Biology at Washington & Jefferson College

Biology at Washington & Jefferson College

 

Amy Seman
Matt Valosen
Walter Velosky
Biology 311
24 November 1997

 b Chemokines and the Suppression of HIV Entry into T-Lymphocytes

I. Specific Aims

The proponent of HIV latency in infected individuals may be attributed to the presence of certain b -chemokines such as RANTES (regulated on activation, normal T-cell expressed and secreted), macrophage inflammatory protein (MIP)-1a , and MIP-1b (Cocchi, DeVico, Garzino-Demo, Arya, Kgallo, and Lusso, 1995). These chemokines can inhibit viral fusion with the cell by blocking an essential chemokine coreceptor, CC-CKR-5, thereby preventing it from binding to the viral envelope glycoprotein gp120 (Cocchi et al., 1995). This can have possible clinical applications; if an appropriate concentration of b-chemokines can be determined to inhibit HIV infection, the possibilities are endless. We will determine the concentrations of RANTES, MIP-1a , and MIP-1b in uninfected individuals, HIV-infected individuals, individuals exposed to HIV but not infected, and symptomatic AIDS patients. The goal of this research is to determine the optimal protein concentration of the chemokines that will inhibit viral infection. Also, the presence and quantity of mRNA for the three chemokines will determined. We will use ELISAs and western blots to determine protein size and concentration, and we will use northern blots and dot blots to detect mRNA.

Human immunodeficiency virus (HIV) pathogenesis involves a complex combination of viral replication, viral-induced killing of lymphocytes, and the host’s immune response (Dewhurst and Whetter, 1997). The typical pattern of HIV-1 infection is illustrated in Figure 1 and consists of three major phases: virus dissemination; clinical latency; and opportunistic infection (Dewhurst et al., 1997). It has been shown that during all three phases of virus infection, the level of HIV RNA remains high, even during the latency period (Dewhurst et al., 1997). The balance of the phases is based on interactions between viral replication and the immune response of the host.

HIV enters into T helper cells and CD4+ macrophages by using the viral envelope glycoprotein (gp120) to bind to expressed CD4 receptors on the cell surface (Deng, Liu, Ellmeier, Choe, Unutmaz, Burkhart, Marzio, Marmon, Sutton, Hill, Davis, Peiper, Schall, Littman, and Landau, 1996). However, HIV entry into immune cells may require additional cofactors. Feng, Broder, Kennedy, and Berger (1996) identified the protein fusin, which is believed to be a cofactor necessary for the entry of HIV into primary T-cells and transformed T-cell lines (Figure 2). In addition, a recently identified coreceptor for entry into primary T-cells and macrophages is CC-CKR-5 (also called CCR5), a cell surface receptor for the chemokines RANTES, MIP-1a , and MIP-1b (Deng et al., 1996). A model of HIV entry into T cells is shown in Figure 3a. RANTES, MIP-1a , and MIP-1b belong to a class of intracellular messenger molecules known as the CC or b-chemokine family, indicating that they have two N-terminal conserved cysteine residues next to each other (Alam, 1996). Some of the known functions of chemokines are to attract immune cells to sites of infection; they also play various roles in antiviral immunity, regulation of hematopoiesis, cell growth, and cell metabolism (Alam, 1996). RANTES, MIP-1a , and MIP-1b are primarily secreted by CD8+ T lymphocytes (Cocchi et al., 1995).

In 1995, Cocchi et al. found evidence that RANTES, MIP-1a , and MIP-1b acted to suppress HIV infection of CD4+ T cells. Since the CC-CKR-5 receptor is necessary for the virus to infect a cell, the presence of b -chemokines may competitively prevent fusion of the virus with the cell membrane, as shown in Figure 3b (Trkola, Dragic, Arthos, Binley, Olson, Allaway, Cheng-Mayer, Robinson, Maddon, and Moore, 1996). Gilden (1995) found that the presence of all three chemokines practically eliminated HIV entry into cells. However, when only one or two of these chemokines is present, the viral entry was reduced, but not completely eliminated. All three peptides do not suppress HIV infection with the same efficiency; RANTES has the most suppressive capabilities, followed by MIP-1b , and then MIP-1a (Shmidtmayerova, Sherry, and Bukrinsky, 1996). The chemokines are graphically depicted in Figure 4 during the process of receptor binding and HIV blocking.

