The innate HIV-1 immune response is composed of cellular and soluble factors, which limit the spread of the virus prior to development of a HIV-specific adaptive immune response . Furthermore, the innate immune response persists throughout the course of HIV-1 infection and continues to influence the adaptive immune control .
We have previously shown that leukemia inhibitor factor (LIF) inhibits HIV-1 replication at pg concentrations in vitro as well as in placenta and thymus explant models . It occurred in a virus tropism-independent manner, but was dependent upon gp130 cell surface expression, the signalling receptor for LIF . Furthermore, LIF was shown to be upregulated in the placenta of HIV-1 infected pregnant women who did not transmit virus to the fetus as compared with transmitting mothers . This LIF-mediated anti-HIV-1 activity occurred prior to reverse transcription . Here we have investigated the potential systemic clinical importance of LIF and its receptors (gp130 and LIFR-α) by examining their expression at the site of the majority of HIV-1 replication, the tonsils and lymph nodes.
Material and methods
Patients and controls
LT biopsies (tonsils and lymph nodes) were collected from 19 HIV-1 seropositive and seven HIV-1 seronegative patients, (Table 1). Six patients had primary HIV-1 infection (PHI), including five who were recruited from a multinational study, the Quest/probe3005 study, plus one patient recruited from the University of Colorado, USA. They were enrolled within 2 days to 4 months from onset of acute retroviral syndrome (ARS). Six patients had chronic HIV-1 infection (cHI) (infected > 2 years). Biopsies from these cHI patients were obtained from the University of Colorado, USA. Seven long-term non-progressors (LTNP) were recruited from the Infectious Diseases HIV Unit, Huddinge University Hospital, Stockholm, Sweden. Control tonsil biopsies from HIV-1 seronegative individuals with tonsillar hypertrophy were obtained from Huddinge University Hospital. The study was performed after approval from the Institutional Review Boards and Ethical Committees at each participating site. The CD4 T cell counts in peripheral blood were determined by routine flow cytometric analysis. Plasma HIV-1 RNA concentration was determined using Amplicor HIV monitor test or ultra sensitive test (Roche molecular Systems, Sommersville, New Jersey, USA).
Eight-μm thick sections from cryopreserved LT biopsies were fixed, permeabilized and stained as described previously [3,4]. Anti-LIF antibody (AB1886 LIF; Silenius, Victoria, Australia) was used to detect LIF and anti-LIFR-α (AF-249-NA, goat IgG; R & D systems, Abingdon, UK) and anti-gp130 (AM64; Pharmingen, Palo Alto, California, USA) were used to detect LIF receptors.
Quantification of LIF and its receptors, gp130 and LIFR-α, by in situ image analysis
The total stained tissue area was measured in a quantitative manner by a specialized software program . The frequency of positively stained cells was expressed as percentage of stained area out of the total tissue area. Each parameter was evaluated in duplicate.
Two colour staining for phenotyping cells expressing LIF and its receptors, gp130 and LIFR-α
Sections were incubated with antibodies against CD3 (S4.1; Caltag Laboratories, Burlingame, California, USA), CD4 and CD8 (RPA-T4 and RPA-T8, Pharmingen) followed by addition of an alkaline phosphatase substrate (kit from Vector Laboratories Inc., Burlingame, CA, USA) to develop a red colour. The sections were subsequently stained for LIF or the LIF receptors as described above.
Triple fluorescent staining
LT sections were stained with mouse anti-gp130 and biotinylated goat anti-mouse antibodies followed by Alexa 645-labelled streptavidin (Molecular Probes Inc., Eugene, Oregon, USA). Thereafter the fluorsceine isothiocyanate-labeled KCF7 antibody was added (Coulter Corp, Miami, Florida, USA) to detect four different HIV core-proteins (55, 39, 33 and 24 kDa). The sections were subsequently avidin–biotin blocked (kit from Vector Laboratories Inc.) before addition of a phycoerythrin-conjugated mouse anti-CD4 antibody (BD Bioscience, San Jose, California, USA) or a rabbit anti-LIF antibody, followed by a biotinylated goat anti-rabbit antibody and Alexa 633-labelled streptavidin (Molecular Probes). All sections were evaluated by sequential spectrophotometric separation in a laser scanning confocal microscope (SP102, Leica).
Real time quantitative reverse transcription (RT)–PCR for LIF
RNA was extracted and purified from snap frozen OCT embedded LT (Tissue-Tek, Miter, Elkhart, IN, USA) and quantitative kinetic RT–PCR was performed as described previously . LIF upstream primer: 5’-GGGCCACACTCACCCTTGT-3’; LIF downstream primer: 5’-TTCTCGAAGCCCATCCT GG-3’ and LIF Probe: 5’-5-carboxyfluorescein (FAM)-CCTTCCTGCTTCATCCGGCTTAGCTTG-5-carboxy-tetramethylrodamine (TAMRA)-3’.
