Macrophage Inhibitory Protein Research Articles

Genetic deficiency in the chemokine receptor CCR1 protects against acute Clostridium difficile toxin A enteritis in mice

The role of the CC chemokine receptor (CCR) 1 in acute enteritis was investigated by subjecting CCR1 knockout mice to Clostridium difficile toxin A treatment. Toxin A or vehicle was injected into ileal loops in anesthetized wild-type, CCR1-/- and macrophage inhibitory protein (MIP)-1alpha-/- mice. After 1-4 hours, fluid accumulation was calculated, and the loops were processed for histology, myeloperoxidase activity, regulated on activation, normal T cell expressed and secreted (RANTES) production, and messenger RNA measurements. Toxin A induced in all mice a significant (P < 0.05) increase in ileal fluid accumulation, epithelial damage, and neutrophil infiltration, with all parameters being significantly (P < 0.01) lower in CCR1-/- and MIP-1alpha-/- mice. Ileal messenger RNA expression of the CCR1 ligands MIP-1alpha and RANTES and RANTES synthesis were increased in toxin A-treated wild-type mice. The RANTES antagonist Met-RANTES significantly (P < 0.01) reduced the toxin A-induced increases in ileal fluid accumulation and myeloperoxidase activity in wild-type mice. C. difficile toxin A-induced murine enteritis involves CCR1 and its ligands MIP-1alpha and RANTES, which may be important mediators of the neutrophil recruitment characterizing acute, enterotoxin-mediated enteritis.

Evaluation and management of pulmonary fibrosis in scleroderma.

Pulmonary fibrosis causes significant morbidity and mortality in patients with scleroderma. Lung inflammation identifies patients at greater risk for decline in forced vital capacity and diffusing capacity for carbon monoxide. Factors that are increased in patients with scleroderma with lung fibrosis include connective tissue growth factor, KL-6, pulmonary surfactant-D, tissue inhibitor of metalloproteinase 2, monocyte chemotactic protein-1, macrophage inhibitory protein-1 alpha, soluble interleukin-6 receptors, anti-endothelial cell antibodies, and anti-DNA topoisomerase I antibodies. Potential mechanisms of lung damage in scleroderma include increased production of profibrotic type 2 cytokines and abnormal signaling by thrombin of tenascin-C production by lung fibroblasts, with protein kinase C epsilon as an intermediate in the signaling pathway. Treatment of scleroderma lung disease with cyclophosphamide may have a beneficial effect on pulmonary function and survival. Lung transplantation provides a therapeutic option for patients with scleroderma with end-stage lung disease.

Enhanced maturation and functional capacity of monocyte-derived immature dendritic cells by the synthetic immunomodulator Murabutide.

Murabutide is a safe synthetic immunomodulator derived from muramyl dipeptide, the smallest bioactive unit of bacterial peptidoglycan. Although it is well known that muramyl peptides modulate the functions of monocytes/macrophages, their activity on dendritic cells is poorly documented. We thus investigated the effects of Murabutide on immunophenotype, endocytosis, T-cell stimulatory capacity, and cytokine secretion of human monocyte-derived immature dendritic cells (iDCs). We found that Murabutide triggers immunophenotypic changes as upon treatment, iDCs up-regulate the surface expression of the major histocompatibility complex type II molecule human leucocyte antigen-DR, the co-stimulatory molecules CD80, CD86 and CD40 and the differentiation marker CD83, and down-regulate the expression of the mannose receptor. These phenotypic changes are also mirrored by changes in their biological activity. Subsequent to treatment with the synthetic immunomodulator, DC have a decreased endocytic capacity but exhibit enhanced stimulatory capacity for both allogeneic and autologous T cells. In addition, Murabutide-stimulated iDCs have a greater cytostatic activity toward the tumour cell line THP-1. Furthermore, in the presence of Murabutide, DCs transiently increased the release of macrophage inhibitory protein-1 beta, tumour necrosis factor-alpha and interleukin-10, whereas the enhanced production of macrophage-colony stimulating factor was sustained over the 3-day period analysed. In addition, Murabutide triggers the phosphorylation of the three classes of mitogen-activated protein kinases in iDCs. Altogether our results demonstrate that Murabutide triggers the maturation and activation of monocyte-derived iDCs. As this immunomodulator is approved for administration in humans, it could be a useful adjunct to boost the efficacy of DC-based vaccines designed against tumours or virus-infected cells.

