Essential role of sphingosine-1-phosphate receptor 1-bearing CD8+CD44+CCR7+ T cells in acute skin allograft rejection.

A subset of naturally formed sphingosine-1-phosphate receptor 1 (S1P1)-bearing CD8(+)CD44(+)CCR7(+) memory T cells has been identified in transplant recipient BALB/c (H-2(d)) mice. The frequency of this subset of memory T cells is significantly increased in the spleen, lymph nodes and skin grafts in the recipient BALB/c mice during acute skin allograft rejections. The immune-reconstitution with CD8(+)CD44(+)CCR7(+)S1P1(+) memory T cells facilitates acute skin allograft rejection in SCID mice. Being Th1-polarized and cytotoxic, CD8(+)CD44(+)CCR7(+)S1P1(+) memory T cells proliferate and differentiate immediately into effectors upon encountering allo-antigens. A siRNA against S1P1 inhibits CD8(+)CD44(+)CCR7(+)S1P1(+) memory T cell-mediated acute skin allograft rejection in SCID mice by means of knocking-down S1P1-expression. CCL21 mutant (CCL21-DeltaCT) has been used to compete with wild-type CCL21 in the course of binding to CCR7. Combined administration of siRNA S1P1 and CCL21-DeltaCT significantly prolongs the survival of skin allograft in the recipient BALB/c mice by means of inhibiting accumulation of CD8(+)CD44(+)CCR7(+)S1P1(+) memory T cells in the spleen and the skin grafts. Our data provide direct evidence that S1P1 and CCR7 are involved in the proliferation and trafficking of CD8(+)CD44(+)CCR7(+)S1P1(+) memory T cells. S1P1 may serve as a functional marker for CD8(+)CD44(+)CCR7(+) memory T cells. Targeting CD8(+)CD44(+)CCR7(+)S1P1(+) T cells may be a useful strategy to prolong the survival of allograft transplant.

Androgen activates PEG10 to promote carcinogenesis in hepatic cancer cells.

The molecular mechanism of striking higher prevalence of hepatocellular carcinoma (HCC) in male subjects has not yet been fully elucidated. Here, we report that androgen receptor (AR) is differentially expressed in different HCC cell lines. AR agonist dihydrotestosterone (DHT) enhances HCC cell growth and apoptotic resistance. Antagonist flutamide (FLU) blocks the effects of DHT on the HCC cell lines. Paternally expressed gene 10 (PEG10) is expressed in HCC cell lines at substantial high level. Using small interfering RNAs against AR and PEG10 in AR- and PEG10-expressing BEL-7404 hepatoma cells and HuH7 hepatoma cells (HuH7) cells, and AR-transfection technique in AR-lacking and PEG10-expressing HepG2 cells, we have confirmed that through upregulation and activation of PEG10, DHT enhances HCC cell growth and apoptotic resistance. We have further demonstrated that DHT upregulates expression of human telomerase reverse transcriptase (hTERT) in HCC cell lines in a PEG10-dependent manner. Moreover, AR directly interacts in vivo with androgen-responsive elements in the regions of promoter and exon 2 of PEG10 gene in HCC cell lines. DHT promotes the hepatoma formation in vivo nude mice through PEG10 activation. AR antagonists (FLU and valproate) inhibit the hepatoma formation. These findings suggest that PEG10 plays an essential role in hepatocarcinogenesis. The PEG10 inhibition can be a novel approach for therapy of HCC.

LPS-induced cytokine production in the monocytic cell line THP-1 determined by multiple quantitative competitive PCR (QC-PCR).

