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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 12, 2002 as doi:10.1096/fj.01-0655fje. |
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Division of Immunologic and Infectious Diseases, The Children’s Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
3Correspondence: Division of Immunologic and Infectious Diseases, The Children’s Hospital of Philadelphia, 34th Street & Civic Center Blvd., Philadelphia, PA 19104, USA. E-mail: douglas{at}email.chop.edu
SPECIFIC AIMS
The neuropeptide substance P (SP) is a potent mediator of neuroimmune regulation. We previously observed increased levels of plasma SP in HIV-infected men vs. uninfected control subjects. In this study, we investigated the possible cellular source of the plasma SP. We determined whether HIV infection up-regulates SP expression in mononuclear phagocytes and T lymphocytes isolated from human peripheral and placental cord blood, and whether activation and replication of HIV in latently infected immune cells affect SP gene expression.
PRINCIPAL FINDINGS
1. HIV infection enhances SP expression in human immune cells
To understand the interaction between SP and HIV in the human immune cells, we studied the effect of HIV infection on the ability of human monocytes and lymphocytes to synthesize SP mRNA as determined by real-time RT-PCR. HIV Bal strain infection significantly enhanced SP mRNA expression (3- to 
18-fold) in cultured human blood macrophages (Fig. 1
A). The increased SP mRNA expression correlated with HIV RT activity, as the SP mRNA level steadily increased during the time course of HIV replication in macrophages. HIV RPV strain-infected peripheral blood lymphocytes (PBL) and cord blood lymphocytes (CBL) manifested increased levels of SP mRNA vs. uninfected PBL and CBL (Fig. 1B
). Mononuclear phagocytes were tested for their ability to produce SP peptide in response to HIV Bal strain infection. Seven day cultured monocytes were challenged with HIV Bal strain at different concentrations. Levels of SP and RT in the culture supernatants were analyzed by EIA and RT assays 7 days postinfection. Increased SP levels were observed in the macrophages infected with HIV Bal strain vs. mock-infected macrophages. SP peptide production in HIV Bal-infected macrophage cultures positively correlated with the viral RT activity.
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2. Purified recombinant HIV gp120 increases SP expression
To determine whether HIV gp120 protein binding to the cell membrane has a direct effect on SP mRNA expression in human blood mononuclear phagocytes, we used gp120 protein derived from M- (MN) and T-tropic strain (IIIB). The addition of either MN- or IIIB-derived gp120 to macrophages significantly increased SP mRNA expression compared with untreated macrophages (Fig. 1C
). This effect of the gp120 was abrogated by preincubation with soluble CD4 (Fig. 1C
), indicating a specific gp120-CD4 receptor-mediated effect on SP mRNA expression in macrophages.
3. Activation of HIV in latently infected U1 and ACH-2 cells up-regulates SP expression
To determine whether HIV replication influence SP expression in latently infected human immune cells, we investigated whether SP mRNA expression is affected by activation and replication of HIV in latently infected promonocytic (U1) and T lymphocytic (ACH-2) cells. U1 and ACH-2 cells were treated with tumor necrosis factor 
(TNF-) (2 ng/ml) plus PMA (25 ng/ml) for 24 h, then subjected to real-time RT-PCR for SP mRNA expression. As expected, TNF- dramatically increased HIV replication in U1 and ACH-2 cells (Fig. 2
). TNF--induced HIV replication significantly enhanced SP mRNA expression in U1 and ACH-2 cells (Fig. 2)
. To rule out the possibility that the increased SP mRNA expression was the direct effect of TNF- and PMA added to the cultures, we performed a similar experiment using the U937 cell line (a parental cell line of U1), which does not have latent HIV virions. TNF- plus PMA did not affect SP gene expression in U937 cells.
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CONCLUSIONS
SP is a potent mediator of neuroimmune regulation. SP is produced mainly in the primary sensory neurons and intrinsic enteric neurons. Our studies, however, have demonstrated that human immune cells express SP and its receptor. Since SP modulates the function of human immune cells that are also targets for HIV infection, we postulated that SP promotes HIV infection of these immune cells. In support of our hypothesis, we demonstrated that SP enhanced HIV expression in macrophages isolated from healthy individuals and that SP activates HIV replication in latently infected immune cells. We recently observed that a SP receptor antagonist (CP-96,345) inhibits HIV replication in human mononuclear phagocytes. These findings indicate that SP and its receptor (NK-1R) are both indeed involved in HIV infection of macrophage. In the present study, we demonstrate (Fig. 1A, B
) that HIV infection (macrophage tropic and T cell tropic strains) induces SP expression in human mononuclear phagocytes and lymphocytes at both mRNA and protein levels. In addition, binding of HIV gp120 to CD4 receptors on macrophages was sufficient for up-regulation of SP mRNA expression (Fig. 1C
), suggesting that gp120 shed from HIV-infected macrophages may play an important role in HIV-induced SP expression in these cells. Our observation that the soluble CD4 blocks the effect of gp120 on SP induction by macrophages (Fig. 1C
) suggests a CD4 receptor-dependent pathway by gp120. These data indicate that the interaction of gp120 and SP plays an important role in immunopathogenesis of HIV infection and neuro AIDS.
We observed that the activation of HIV by TNF- in latently infected promonocytic (U1) and T (ACH-2) cells promoted SP mRNA expression (Fig. 2)
. This increased SP mRNA expression was not due to the direct effect of TNF-, since the addition of TNF- to macrophage and U937 (the parent cell line of U1 which lacks HIV) cell cultures had no effect on SP mRNA expression (data not shown). Although the precise mechanism(s) by which HIV induces SP expression in the immune cells remains to be determined, the markedly enhanced capacity of HIV-infected mononuclear phagocytes and lymphocytes to produce SP reflects a specific transcription stimulation, such as activation of NF-
B, a transcription factor involved in the control of cytokine expression, through which HIV replication in these cells is affected (Fig. 3
). Based on our data showing that HIV enhances SP expression in macrophages and T lymphocytes, it is reasonable to speculate that SP levels in circulation and peripheral tissues may increase as a result of HIV infection of the immune cells, such as macrophages and CD4+ T lymphocytes, the primary target cells for HIV. SP released from these infected immune cells may up-regulate HIV by directly facilitating HIV replication within macrophages and T cells and/or by indirectly affecting HIV proliferation through induction of inflammatory cytokines such as IL-1, IL-6, and TNF- and via alteration of expression of ß-chemokines and their receptors, which may reciprocally enhance HIV replication in these cells (Fig. 3)
.
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With our previous findings that SP modulates HIV replication in human blood mononuclear phagocytes and that HIV-infected men have higher levels of plasma SP than uninfected individuals, the present data showing that SP mRNA expression and synthesis are enhanced by HIV infection and gp120 treatment of human immune cells indicate a reciprocal relationship between SP and HIV in the infected immune cells. Although the direct clinical significance of our data remains to be determined, the effects of HIV infection on SP expression in human immune cells are likely to have in vivo relevance to HIV infection of human immune cells. Since macrophages and CD4+ T cells are important immune cells in inflammatory reactions and primary target cells for HIV infection, the data from our studies imply that the interaction between SP and HIV in these immune cells may be involved in the immunopathogenesis of HIV infection and AIDS.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0655fje; to cite this article, use FASEB J. (February 12, 2002) 10.1096/fj.01-0655fje 
2 W.-Z.H. and J.-P.L. contributed equally to this work.
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