Local distribution of microglia in the normal adult human central nervous system differs by up to one order of magnitude.

Although microglia are considered to be a sensitive sensor for pathological processes in the central nervous system, there are only a few studies about the distribution and density of microglia in the normal human brain. Therefore, a study of local density of microglial cells was conducted by investigating 20 normal human brains with no clinical neurological symptoms or diseases and no neuropathological alterations. Microglial cells were visualized by immunolabeling of proteins which are known to be expressed either constitutively or facultatively, such as CD68, major histocompatibility complex class II (MHC-II), leukocyte common antigen (LCA), leukocyte chemotactic factor (LCF), macrophage inhibitory factor-related protein (MRP) 8, MRP14, CD4 and allograft-inflammatory factor-1 (AIF-1). CD68, MHC-II and AIF-1 showed the highest densities with significant regional differences ranging from 0.5% to 16.6% of all cells in the brain parenchyma with significantly more microglia in white than in gray matter. LCF and LCA showed a similar pattern of distribution as the proteins described above, but with lower percentages of microglial cells. CD4 was not found in the brain parenchyma. We conclude that CD68, MHC-II and AIF-1 define the main microglial cell population, whereas LCF and LCA are expressed by a subpopulation of microglial cells. The brains showed no or a negligible vascular expression of MRP8 and MRP14. Information about the local microglia density in the normal human brain can serve as a reference for the evaluation of pathological microglial responses.

Molecular and cellular mediators of interleukin-1-dependent acute inflammatory arthritis.

To examine the molecular and cellular mechanisms in a model of acute inflammatory monarticular arthritis induced by methylated bovine serum albumin (mBSA) and interleukin-1 (IL-1). Mice were injected intraarticularly with mBSA on day 0 and subcutaneously with recombinant human IL-1beta on days 0-2. At day 7, knee joints were removed and assessed histologically. Flow cytometry and RNase protection were used to analyze IL-1-dependent events. C57BL/6 (B6), 129/Sv, and (B6 x 129/ Sv)F1 hybrid mice, all H-2b strains, were susceptible to mBSA/IL-1-induced arthritis, whereas C3H/HeJ (H-2k) mice were not. B6 mice lacking T and B cells (RAG1-/-) or major histocompatibility complex (MHC) class II antigens (MHCII-/-), and B6 mice treated with a CD4+ T cell-depleting monoclonal antibody, were resistant to disease. In contrast, B cell-deficient (muMT/ muMT) mice developed arthritis at an incidence and severity similar to that of controls. RelB-deficient (RelB-/-) bone marrow chimeric mice had arthritis that was significantly reduced in incidence and severity. In B6 mice, flow cytometry demonstrated an IL-1-dependent leukocyte infiltration into the synovial compartment and RNase protection assays revealed induction of messenger RNA (mRNA) for the chemokines monocyte chemoattractant protein 1, macrophage inhibitory protein 2 (MIP-2), RANTES, MIP-1alpha, and MIP-1beta, in vivo and in vitro. Arthritis induced by mBSA/IL-1 is strain specific and dependent on CD4+ T lymphocytes and at least partially on RelB, but not on B lymphocytes or antibody. IL-1 contributes to leukocyte recruitment to the synovium and directly induces chemokine mRNA production by synovial cells. This model of acute monarticular arthritis is particularly suitable for further investigations into cell-mediated immunity in arthritis and the role of IL-1.

Interleukin-1 beta induces production of macrophage inhibitory protein-2 by brain microvascular endothelial cells

Interleukin-1 beta induces production of macrophage inhibitory protein-2 by brain microvascular endothelial cells

Macrophage inflammatory protein 1-alpha is a potential osteoclast stimulatory factor in multiple myeloma