BACKGROUND: Quantifying cytokines on the protein level can be problematic because of low concentrations or degradation during sample handling. Aiming towards finding a simple method by which to quantify cytokines on the mRNA level, we combined existing and established molecular biology techniques. Based on the principle of quantitative competitive RT-PCR with a DNA-competitor, IL-1beta, IL-6, IL-12alpha and the housekeeping enzyme GAPDH are measured at levels down to 200 copies of mRNA. METHODS: As a source of mRNA, the total RNA from 4 samples of 5 x 10(6) THP-1 cells stimulated with LPS (1 microg/ml for 24 h) was isolated. For competitors, we constructed sequences similar to the target sequences, but with deletion or insertion of 10-15% of the target length. For validating this method, we performed first strand synthesis on different days using different amounts of RNA (1-4 microg) isolated from the same pool of cells. Quantitative competitive PCR was accomplished using different amounts of cDNA (0.125-4 microL). Using IL-1beta as an example, the assay was validated for a dynamic range of 5-300 x 10(3) copies. RESULTS: A linear correlation was found between output and amount of RNA for cDNA synthesis, signifying that the final result of the analysis was linearly related to the amount of RNA or cDNA when operating within the range 1-4 microg (RNA isolation). CONCLUSION: The quantitative, competitive RT-PCR produces highly reproducible results within a 60-fold dynamic range.

In vitro effects of monophthalates on cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells from allergic and non-allergic donors.

It has recently been shown that plasticizers are present in indoor air dust, which may lead to human exposure via the inhalation route. Moreover, studies have indicated that plasticizers may possess adjuvant effects increasing the health damaging potential of allergens. The aim of this study was to investigate the in vitro effect of metabolites of phthalate plastisizers, such as whether an adjuvant effect is paralleled by changes of the cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells (PBMCs) from allergics and non-allergics. The toxicity monitored by cell viability was determined by incubating THP-1 cells with a 10-fold dilution series of monophthalates for 24 h. At different points in time cytokine expression (IL-1beta, IL-6, IL-12alpha (p35)) in THP-1 cells incubated with non-toxic concentrations of monophthalate (2-20 microg/ml)+/-LPS (1 microg/ml) were determined using Quantitative Competitive RT-PCR. PBMCs from allergics and non-allergics were incubated with monophthalate 220 microg/ml) for up to 48 h and cytokine expression (IL-4, IL-5, IFN-gamma) was measured using real-time PCR. The cytotoxic level of monophthalates is 20-200 microg/ml, depending on the individual monophthalate. There seems to be a correlation between increasing side-chain length and toxicity. Monophthalates did not induce changes in cytokine expression in THP-1 cells, though there is an increase when co-incubating with LPS. Cytokine expression in PBMC seems virtually unchanged when co-incubated with monophthalate, though mono-n-butyl phthalate (MBUP) tends to increase the level of IL-4 in PBMCs from allergic individuals. The two cellular models demonstrated the dynamics of regulated cytokine mRNA and are applicable for in vitro immunotoxicological investigations. The results regarding monophthalates suggest these to have a limited effect on cytokine expression in the monocytic cell line THP-1 and weak effect on cytokine expression in PBMCs from allergic and non-allergic individuals.

CXCR3 expression on CD34(+) hemopoietic progenitors induced by granulocyte-macrophage colony-stimulating factor: II. Signaling pathways involved.

CXCR3, known to have four ligands (IFN-gamma inducible protein 10 (gamma IP-10), monokine induced by IFN-gamma (Mig), I-TAC, and 6Ckine), is predominantly expressed on memory/activated T lymphocytes. We recently reported that GM-CSF induces CXCR3 expression on CD34(+) hemopoietic progenitors, in which gamma IP-10 and Mig induce chemotaxis and adhesion. Here we further report that stimulation with GM-CSF causes phosphorylation of Syk protein kinase, but neither Casitas B-lineage lymphoma (Cbl) nor Cbl-b in CD34(+) hemopoietic progenitors can be blocked by anti-CD116 mAb. Specific Syk blocking generated by PNA antisense completely inhibits GM-CSF-induced CXCR3 expression in CD34(+) progenitors at both mRNA and protein as well as at functional levels (chemotaxis and adhesion). Cbl and Cbl-b blocking have no such effects. Thus, GM-CSF binds to its receptor CD116, and consequently activates Syk phosphorylation, which leads to induce CXCR3 expression. gamma IP-10 and Mig can induce Syk, Cbl, and Cbl-b phosphorylation in CD34(+) progenitors by means of CXCR3. gamma IP-10 or Mig has induced neither chemotaxis nor adhesion in GM-CSF-stimulated Cbl-b-blocked CD34(+) hemopoietic progenitors, whereas SDF-1alpha induces both chemotaxis and adhesion in these cells. Interestingly, gamma IP-10 and Mig can induce chemotaxis and adhesion in GM-CSF-stimulated Syk- or Cbl-blocked CD34(+) hemopoietic progenitors. Thus, Cbl-b, but not Syk and Cbl phosphorylation, is essential for gamma IP-10- and Mig-induced chemotaxis and adhesion in CD34(+) hemopoietic progenitors. This study provides a useful insight into novel signaling transduction pathways of the functions of CXCR3/gamma IP-10 and Mig, which may be especially important in the cytokine/chemokine environment for mobilization, homing, and recruitment during proliferation, differentiation, and maturation of hemopoietic progenitor cells.

