midline laparotomy, mean blood pressure 35 +/- 5 mmHg for 90 min, followed by resuscitation) and sacrificed 2 h thereafter. A marked increase in serum alpha-glutathione transferase, TNF-alpha, IL-6, IL-10, MCP-1, and MIP-1alpha and Kupffer cell cytokine production was observed in WT T-H mice compared with shams or control. In addition lung and liver tissue edema and neutrophil infiltration (myeloperoxidase (MPO) content) was also increased following T-H in WT animals. These inflammatory markers were markedly attenuated in the MIP-1alpha KO mice following T-H. Furthermore, compared with 2 h, MPO activities at 24 and 48 h after T-H declined steadily in both WT and KO mice. However, normalization of MPO activities to sham levels within 24 h was seen in KO mice but not in WT mice. Thus, MIP-1alpha plays an important role in mediating the acute inflammatory response following T-H. In the absence of MIP-1alpha, acute inflammatory responses were attenuated; rapidly recovered and less remote organ injury was noted following T-H. Thus, interventions that reduce MIP-1alpha levels following T-H should be useful in decreasing the deleterious inflammatory consequence of trauma.     

DNA vaccines encoding interleukin-8 and RANTES enhance antigen-specific Th1-type CD4(+) T-cell-mediated protective immunity against herpes simplex virus type 2 in vivo

Chemokines are inflammatory molecules that act primarily as chemoattractants and as activators of leukocytes. Their role in antigen-specific immune responses is of importance, but their role in disease protection is unknown. Recently it has been suggested that chemokines modulate immunity along more classical Th1 and Th2 phenotypes. However, no data currently exist in an infectious challenge model system. We analyzed the modulatory effects of selected chemokines (interleukin-8 [IL-8], gamma interferon-inducible protein 10 [IP-10], RANTES, monocyte chemotactic protein 1 [MCP-1], and macrophage inflammatory protein 1 alpha [MIP-1 alpha]) on immune phenotype and protection against lethal challenge with herpes simplex virus type 2 (HSV-2). We observed that coinjection with IL-8 and RANTES plasmid DNAs dramatically enhanced antigen-specific Th1 type cellular immune responses and protection from lethal HSV-2 challenge. This enhanced protection appears to be mediated by CD4(+) T cells, as determined by in vitro and in vivo T-cell subset deletion. Thus, IL-8 and RANTES cDNAs used as DNA vaccine adjuvants drive antigen-specific Th1 type CD4(+) T-cell responses, which result in reduced HSV-2-derived morbidity, as well as reduced mortality. However, coinjection with DNAs expressing MCP-1, IP-10, and MIP-1 alpha increased mortality in the challenged mice. Chemokine DNA coinjection also modulated its own production as well as the production of cytokines. These studies demonstrate that chemokines can dominate and drive immune responses with defined phenotypes, playing an important role in the generation of protective antigen-specific immunity.     

The chemokine macrophage-inflammatory protein-1 alpha and its receptor CCR1 control pulmonary inflammation and antiviral host defense in paramyxovirus infection

In this work, we explore the responses of specific gene-deleted mice to infection with the paramyxovirus pneumonia virus of mice (PVM). We have shown previously that infection of wild type mice with PVM results in pulmonary neutrophilia and eosinophilia accompanied by local production of macrophage-inflammatory protein-1 alpha (MIP-1 alpha). Here we examine the role of MIP-1 alpha in the pathogenesis of this disease using mice deficient in MIP-1 alpha or its receptor, CCR1. The inflammatory response to PVM in MIP-1 alpha-deficient mice was minimal, with approximately 10-60 neutrophils/ml and no eosinophils detected in bronchoalveolar lavage fluid. Higher levels of infectious virus were recovered from lung tissue excised from MIP-1 alpha-deficient than from fully competent mice, suggesting that the inflammatory response limits the rate of virus replication in vivo. PVM infection of CCR1-deficient mice was also associated with attenuated inflammation, with enhanced recovery of infectious virus, and with accelerated mortality. These results suggest that the MIP-1 alpha/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of infection.     

CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A

The angiogenic activity of CXC-ELR(+) chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-alpha in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck(-/-)fgr(-/-)), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck(-/-)fgr(-/-) mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck(-/-)fgr(-/-) mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck(-/-)fgr(-/-) mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.     

Induction of the chemokine beta peptides, MIP-1 alpha and MIP-1 beta, by lipopolysaccharide is differentially regulated by immunomodulatory cytokines gamma-IFN, IL-10, IL-4, and TGF-beta.

The macrophage occupies a central role in the host response to invasion, exerting its control over the developing inflammatory response largely through the elaboration of an assortment of endogenous mediators including many cytokines. The beta chemokine peptides, macrophage inflammatory protein [MIP]-1 alpha and MIP-1 beta, are two such effectors markedly up-regulated in macrophages following exposure to bacterial lipopolysaccharide (LPS). These highly homologous peptides, like the other members of the beta chemokine family, exhibit diverse but partially overlapping biological activity profiles, suggesting that the cellular participants and intensity of an inflammatory response may in part be regulated by selective expression of these chemokines. Studies reported here demonstrate that, in contrast to the "balanced" MIP-1 alpha/MIP-1 beta chemokine responses of LPS-stimulated macrophage cultures in vitro, circulating levels of MIP-1 beta are significantly higher than those of MIP-1 alpha following LPS administration in vivo. Further studies have revealed that several immunomodulatory cytokines known to be up-regulated in vivo as a consequence of exposure to an invasive stimulus (gamma-IFN, IL-10, IL-4, and transforming growth factor [TGF]-beta) down-regulated the LPS-induced release of MIP-1 alpha by macrophages in vitro, but spared the MIP-1 beta response. This altered pattern of secretion may explain, at least in part, the high circulating levels of MIP-1 beta relative to MIP-1 alpha observed in vivo in response to LPS challenge.     

Biological and structural properties of MIP-1 alpha expressed in yeast.

The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).     

Increased expression of MIP-1 alpha and MIP-1 beta mRNAs in the brain correlates spatially and temporally with the spongiform neurodegeneration induced by a murine oncornavirus

The chimeric murine oncornavirus FrCas(E) causes a rapidly progressive paralytic disease associated with spongiform neurodegeneration throughout the neuroaxis. Neurovirulence is determined by the sequence of the viral envelope gene and by the capacity of the virus to infect microglia. The neurocytopathic effect of this virus appears to be indirect, since the cells which degenerate are not infected. In the present study we have examined the possible role of inflammatory responses in this disease and have used as a control the virus F43. F43 is an highly neuroinvasive but avirulent virus which differs from FrCas(E) only in 3' pol and env sequences. Like FrCas(E), F43 infects large numbers of microglial cells, but it does not induce spongiform neurodegeneration. RNAase protection assays were used to detect differential expression of genes encoding a variety of cytokines, chemokines, and inflammatory cell-specific markers. Tumor necrosis factor alpha (TNF-alpha) and TNF-beta mRNAs were upregulated in advanced stages of disease but not early, even in regions with prominent spongiosis. Surprisingly there was no evidence for upregulation of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6 or of the microglial marker F4/80 at any stage of this disease. In contrast, increased levels of the beta-chemokines MIP-1 alpha and -beta were seen early in the disease and were concentrated in regions of the brain rich in spongiosis, and the magnitude of responses was similar to that observed in the brains of mice injected with the glutamatergic neurotoxin ibotenic acid. MIP-1alpha and MIP-1beta mRNAs were also upregulated in F43-inoculated mice, but the responses were three- to fivefold lower and occurred later in the course of infection than was observed in FrCas(E)-inoculated mice. These results suggest that the robust increase in expression of MIP-1 alpha and MIP-1 beta in the brain represents a correlate of neurovirulence in this disease, whereas the TNF responses are likely secondary events.     

Cytokine and chemokine mRNA expression in neutrophils from CBA/NSlc mice infected with Plasmodium berghei ANKA that induces experimental cerebral malaria.