This study was designed to determine if macrophage inhibitory protein-1 (MIP-1), a recently described osteoclast (OCL) stimulatory factor,1 was present in marrow from patients with multiple myeloma (MM) and possibly involved in the bone destructive process. MIP-1, but not interleukin-1β (IL-1β), tumor necrosis factor-β (TNF-β), or interleukin-6 (IL-6), messenger RNA was elevated in freshly isolated bone marrow from 3 of 4 patients with MM compared to normal controls. Furthermore, enzyme-linked immunosorbent assays of freshly isolated bone marrow plasma detected increased concentrations of hMIP-1 (range, 75-7784 pg/mL) in 8 of 13 patients (62%) with active myeloma, in 3 of 18 patients (17%) with stable myeloma (range, 75-190.3), as well as in conditioned media from 4 of 5 lymphoblastoid cell lines (LCLs) derived from patients with MM. Mildly elevated levels of MIP-1 were detected in 3 of 14 patients (21%) with other hematologic diagnoses (range, 80.2-118.3, median value of 96 pg/mL) but not in normal controls (0 of 7). MIP-1 was not detected in the peripheral blood of any patients with MM. In addition, recombinant hMIP-1 induced OCL formation in human bone marrow cultures. Importantly, addition of a neutralizing antibody to MIP-1 to human bone marrow cultures treated with freshly isolated marrow plasma from patients with MM blocked the increased OCL formation induced by these marrow samples but had no effect on control levels of OCL formation. Thus, high levels of MIP-1 are expressed in marrow samples from patients with MM, but not in marrow from patients with other hematologic disorders or controls, and support an important role for MIP-1 as one of the major factors responsible for the increased OCL stimulatory activity in patients with active MM.

Extracellular matrix proteins in organ transplantation.

Extracellular matrix proteins in organ transplantation.

CD8+,CD57+ T cells from healthy elderly subjects suppress neutrophil development in vitro: Implications for the neutropenia of Felty's and large granular lymphocyte syndromes

To investigate the ability of CD8+,CD57+ large granular lymphocytes (LGL) from normal individuals and from Felty's syndrome (FS) or LGL syndrome patients to suppress allogeneic neutrophil precursor development. Six FS patients, 5 LGL syndrome patients, and 13 elderly controls were studied. CD8+,CD57+ T cells were cocultured with cord blood-derived stem cells, and percentage inhibition was calculated. Recombinant chemokines and Fas-stimulating molecules were used in separate cultures to address possible mechanisms of suppression. Proliferation after stimulation with interleukin-2 (IL-2) and anti-CD3 was assessed. Significant (79%) suppression of colony-forming unit-granulocyte-macrophage (CFU-GM) by the CD8+,CD57+ subset was shown by 1 FS patient. None of the CD8+,CD57+ cells from LGL syndrome patients had any effect. Six of 13 controls studied showed >40% inhibition of CFU-GM, and all but 2 showed at least some suppression. The suppressive effect was not mediated by Fas/Fas ligand interactions or by the chemokines macrophage inhibitory protein 1alpha or IL-8. LGL from both patients and controls were largely CD28- and had reduced proliferative capacity. In a subset of FS patients, expansion of CD8+,CD57+ T cells in the bone marrow may be responsible for neutropenia by suppressing neutrophil precursors. This effect is also seen with normal LGL, which are likely to have an important function in neutrophil homeostasis.

Activation of inflammatory mediators in rat renal isografts by donor brain death.

Brain death (BD) has been thought to influence the early course of transplanted organs by triggering a series of nonspecific inflammatory events that in turn may increase the kinetics and intensity of the immunological host responses. In this study early nonspecific, cellular, and molecular changes occurring in kidney isografts from BD donors are compared with those from normal anesthetized, ventilated controls. After induction of brain death, the animals were mechanically ventilated for 6 hr before organ removal. Only rats with stable blood pressure (mean arterial pressure >80 mmHg) were included. Serum creatinines were measured daily. Representative grafts were harvested 6 hr after brain death and between 1 hr and 5 days after engraftment for morphology, immunohistology, and reverse transcriptase-polymerase chain reaction. The presence of serum cytokines was assessed by enzyme linked immunoabsorbant assay. Serum creatinine levels rose slightly in recipients from BD donors. Serum interleukin-1beta levels increased within 6 hr versus controls (P<0.05). mRNA levels of interleukin-1beta and macrophage inhibitory protein-1 in the kidneys were up-regulated transiently before engraftment (6 hr after BD) and 1 hr after revascularization (P<0.05). By immunohistology, numbers of infiltrating polymorphonuclear leukocytes peaked at 24 hr in parallel with intragraft induction of P- and E-selectin, complement, and other proinflammatory chemokines and cytokines. At 5 days, the isografts from BD donors were highly infiltrated by host leukocyte populations associated with intense up-regulation of their products. In contrast, those from control donors remained relatively normal through this initial follow-up period. The intense nonimmune inflammation produced in isografts after donor BD may represent the initial stages of a continuum between an initial nonspecific and later immune reactivity, when placed in the context of allotransplantation.