Detergents in the indoor environment - what is the evidence for an allergy promoting effect? Known and postulated mechanisms.

IgE-mediated allergic diseases, such as asthma and rhinitis seem to be increasing in industrialised societies. One possible explanation for this could be the increased use of more effective and aggressive detergents. The surfactants from these could interfere with the sensitisation process in which specific IgE is formed to ubiquitously occurring environmental allergens. Only sparse data exist in relation to surfactants and allergic sensitization. However, it can be speculated that the strong surfactant properties of some of ingredients used in modem detergents may interfere with some of the intricate cellular interactions taking place along the immunological pathways. These include formation of IL-4 and IL-5 producing T helper lymphocytes type 2 and the B-lymphocyte isotype switch, which leads to production of specific IgE. Candidates for experimental studies of such phenomena on the cellular level are proposed.

Chemokine stromal cell-derived factor 1alpha activates basophils by means of CXCR4.

BACKGROUND: The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive T lymphocytes, B lymphocytes, dendritic cells, and endothelial cells. CXC chemokine stromal cell-derived factor 1alpha (SDF-1alpha) is the only known ligand for CXCR4. To date, the CXCR4 expression and function of SDF-1alpha in basophils are unknown. OBJECTIVE: The purpose of this study was to investigate the expression of CXCR4 and functions of SDF-1alpha in basophils and to characterize the role of the CXCR4-SDF-1alpha receptor ligand pair in the allergic inflammation. METHODS: Basophil purification, flow cytometry, real-time quantitative RT-PCR assay, Northern blotting, intracellular free Ca(2+) change, chemotaxis assay, and histamine release assay were used. RESULTS: CXCR4 is abundantly expressed on peripheral blood resting basophils (91%). Likewise, CXCR4 messenger (m)RNA is expressed in resting basophils (3.2 x 10(3) copies per 2 x 10(2) cells). The existence of CXCR4 mRNA was also confirmed in basophils by means of Northern blot analysis. SDF-1alpha induces an increase in intracellular free Ca(2+) in basophils. SDF-1alpha activates basophils to chemotaxis (chemotactic index = 3.8) and histamine release (36% of total content) through CXCR4 on the cells. The chemokines SDF-1alpha, eotaxin, RANTES, monocyte chemoattractant protein (MCP) 1, and macrophage inflammatory protein (MIP) 1alpha have been demonstrated at different potencies in induction of chemotaxis (eotaxin > SDF-1alpha > RANTES congruent with MCP-1 >> MIP-1alpha) and histamine release (MCP-1 congruent with SDF-1alpha > eotaxin > RANTES > MIP-1alpha). The optimal concentration seen for SDF-1alpha effects (chemotaxis and histamine release) on basophils was 100 ng/mL. CONCLUSION: These results indicate that the CXCR4-SDF-1alpha receptor ligand pair may be important for the recruitment and activation of the basophils, which is a characteristic effector cell of the allergic inflammation.

CXC chemokine receptor 3 expression on CD34(+) hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor: chemotaxis and adhesion induced by its ligands, interferon gamma-inducible protein 10 and monokine induced by interferon gamma.

CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon gamma (IFN-gamma)-inducible protein 10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report the novel finding that CXCR3 is also expressed on CD34(+) hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34(+) progenitors. Freshly isolated CD34(+) progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase-polymerase chain reaction technique. gamma IP-10 and Mig induced chemotaxis of GM-CSF-stimulated CD34(+) progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block gamma IP-10-induced and Mig-induced CD34(+) progenitor chemotaxis. These chemotactic attracted CD34(+) progenitors are colony-forming units-granulocyte-macrophage. gamma IP-10 and Mig also induced GM-CSF-stimulated CD34(+) progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 mAb blocked these functions of gammaIP-10 and Mig but not of chemokine stromal cell-derived factor 1 alpha. gamma IP-10-induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF-stimulated CD34(+) progenitors. Moreover, gamma IP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF-stimulated CD34(+) progenitors. These results indicate that CXCR3-gamma IP-10 and CXCR3-Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment for the physiologic and pathophysiologic events of differentiation of CD34(+) hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34(+) hematopoietic progenitors. (Blood. 2000;96:1230-1238)

CXCR3 expression and activation of eosinophils: role of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma.

CXC chemokine receptor 3 (CXCR3), predominately expressed on memory/activated T lymphocytes, is a receptor for both IFN-gamma-inducible protein-10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report a novel finding that CXCR3 is also expressed on eosinophils. gamma IP-10 and Mig induce eosinophil chemotaxis via CXCR3, as documented by the fact that anti-CXCR3 mAb blocks gamma IP-10- and Mig-induced eosinophil chemotaxis. gamma IP-10- and Mig-induced eosinophil chemotaxis are up- and down-regulated by IL-2 and IL-10, respectively. Correspondingly, CXCR3 protein and mRNA expressions in eosinophils are up- and down-regulated by IL-2 and IL-10, respectively, as detected using flow cytometry, immunocytochemical assay, and a real-time quantitative RT-PCR technique. gamma IP-10 and Mig act eosinophils to induce chemotaxis via the cAMP-dependent protein kinase A signaling pathways. The fact that gamma IP-10 and Mig induce an increase in intracellular calcium in eosinophils confirms that CXCR3 exists on eosinophils. Besides induction to chemotaxis, gamma IP-10 and Mig also activate eosinophils to eosinophil cationic protein release. These results indicate that CXCR3-gamma IP-10 and -Mig receptor-ligand pairs as well as the effects of IL-2 and IL-10 on them may be especially important in the cytokine/chemokine environment for the pathophysiologic events of allergic inflammation, including initiation, progression, and termination in the processes.

Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases.

Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3 and the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to assess the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay. Chemotaxis was assessed using Boyden microchambers. Eotaxin (10(-11) to 10(-7) mol/L) induced concentration-dependent phosphorylation of ERK2 and p38. Phosphorylation was detectable after 30 seconds, peaked at about 1 minute, and returned to baseline after 2 to 5 minutes. Phosphorylation of JNK above baseline could not be detected. The kinase activity of ERK2 and p38 paralleled phosphorylation. PD980 59, an inhibitor of the ERK2-activating enzyme MEK (MAP ERK kinase), blocked phosphorylation of ERK2 in a concentration-dependent manner. The functional relevance of ERK2 and p38 was studied using PD98 059 and the p38 inhibitor SB202 190. PD98 059 and SB202 190 both caused inhibition of eotaxin-induced ECP release and chemotaxis. We conclude that eotaxin induces a rapid concentration-dependent activation of ERK2 and p38 in eosinophils and that the activation of these MAP kinases is required for eotaxin-stimulated degranulation and directed locomotion. (Blood. 2000;95:1911-1917)

CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10.