To investigate the role of neutrophils in experimental cerebral malaria (ECM), in a previous study we found that early neutrophil depletion prevented the development of ECM and down regulated the expression of Th1 cytokines in the brain. To further clarify the mechanisms responsible for these findings, in the present study, using RT-PCR, we examined the expression of cytokine and chemokine mRNAs in neutrophils and macrophages after PbA infection. We found that, after infection, neutrophils not only expressed cytokines IL-2, IL-12p40, IL-18, IFN-gamma and TNF-alpha mRNAs, but also mRNAs for Th1 chemoattractive chemokines, monokine-induced by IFN-gamma (MIG), macrophage-inflammatory protein-1alpha (MIP-1alpha) and IFN-gamma inducible protein-10 (IP-10). Neutrophil depletion down regulated the expression of IL-18 and MIG mRNAs in macrophages, but did not affect the expression of IFN-gamma, TNF-alpha, MIP-1alpha and IP-10 mRNAs. Therefore, this study confirms our hypothesis that neutrophils may play a role in the pathogenesis of ECM via their expression of cytokines or chemokines.     

Neutrophils promote inflammatory angiogenesis via release of preformed VEGF in an in vivo corneal model

We investigated the role of neutrophilic cells (neutrophils) in inflammatory angiogenesis and explored the possible mechanisms involved. Corneal angiogenesis was induced in vivo with a 75% silver nitrate applicator. Depletion of neutrophils was accomplished by the intraperitoneal administration of RB6-8C5, a neutrophil-depleting antibody. Angiogenesis, neutrophil infiltration, and the localization of vascular endothelial growth factor (VEGF) were evaluated by biomicroscopic observations, histology, and immunohistochemistry in control and RB6-8C5 treatment groups. Protein levels of VEGF, macrophage inflammatory protein-1alpha (MIP-1α), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor alpha in the cornea were determined by enzyme-linked immunosorbent assay. An in vitro model of neutrophil activation was also used to examine the ability of neutrophils to produce and release VEGF, MIP-1α, and MIP-2. At day 1 after injury, neutrophil infiltration in the cornea was highest, and VEGF was expressed in the infiltrating neutrophils. The enhanced protein levels of VEGF, MIP-1α, and MIP-2 correlated with the degree of neutrophil infiltration. Neutrophil depletion significantly inhibited corneal angiogenesis and reduced the protein levels of VEGF, MIP-1α, and MIP-2 in the cornea. Upon stimulation, isolated neutrophils released VEGF from preformed stores and MIP-1α and MIP-2 by de novo synthesis. Neutrophil depletion thus significantly impaired inflammatory angiogenesis, identifying neutrophils as an important player in inflammatory angiogenesis. Neutrophils may exercise their angiogenic function by releasing proangiogenic factors such as VEGF. Intervention measures targeting neutrophils may therefore help to deal with abnormal angiogenesis involved in chronic inflammatory diseases.     

Distinct temporal patterns of macrophage-inflammatory protein-2 and KC chemokine gene expression in surgical injury

In the present study the regulation of CXC chemokine expression was evaluated in full-thickness abdominal wounds in mice. During the first 24 h after injury, IL-1alphabeta, KC, macrophage-inflammatory protein (MIP)-2, and monocyte chemoattractant protein-1 were the predominant cytokines and chemokines produced; TNF-alpha was not detected. Chemokine mRNA expression and protein secretion occurred in two temporal stages. The first, which reached a maximum at 6 h, was associated with high levels of IL-1alpha and KC and low levels of MIP-2. This stage could be reproduced by intradermal injection of IL-1alpha or IL-1beta and was partially blocked by injection of neutralizing Ab against IL-1alpha but not IL-1beta. In animals depleted of circulating neutrophils, chemokine expression was reduced by nearly 70% during this stage. In the second stage, which peaked at 24 h after injury, modest but significant levels of IL-1beta were detected in association with low levels of KC and high levels of MIP-2. This pattern of chemokine expression could not be mimicked by injection of IL-1alpha or IL-1beta (even with prolonged exposure), although MIP-2 expression could be partially inhibited by intradermal injection of neutralizing Ab against IL-1beta. Surprisingly, neutrophil depletion before injury resulted in sustained high levels of both KC and MIP-2 expression. These observations demonstrate that these two closely related chemokines are under distinct regulatory controls in vivo that are likely to reflect the temporally ordered participation of different cell types and/or extracellular stimuli and inhibitors.     