The HIV suppressive action of the b -chemokines is thought to play a critical role in clinical latency of the HIV virus and viral neutralization (Cocchi et al., 1995). One research group from the Aaron Diamond AIDS Research Center in New York looked at individuals that were exposed to but not infected with the virus and found an early indication of unusually high levels of the chemokines RANTES, MIP-1a , and MIP-1b (Gilden, 1995).  

The purpose of these experiments is to determine the concentration of RANTES, MIP-1a , and MIP-1b in the serum of the following four subject-types: normal individuals, HIV infected individuals, symptomatic AIDS patients, and individuals exposed to HIV but not infected. The goal is to see if there is a correlation between the stage of infection and the amount of HIV-suppressive chemokines found in the serum.

In addition, we will determine the concentration of mRNA for RANTES, MIP-1a , and MIP-1b in each subject type. If we find significant differences in protein concentration between the subject types, the concentration of mRNA will indicate whether the difference in protein production is the result of a change in the transcription of the genes for the proteins, or if it is caused by a change in the rate of mRNA translation by the cell.

These experiments have definite clinical applications; it may be possible to determine a concentration of specific b-chemokines that can be used to inhibit HIV infection in vivo. This can lead to possible effective therapeutic approaches for the treatment of AIDS. In addition, it will help to more fully understand the mechanism of HIV infection and also the complex array of host immune responses to the virus and how they operate.

Figure 1: Schematic representation of the course of HIV-1 infection in vivo (from Dewhurst and Whetter, 1997).

 Figure 2: Model for HIV-1 co-receptor usage. Macrophage-tropic viruses (dark shading), analogous to the NSI viruses that predominate early in infection, are proposed to use CC-CKR-5, which is expressed in macrophage and primary T-cells. T-tropic viruses (light shading), analogous to SI viruses, use fusin, which is presumed to be expressed on primary T-cells and transformed T-cell lines (from Deng et al., 1996).

Figure 3: Model of HIV entry into T-cells. (a) HIV gp120 first binds to cellular CD4. This results in a conformational change in gp120, allowing it to bind to the chemokine receptors CCR5, thereby forming a tri-molecular complex. (b) An excess of the natural ligands for CCR5 can competitively inhibit this step of infection (as noted). After binding to the chemokine receptor, gp120 is thought to become stripped off the virion, thereby exposing a hydrophobic domain at the N-terminus of gp41, which mediates fusion of the host cell and virus membranes, thereby allowing the virus core to enter the host cell cytoplasm (from Dewhurst and Whetter, 1997). 

 Figure 4: Beta-chemokines block entry of HIV into cells (from Dewhurst and Whetter, 1997). 

III. Experimental Design and Methods

The research will take place in Boston, Massachusetts at either the Boston University Medical Center or the Pulmonary Center. Dr. William W. Cruikshank has volunteered the use of his lab for this research (Cruikshank, 1997). Peripheral blood mononuclear cells (PBMC’s) will be isolated from normal (non-HIV infected) individuals, HIV-infected individuals, individuals exposed to HIV but not infected, and symptomatic AIDS patients. All blood samples needed will be available to us at the University Hospital. To detect for the presence of RANTES, MIP-1a , and MIP-1b in the serum of all patient types, enzyme-linked immunosorbent assays (ELISAs) and western blots will be performed. All monoclonal antibodies needed for this research will be purchased from Research Diagnostics Incorporated (Richmond, VA).

The ELISA assays will detect the concentration of the RANTES, MIP-1a , and MIP-1b in the serum of the patients. This will be accomplished by attaching at the bottom of different wells an antibody specific for each individual chemokine. Patient’s serum will then be added to the wells. If chemokines are present, they will bind to the antibody. The well is then washed and an antibody to the specific chemokine will be added. This antibody is linked to the color forming enzyme horseradish peroxidase. When the enzyme binds to the chemokine, the color formed will allow for concentration to be detected (Grier, Schultz, and Vogel, 1987).