We report that interleukin (IL)-4 and IL-10 can significantly up- or down-regulate CXC chemokine receptor 4 (CXCR4) expression on CD4+ T lymphocytes, respectively. Stromal cell-derived factor-1alpha (SDF-1alpha)-induced CD4+ T-lymphocyte chemotaxis was also correspondingly regulated by IL-4 and IL-10. IL-4 and IL-10 up- or down-regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd approximately 6.3 nM), and approximately 70,000 SDF-1alpha-binding sites per cell, among freshly isolated CD4+ T lymphocytes, and two types of CXCR4 with different affinities (Kd1 approximately 4.4 nM and Kd2 approximately 14.6 nM), and a total of approximately 130,000 SDF-1alpha-binding sites per cell, among IL-4-stimulated CD4+ T lymphocytes. The regulation of CXCR4 expression in CD4+ T lymphocytes by IL-4 and IL-10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP- and cGMP-dependent protein kinase (H-8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both cAMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL-4 and IL-10 on CXCR4 expression implied that the capacity of IL-4 and IL-10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium-mobilization stimulation. These results indicate that the effects of IL-4 and IL-10 on the CXCR4-SDF-1 receptor-ligand pair may be of particular importance in the cytokine/chemokine environment concerning the inflammatory processes and in the progression of human immunodeficiency virus (HIV) infection.

Histamine immunocytochemistry: a new method for detection of basophils in peripheral blood.

We report that basophils in peripheral blood can be stained using histamine immunocytochemistry. The staining is based on the fixation of leucocytes with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (CDI) and the subsequent incubation of these cells with antisera raised against histamine conjugated to different carrier proteins using CDI. The staining appears to be specific for basophils and stained cells can be examined using both fluorescence microscopy and flow cytometry. In addition, histamine immunocytochemistry can be combined with conventional immunocytochemistry by incubating leucocytes with antibodies to cell surface antigens prior to or following fixation of the cells with CDI. Thus, histamine immunocytochemistry may be a valuable tool in future studies of human basophils.

Cutting edge: expression of the NF of activated T cells in eosinophils: regulation by IL-4 and IL-5.

We report that NF-AT1 and NF-AT4 are expressed cytoplasmically in resting eosinophils, whereas NF-AT2 and NF-AT3 have not been seen. Likewise, NF-AT1 mRNA and NF-AT4 mRNA have been detected in resting eosinophils, and their levels can be significantly up-regulated by the Th2-associated cytokines IL-4 and IL-5. There is no detectable NF-AT protein expression in the nuclei of resting eosinophils. However NF-ATs appear in the nuclei of IL-4-, IL-5-, or ionomycin-stimulated eosinophils. Only NF-AT1 and NF-AT4, but not NF-AT2 and NF-AT3, have translocated into the nuclei in IL-4- or IL-5-stimulated eosinophils. These findings delineate a novel pathway in the cytokine network in which Th2 lymphocytes "control" eosinophils via the release of IL-4 and IL-5, and activation of NF-AT in eosinophils. The findings also suggest that a later feedback "talking" may exist between eosinophils and Th2 lymphocytes.

Eotaxin activates T cells to chemotaxis and adhesion only if induced to express CCR3 by IL-2 together with IL-4.

The transmigration and adherence of T lymphocytes through microvascular endothelium are essential events for their recruitment into inflammatory sites. In the present study, we investigated the expression of CC chemokine receptor CCR3 on T lymphocytes and the capacities of the CC chemokine eotaxin to induce chemotaxis and adhesion in T lymphocytes. We have observed a novel phenomenon that IL-2 and IL-4 induce the expression of CCR3 on T lymphocytes. We also report that CC chemokine eotaxin is a potent chemoattractant for IL-2- and IL-4-stimulated T lymphocytes, but not for freshly isolated T lymphocytes. Eotaxin attracts T lymphocytes via CCR3, documented by the fact that anti-CCR3 mAb blocks eotaxin-mediated T lymphocyte chemotaxis. In combination with IL-2 and IL-4, eotaxin enhances the expression of adhesion molecules such as ICAM-1 and several integrins (CD29, CD49a, and CD49b) on T lymphocytes and thus promotes adhesion and aggregation of T lymphocytes. The eotaxin-induced T lymphocyte adhesion could be selectively blocked by a specific cAMP-dependent protein kinase inhibitor, H-89, indicating that eotaxin activates T lymphocytes via a special cAMP-signaling pathway. Our new findings all point toward the fact that eotaxin, in association with the Th1-derived cytokine IL-2 and the Th2-derived cytokine IL-4, is an important T lymphocyte activator, stimulating the directional migration, adhesion, accumulation, and recruitment of T lymphocytes, and paralleled the accumulation of eosinophils and basophils during the process of certain types of inflammation such as allergy.