Human osteoblast‐like cells and osteosarcoma cell lines synthesize macrophage inhibitory protein 1α in response to interleukin 1β and tumour necrosis factor α stimulation in vitro

Recent investigations have demonstrated that macrophage inhibitory protein 1alpha (MIP-1alpha) plays a critical role in haematopoiesis. In part, MIP-1alpha limits the differentiation of early haematopoietic cells, thereby ensuring that sufficient quantities of blood precursors are available to meet haematopoietic demands. MIP-1alpha is produced by cells of the marrow microenvironment (marrow stromal cells) in response to a variety of stimuli, including interleukin 1beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha). Our recent investigations demonstrated that normal human osteoblast-like cells (HOBs) maintain the early phenotype of haematopoietic precursors, like other members of the bone marrow stroma. Although the precise molecular mechanisms for these observations have not been determined, the production of MIP-1alpha remains one such possibility. In the present study, we investigated whether cells of the osteoblast lineage under basal, IL-1beta and/or TNF-alpha stimulation produce MIP-1alpha. We observed that IL-1beta and TNF-alpha stimulated HOBs and human osteosarcoma cells to rapidly express MIP-1alpha mRNA and to secrete large quantities of the protein. MIP-1alpha mRNA and protein was not, however, detected under basal conditions. Perhaps more importantly, enriched human CD34+ bone marrow cells in co-culture may be capable of stimulating the expression of MIP-1alpha mRNA by HOBs in vitro. These findings suggest that human osteoblast-like cells may produce MIP-1alpha in vivo to support haematopoiesis at sites where osteoblasts and haematopoietic cells are closely associated.

Semiquantitation of human chemokine mRNA levels with a newly constructed multispecific competitor fragment

Chemokines are a group of inducible, locally acting proinflammatory cytokines which have been implicated in the pathogenesis of a variety of diseases. Important members of the group include monocyte chemoattractant protein (MCP)-1, -2, -3, macrophage inhibitory protein (MIP)-1α, -1β and RANTES (regulated upon activation, normal T expressed and secreted). To facilitate further investigation of the human chemokines, we have constructed a novel multispecific competitor fragment containing primer binding sites for the CC-chemokines MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β and RANTES, the CXC-chemokines MIP-2α, MIP-2β as well as for the housekeeping gene β-actin. Using this competitor fragment we can demonstrate reliable semiquantitation of reverse transcribed chemokine mRNAs. The assay should be useful for further studies, in particular for the semiquantitation of chemokine mRNA species from small cell or tissue specimens.

Murine T Lymphocytes Incapable of Producing Macrophage Inhibitory Protein-1 Are Impaired in Causing Graft-Versus-Host Disease Across a Class I But Not Class II Major Histocompatibility Complex Barrier

The routine use of bone marrow transplantation is limited by the occurrence of acute and chronic graft-versus-host disease (GVHD). Current approaches to decreasing the occurrence of GVHD after allogeneic transplantation use T-cell depletion, use immunosuppressive agents, or block costimulatory molecule function. The role of proteins in the recruitment of alloreactive lymphocytes has not been well characterized. Chemokines are a large family of proteins that mediate recruitment of mononuclear cells in vitro and in vivo. To investigate the role of T-cell production of the chemokine macrophage inhibitory protein-1 (MIP-1) in the occurrence of GVHD, splenocytes either from wild-type or from MIP-1−/− mice were administered to class I (B6.C-H2bm1) and class II disparate mice (B6-C-H2bm12). The incidence and severity of GVHD was markedly reduced in bm1 mice receiving splenocytes from MIP-1−/− mice as compared with mice receiving wild-type splenocytes. Bm1 mice receiving MIP-1−/− splenocytes had significantly less weight loss and markedly reduced inflammatory responses in the lung and liver than mice receiving C57BL/6 splenocytes. Bm1 mice receiving MIP-1−/− splenocytes had a markedly decreased production of antichromatin autoantibodies and impaired generation of bm1-specific T lymphocytes versus wild-type mice. However, MIP-1−/− splenocytes easily induced GVHD when administered to bm12 mice. This data show that blockade of chemokine production or function may provide a new approach to the prevention or treatment of GVHD but that chemokines that recruit both CD4+ and CD8+ lymphocytes may need to be targeted.