内源性IL-17诱导MIP-2与IL-6表达在衣原体肺炎中促进中性粒细胞循环

目的 探讨衣原体肺炎中白细胞介素-17（interleukin -17,IL-17）对中性粒细胞（polymorphonuclear leucocyte,PMN）循环的调节作用及机制.方法 用40 μl含1×10^3包涵体形成单位（inclusion-forming units,IFU）的衣原体鼠肺炎株（Chlamydia muridarum,Cm）呼吸道感染BALB/c小鼠,诱导鼠衣原体肺炎.用抗鼠IL-17单克隆抗体吸入中和内源性IL-17,以相应独特型抗体（IgG2α）作为对照.用RT-PCR检测小鼠肺组织及肺上皮细胞系巨噬细胞炎性蛋白-2 （macrophage inflammatory protein-2,MIP-2）和IL-6 mRNA的表达.取小鼠支气管肺泡灌洗液细胞染色计数PMN,感染肺组织进行病理染色.结果 衣原体肺炎中,内源性IL-17中和小鼠肺组织PMN浸润显著降低,支气管肺泡灌洗液PMN数量显著低于对照组.IL-17与TNF-α协同可上调肺上皮细胞MIP-2和IL-6 mRNA表达,且内源性IL-17中和小鼠肺组织MIP-2和IL-6表达显著降低.结论 衣原体肺炎中IL-17通过促进肺组织细胞分泌趋化性细胞因子MIP-2和前症性细胞因子IL-6,诱导PMN循环,参与宿主抗衣原体炎性应答.     

Involvement of MIP-2 and CXCR2 in neutrophil infiltration following injection of late apoptotic cells into the peritoneal cavity

Apoptotic cells are cleared by phagocytes, such as macrophages, as soon as they appear in vivo. If apoptosis occurs acutely, however, macrophages may be outnumbered by apoptotic cells, which causes late apoptosis. We previously showed that injection of late apoptotic cells into the peritoneal cavity led to transient infiltration of neutrophils. In this study, we examined the involvement of MIP-2 and CXCR2 in the neutrophil infiltration. We first produced a recombinant MIP-2 protein, and a fusion protein between CXCR2 and GST in E. coli, and then generated anti-MIP-2 antibodies and anti-CXCR2 antibodies in rabbits. We then confirmed their specificity by Western blotting analysis and flow cytometry. Injection of late apoptotic cells, such as P388 cells treated with etoposide for 24 hours and CTLL-2 cells cultured in IL-2-free medium for 28 hours, induced neutrophil infiltration into the peritoneal cavity, as expected. The antibodies, but not control antibodies against GST, suppressed the neutrophil infiltration to the level caused by injection of normal (viable) cells, suggesting that MIP-2 and CXCR2 are mainly involved in the neutrophil infiltration caused by late apoptotic cells.     

Regulation of macrophage inflammatory protein-1 alpha expression and function by endogenous interleukin-10 in a model of acute inflammation

In this study we have determined the role of endogenous interleukin (IL)-10 on leucocyte recruitment and production of the CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) in a murine model of acute inflammation. Intraperitoneal injection of zymosan produced a dose-dependent cellular infiltration which was concomitant with MIP-1alpha release in the lavage fluids. Release of this chemokine had a functional role since treatment of mice with a specific anti-MIP-1alpha antibody reduced both neutrophil and monocyte accumulation into the peritoneal cavity. An unexpected increase in cell influx and MIP-1alpha production was measured following depletion of resident peritoneal macrophages, as achieved by a 3-day liposome treatment. A similar result was obtained when the zymosan peritonitis response was elicited in IL-10 knock-out mice. In summary we propose a functional cross talk between endogenous IL-10 and this CC chemokine during the host inflammatory response.     

Cutaneous injection of human subjects with macrophage inflammatory protein-1 alpha induces significant recruitment of neutrophils and monocytes

Macrophage inflammatory protein (MIP-1 alpha), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1 alpha have been well documented, the in vivo biological activities of MIP-1 alpha in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1 alpha and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10-24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1 alpha stimulation. These studies substantiate the biological effects of MIP-1 alpha on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1 alpha to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.