Chemotaxis and IL-8 receptor expression in B cells from normal and HIV-infected subjects.

To date, the activities of the alpha chemokines for human peripheral B cells from normal subjects (N-B cells) or from HIV-infected subjects (HIV-B cells) are not well established. No report on the IL-8R expression on N-B cells and HIV-B cells has been seen. We report in this work that the alpha chemokines IL-8 and growth-regulatory oncogene-alpha (GRO-alpha) induce a chemotactic migration of N-B cells and HIV-B cells via stimulating the IL-8RB on these cells. The chemotaxis of N-B cells can be inhibited by IFN-gamma and IL-2, and augmented by IL-4 and IL-13, whereas TNF-alpha and IL-10 have no influence. The chemotaxis of HIV-B cells can be inhibited by IFN-gamma and IL-2, and augmented by TNF-alpha, IL-4, and IL-10, whereas IL-13 has no influence. IL-8R are expressed more abundantly on freshly isolated HIV-B cells than N-B cells (51% and 15%, respectively). The IL-8R on N-B cells can be down-regulated by IFN-gamma, IL-2, and TNF-alpha (selectively on IL-8RA), and up-regulated by IL-4 and IL-13, whereas IL-10 has no influence. The IL-8R on HIV-B cells can be down-regulated by IFN-gamma and IL-2, and up-regulated by TNF-alpha, IL-4, and IL-10, whereas IL-13 has no influence. Importantly, N-B cell and HIV-B cell chemotaxis toward IL-8 and GRO-alpha can be blocked by anti-IL-8RB polyclonal Ab, but not by anti-IL-8RA polyclonal Ab. Our results demonstrate that IL-8 and GRO-alpha are important inflammatory mediators that stimulate the directional migration and recruitment of B lymphocytes. The migratory behavior and the expression of IL-8R on HIV-B cells and some of the reactions to Th1- and Th2-like cytokines are modified significantly during HIV infection.

Identification of functional domains on human interleukin 10.

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein-Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152-160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1beta-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-alpha production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-gamma-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Psoriasin: a novel chemotactic protein.

Inflammatory skin disorders such as psoriasis show a preferential epidermal infiltration of neutrophils and T lymphocytes. This observation raises a question as to which factors determine the appearance and composition of leukocyte tissue infiltrations. Previously, we described a low molecular mass calcium-binding protein (psoriasin, molecular mass 11,457 Da, pI 6.77) belonging to the S1OO family that is highly upregulated in psoriatic keratinocytes and whose expression patterns implied a role in the inflammatory response. Here we report that human psoriasin is a potent and selective chemotactic inflammatory protein for CD4+ T lymphocytes and neutrophils at concentrations of about 10(-11) M. Psoriasin is not structurally related to the alpha or the beta chemokine subfamilies or to lymphotactin, a member of a newly described class of chemokines. Thus, we have observed a chemotactic protein outside the chemokine subfamilies that could be an important new inflammatory mediator.

Recombinant human growth-regulated oncogene-alpha induces T lymphocyte chemotaxis. A process regulated via IL-8 receptors by IFN-gamma, TNF-alpha, IL-4, IL-10, and IL-13.