Inhibition of simian immunodeficiency virus (SIV) replication by CD8(+) T lymphocytes from macaques immunized with live attenuated SIV.

Characterization of immune responses induced by live attenuated simian immunodeficiency virus (SIV) strains may yield clues to the nature of protective immunity induced by this vaccine approach. We investigated the ability of CD8(+) T lymphocytes from rhesus macaques immunized with the live, attenuated SIV strain SIVmac239Deltanef or SIVmac239Delta3 to inhibit SIV replication. CD8(+) T lymphocytes from immunized animals were able to potently suppress SIV replication in autologous SIV-infected CD4(+) T cells. Suppression of SIV replication by unstimulated CD8(+) T cells required direct contact and was major histocompatibility complex (MHC) restricted. However, CD3-stimulated CD8(+) T cells produced soluble factors that inhibited SIV replication in an MHC-unrestricted fashion as much as 30-fold. Supernatants from stimulated CD8(+) T cells were also able to inhibit replication of both CCR5- and CXCR4-dependent human immunodeficiency virus type 1 (HIV-1) strains. Stimulation of CD8(+) cells with cognate cytotoxic T-lymphocyte epitopes also induced secretion of soluble factors able to inhibit SIV replication. Production of RANTES, macrophage inhibitory protein 1alpha (MIP-1alpha), or MIP-1beta from stimulated CD8(+) T cells of vaccinated animals was almost 10-fold higher than that from stimulated CD8(+) T cells of control animals. However, addition of antibodies that neutralize these beta-chemokines, either alone or in combination, only partly blocked inhibition of SIV and HIV replication by soluble factors produced by stimulated CD8(+) T cells. Our results indicate that inhibition of SIV replication by CD8(+) T cells from animals immunized with live attenuated SIV strains involves both MHC-restricted and -unrestricted mechanisms and that MHC-unrestricted inhibition of SIV replication is due principally to soluble factors other than RANTES, MIP-1alpha, and MIP-1beta.

Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies.

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1beta, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.

Kinetics of β-Chemokine Levels during Anti-HIV Therapy

Chemokines are pro-inflammatory cytokines that inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. We studied the kinetics of the β-chemokines, macrophage inhibitory protein (MIP)-1α, MIP-1β, RANTES, and monocyte chemotactic protein (MCP)-1 in plasma during 12 months of antiretroviral therapy in 26 HIV-1-infected patients and in 11 untreated subjects. Eleven patients with moderate immunodeficiency had HIV-1 RNA levels &lt;50 copies/ml after 1 year, whereas 12 out of 15 patients with severe immunodeficiency had detectable virus. At baseline, MCP-1 levels correlated positively with HIV-1 RNA and DNA levels and inversely with CD4 cell counts. A reverse pattern was seen for the MIP-1β levels. No correlation was seen between MIP-1α or RANTES and any of the parameters. Also, there was a dichotomy between the different β-chemokines in response to therapy. Decreases of MCP-1 and RANTES levels were found, but no durable changes of MIP-1α and MIP-1β. The MCP-1 levels rebounded back to baseline after 1 year in the patients who responded virologically, which could possibly reflect an increased immune activation. The biological consequences of the changes in β-chemokines levels during antiretroviral treatment are still unknown and deserve further studies.

Genetic Subtype-Independent Inhibition of Human Immunodeficiency Virus Type 1 Replication by CC and CXC Chemokines

We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1alpha against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4+ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1beta, MIP-1alpha. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1beta, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1beta, MIP-1alpha.

Plasma levels of monocyte chemoattractant protein-1 but not those of macrophage inhibitory protein-1alpha and RANTES correlate with virus load in human immunodeficiency virus infection.

Plasma levels of proinflammatory cytokines, cytokine inhibitors, and the beta chemokines RANTES, macrophage inhibitory protein (MIP)-1alpha, and monocyte chemoattractant protein (MCP)-1 were studied in relationship with virus load in 40 patients exhibiting plasma levels of HIV RNA ranging between undetectable and levels >10(6) copies/mL. Mean plasma levels of MCP-1 were increased in patients with high virus load compared with HIV-seropositive subjects with undetectable plasma viral RNA and healthy controls. MCP-1 levels were directly correlated with plasma levels of HIV RNA. No correlation was observed between virus load and plasma concentrations of MIP-1alpha and RANTES. The results suggest that low rates of viral replication in vivo are not dependent on increased production of the suppressive chemokines RANTES and MIP-1alpha. Since MCP-1 upregulates viral replication in vitro, the results may suggest a role for MCP-1 in triggering viral replication in HIV disease.