The western blot will more sensitively detect the concentration of protein and also its size. The serum proteins from all four patient types will be run on SDS polyacrylamide gel electrophoresis (PAGE) to separate the proteins based on molecular weight. The bands will then be transferred to nitrocellulose and incubated with monoclonal antibodies to RANTES, MIP-a , and MIP-1b (Research Diagnostics Inc., Richmond). The bands will be visualized using a secondary antibody attached to a color forming enzyme such as horseradish peroxidase. Therefore, these two methods together will determine concentration and size of the desired chemokines (Grier et al., 1987).

For another aspect, we will look at the presence and concentration of the mRNA message for the three proteins using northern blotting. The mRNA will be isolated from the various cell types; this will be run on SDS-PAGE to separate the fragments by size. The bands will then be transferred to nitrocellulose. We will purchase oligonucleotide probes to each cytokine (from R&D Systems in Minneapolis, Minnesota). The bands on the nitrocellulose will be hybridized with this specific probe, and the mRNA of interest will be located using autoradiography (Sambrook, Fritsch, and Maniatis, 1989). This will indicate the presence and size of the mRNA for the proteins in each patient type.

Following the northern blots, we will use dot blots to determine the concentration of mRNA for the three chemokines. The isolated mRNA from each patient will be spotted on nitrocellulose paper. The nitrocellulose will be hybridized with a specific probe that is labeled with a color-forming enzyme such as horseradish peroxidase. The intensity of the color formation will be determined using spectrophotometry; color intensity correlates with amount of mRNA (Sambrook et al.,1989). This will indicate the concentration of mRNA in each subject type.

In the event that this research does not account for significant differences in the amount of protein and mRNA between the four groups, it would be possible to rule out these three chemokines as possible mediators of symptom suppression and noninfection.

IV. Literature Cited

Alam, R. 1997. Chemokines in allergic inflammation. J. of
     Allergy and Clinical Immunology 99(3):273-277.

Cocchi, F., A. L. DeVico, A. Garzino-Demo, S. K. Arya,
     R C. Gallo, and P. Lusso. 1995. Identification of
     RANTES, MIP-1a , and MIP-1b as the major HIV-
     supressive factors produced by CD8+ T cells. Science
     270:1811-5.

Cruikshank, W.W. 1997. personal communication.

Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M.
     Burkhart, P. DiMarzio, S. Marmon, R. E. Sutton, C. M.
     Hill, C. B. Davis, S. C. Peiper, T. J. Schall, D. R.
     Littman, and N. R. Landau. 1996. Identification of a
     major co-receptor for primary isolates of HIV-1. Nature
     381:661- 666.

Dewhurst, S., and L. Whetter. 1997. Pathogenesis and
     treatment of HIV-1 infection: recent developments.
     Frontiers in Bioscience. Available FTP: Hostname:
     bioscience.org Directory:
    1997/v2/d/dewhurs1/htmls/3.htm.

Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger.
     1996. HIV-1 entry cofactor: functional cDNA cloning of
     a seven-transmembrane G protein-coupled receptor.
     Science 272:872-877.

Gilden, D. (1995). Be kind to your chemokines [5
     paragraphs]. GMHC Treatment Issues.
     http://library.jri.org./library/news/ti/gmti10061.html

Grier, A.H., M. Schultz, and C.W. Vogel. 1987. Cobra
     venom factor and human C3 share carbohydrate
     antigenic determinants. J. of Immunology 139(4):1245-
     1252.

Sambrook, J., E.F. Fritsch, T. Maniatis. 1989. Molecular
     Cloning: a laboratory manual. Cold Spring Harbor
     Laboratory Press, New York.

Schmidtmayerova, H., B. Sherry, and M. Bukrinsky. 1996.
     Chemokines and HIV replication. Nature 382:767.

Trkola, A., T. Dragic, J. Arthos, J. M. Binley, W. C. Olson,
     G.P. Allaway, C. Cheng-Mayer, J. Robinson, P. J.
     Maddon, and J. P. Moore. 1996. CD4-dependent,
     antibody sensitive interactions between HIV-1 and its
     co-receptor CCR-5. Nature 384:184-187.

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