The human cytokine growth-regulated oncogene (GRO)-alpha is a small glycoprotein secreted by monocytes, endothelial cells, glycoprotein secreted by monocytes, endothelial cells, fibroblasts, synovial cells, and some tumor cells such as melanoma cells. It is structurally related to IL-8 and can activate neutrophils, whereas it induces chemotaxis, exocytosis, and a respiratory burst in neutrophils. To date, its functions on T lymphocytes have not been well established. We report here that recombinant human (rh)GRO-alpha is a potent chemoattractant for freshly isolated T lymphocytes, but not for anti-CD3 mAb-activated T lymphocytes. It attracts CD4+ and CD8+ T lymphocyte subsets to an equal extent. The migrating T lymphocytes toward rhGRO-alpha are predominantly CD45RO+ memory CD4+ and CD8+ subsets. The chemotactic migration of T lymphocytes toward rhGRO-alpha is stimulated via the IL-8 receptors on the cells. This process can be augmented by IFN-gamma and TNF-alpha, and inhibited by IL-4, IL-10, and IL-13. In addition, we also document that on T lymphocytes there exist IL-8 receptors that can be up-regulated by IFN-gamma, TNF-alpha, and IL-2. Our results demonstrate that rhGRO-alpha gene encodes for an inflammatory mediator that stimulates the directional migration of T lymphocytes. It may thus be another important mediator in the diseases in which T lymphocytes form the major constituent of the cellular infiltration.

Cetirizine inhibits the in vitro and ex vivo chemotactic response of T lymphocytes and monocytes.

We have studied the effect of a nonsedating antihistamine, cetirizine dihydrochloride, on the in vitro chemotaxis of leukocytes from human peripheral blood. We observed that 0.25 microgram/ml of cetirizine dihydrochloride in vitro significantly inhibited the chemotaxis of monocytes toward N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4. Higher concentrations of cetirizine, 1.0 and 2.5 micrograms/ml, completely inhibited monocyte chemotaxis without affecting cell viability. T-lymphocyte migration was also significantly depressed but not abolished. Pyrilamine (mepyramine) was not inhibitory in equimolar concentrations. According to these in vitro observations, we extended our studies to measure monocyte and T-lymphocyte chemotaxis in an open study, where four healthy volunteers and six patients with atopic dermatitis took 10 and 20 mg/day cetirizine 3 days. We observed a reduction in ex vivo monocyte and T-lymphocyte chemotaxis toward N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4 without a reduction of the blood cell count. The results were confirmed in an ensuing double-blind, placebo-controlled study of 16 healthy subjects and 14 adult patients with atopic dermatitis, where ex vivo monocyte chemotaxis was reduced or abolished during cetirizine therapy. Serum levels of the two eosinophil-derived granule proteins, eosinophilcationic protein P and eosinophil protein X, were not changed during the treatment period of 7 days. The results show that cetirizine dihydrochloride has an inhibitory effect on monocytes and T lymphocytes in vitro and ex vivo. Our findings support the clinical observations that cetirizine dihydrochloride has an antiinflammatory effect besides its H1-blocking activity.

Changes in cellular immune function after immune specific guided imagery and relaxation in high and low hypnotizable healthy subjects.

This article presents the results of two investigations, each measuring cellular immune function on 3 investigation days 1 week apart in 15 high and 15 low hypnotizable healthy subjects randomly selected for three groups: (1) a guided imagery group receiving instructions to enhance cellular immune function: (2) a relaxation group which did not receive instructions regarding the immune system, and (3) a control group. Study 1 investigated changes in monocyte chemotaxis (MC) and lymphocyte proliferative response (LPR) to three mitogens, while natural killer cell activity (NKCA) was measured in study 2. The results show similar patterns of brief decreases in LPR and NKCA immediately after intervention on all investigation days in both the imagery and relaxation groups. Increases in MC were found in both intervention groups on day 1. On a follow-up investigation day in study 2, a brief stress task yielded a slight increase in NKCA. In study 2, the control group showed decreases in NKCA similar to those observed in the two intervention groups. In general there were no significant changes in preintervention immune function throughout the investigation period. When comparing the effects in high and low hypnotizable subjects, we found that high hypnotizables showed greater decreases in LPR and NKCA than low hypnotizables. There are several inconsistencies between the results of the limited number of investigations studying the effects of guided imagery and relaxation on immune function. These differences may in part be explained by differences in methodology, time intervals between blood sampling, and subject characteristics such as age, health status and hypnotizability. The inconsistent results make it premature to make inferences about possible benefits of the application of these techniques in the treatment of immune related diseases, and further investigations are needed.