Elevated serum macrophage inhibitory factor-related protein (MRP) 8/14 levels in advanced HIV infection and during disease exacerbation.

To assess the value of MRP 8, MRP 14, and MRP 8/14 serum concentrations as markers of disease progression in HIV infection and as markers of intercurrent infections. We measured MRP 8, MRP 14, and MRP 8/14 serum concentrations in 184 HIV-infected patients in various stages of disease with or without disease exacerbation and in 50 healthy control subjects. In clinically stable HIV-infection correlations of MRP levels with stage of HIV disease, CD4 counts, p24 antigen, and beta-2 microglobulin levels were studied. In patients with intercurrent illnesses, correlations of MRP levels with type of disease exacerbation and with CRP were calculated and compared with those found in stable HIV infection. MRP 8/14 levels were significantly elevated and MRP 8 levels slightly decreased in stable HIV infection compared with HIV-negative controls. The CD4 cell count and MRP 8/14 levels correlated significantly in patients with AIDS. Despite higher values of MRP 8/14 during advanced disease, these were not significant predictors of progression to death. In patients with acute infections, MRP 8/14 levels were significantly elevated, compared with patients with illnesses of noninfectious origin. Levels of MRP 8/14 associated with acute infections were significantly higher in patients with AIDS than in patients during earlier stages of HIV infection. Both stable HIV infection and advanced immunedeficiency are associated with an elevation of the MRP 8/14 complex and probably with a decline of MRP 8 serum levels. MRP 8/14 is preserved as a marker of acute infection in immunecompromised patients.

Preferential sequestration in vitro of BCR/ABL negative hematopoietic progenitor cells among cytokine nonresponsive CML marrow CD34+ cells.

It is believed that long-term cultures of CML marrow cells favor the outgrowth of BCR/ABL negative hematopoietic progenitor cells (HPC) and that this phenomenon may be enhanced with negative hematopoietic regulators which can maintain primitive HPC in a quiescent state. Proliferation of CML marrow CD34+ cells in primary short-term cultures, maintained in the presence or absence of macrophage inhibitory protein-1 alpha (MIP-1 alpha), was tracked with the membrane dye PKH2. After 7 to 10 days it was possible to distinguish between cytokine responsive (CR) CD34+ cells (cells which had divided thus becoming PKH2dim) and cytokine nonresponsive (CNR) CD34+ cells (cells which had not divided and had therefore remained PKH2bright). CR and CNR CD34+ cells were isolated by flow cytometric cell sorting, seeded in secondary long-term cultures, and their progeny cells assayed weekly for their clonogenic progenitor cell content and expression of BCR/ABL by reverse transcriptase polymerase chain reaction (RT-PCR). Whereas CNR cells isolated from control primary cultures (control/CNR) sustained in vitro hematopoiesis, similar cells from cultures treated with MIP-1 alpha (MIP-1 alpha/CNR) supported a higher and, in some patients, a more extended production of clonogenic HPC, indicating that MIP-1 alpha was able to maintain primitive HPC in a quiescent state. Predominance of BCR/ABL negative progenitors in vitro was more evident in secondary cultures initiated with CNR cells than in those initiated with CR cells, especially those established with MIP-1 alpha/CNR cells. Of interest is the observed decline in the percentage of BCR/ABL+ progenitors in these cultures with time. Whereas up to 100% of progenitors were BCR/ABL+ on day 0, by day 14, only 46% of progenitors in MIP-1 alpha/CNR secondary cultures were BCR/ABL+ and by day 28 and beyond, the percentage of BCR/ABL+ progenitors dropped to below 20%. These results suggest that the quiescent nature of normal HPC present in CML marrow may favor their identification via cell tracking and, subsequently, their isolation from the more actively cycling leukemic cells. These studies also confirm the feasibility of employing negative hematopoietic regulators to augment the sequestration of normal HPC among the cytokine nonresponsive fraction of CD34+ cells, an approach that may be clinically feasible for autotransplantation.