A non-parametric, two-tailed Mann–Whitney test was used to analyse statistical significance between the different patient groups with regards to the expression of LIF protein, LIF mRNA, gp130 protein and LIFR-α protein. The non-parametric, two-tailed Mann–Whitney test was also used to analyse if there was a statistical difference in HIV-1 core antigen content in CD4+/gp130- cells, CD4-/gp130+ cells and CD4+/+gp130 cells in LT tissue. A non-parametric, two-tailed Spearman's’ statistical test, with a 95% confidence interval, was used to analyse the correlation between plasma viral load and LIF protein expression in LT. P < 0.05 was considered significant.
Augmented expression of LIF in lymphoid tissue in primary HIV-1 infection
LIF expression was measured by in situ imaging analysis in cryopreserved LT from patients at different stages of HIV-1 infection and in seronegative controls (Fig. 1a, 1b and 2a). The frequency of LIF expressing cells in the extra follicular LT area was significantly increased in PHI as compared with HIV-1 seronegative controls (P < 0.005). However, in cHI the LIF expression was unchanged as compared with uninfected controls. Furthermore, the LTNP-group showed a significantly decreased expression of LIF versus HIV-1 seronegative controls (P < 0.05).
LIF is a pleiotropic cytokine and is known to be produced by many different cell types . Multicolor staining was performed to identify the phenotype of LIF-expressing cells. This demonstrated that approximately half of the LIF expressing cells were CD3 (mean value 50%). Subsequent staining for CD4/LIF or CD8/LIF co-expression revealed that LIF expression occurred in both CD4 (30–50%) and CD8 (35–60%) phenotypes (Fig. 1c and 1d). This indicated that LIF was not only produced by T cells but also by CD4+CD3- and by CD8+CD3- cells including dendritic/monocyte-linages as well as natural killer (NK)-cells and NK T-cells.
Induction of LIF mRNA and expression in lymphoid tissue in primary HIV-1 infection
LIF mRNA expression was quantified by real-time RT–PCR (Fig. 2b) in individuals from whom LT tissue was available. LIF mRNA levels were elevated in LT sections from the PHI group while cHI, LTNP and HIV-1 seronegative controls showed comparable amounts of LIF mRNA.
Correlation between viral load and LIF expression
HIV-1 RNA copies/ml plasma was quantified in the different HIV-1 cohorts (Fig. 2c). Viral load was significantly higher in PHI and cHI as compared with LTNP (P < 0.005 and P < 0.05, respectively) The non-parametric Spearman's two-tailed statistical test, with a 95% confidence interval showed a significant correlation between plasma viral load and the expression of LIF in LT (r = 0.54 and P = 0.02; Fig. 2d).
Expression of gp130 and LIFR-α in lymphoid tissue in HIV-1 infection
The LIF signalling receptors, gp130 and LIFR-α, were quantified by in situ imaging in cryopreserved LT from patients at different stages of HIV-1 infection and in HIV-1 seronegative controls (Fig. 1e, 1f and Fig. 3). Expression of both gp130 and LIFR-α were significantly upregulated in PHI and cHI patients as compared with the control group (P < 0.005). In contrast, they were significantly downregulated in LTNP [(P < 0.05) for gp130 and (P < 0.005) for LIFR-α]. Two-color staining showed that gp130 and LIFR-α were present on CD3 (30–60% for gp130 and 35–55% for LIFR-α), CD4 (30–60% for gp130 and 20–50% for LIFR-α) and CD8 (10–60% for gp130 and 10–55% for LIFR-α) cells.
HIV core antigen expression was restricted to gp130 negative cells in lymphoid tissue
Multiple fluorescent staining was used to identify HIV-1 replicating cells (cytoplasmic HIV core antigen positivity) either with gp130 and CD4 cell surface expression or gp130 and LIF expression. This analysis was performed only in LT from PHI patients. Confocal microscopy revealed that HIV-1 core antigen was significantly less expressed in CD4+/gp130+ cells and in CD4-/gp130+ cells than in CD4+/gp130- cells (P < 0.05; Fig. 4a and 4c). Thus despite that > 50% of total CD4 cells expressed gp130, they comprised < 5% of the total HIV replicating cells in LT. Furthermore, we could not demonstrate signs of HIV-1 replication in LIF expressing cells (Fig. 4b).
Anti-HIV-1 immunity results in induction of cytokines, chemokines and other soluble factors that may both help the host to protect itself from the virus and increase viral infectivity . LIF is a pleiotropic cytokine that belongs to the interleukin (IL)-6 family. It signals via the gp130/LIFR-α heterodimer which is found on a variety of cells including CD4CD45RO T cells , which are known to be highly susceptible to HIV-1 replication both in vitro  and in vivo . Here we show for the first time that CD3, CD4 and CD8 cells in LT express LIF during HIV-1 infection. A significant correlation was found between plasma viral load and LIF protein expression; high viral load in the plasma was associated with high LIF protein levels in LT. LIF was mainly expressed in the primary phase of HIV-1 infection as indicated by up-regulation of both LIF mRNA and protein levels in LT from PHI patients in comparison to HIV-1 seronegative controls. In fact, LIF protein expression was upregulated in one patient as early as 2 days after the onset of ARS. Thus, LIF expression may occur prior to the emergence of HIV-1 specific cytotoxic T lymphocyte activity [12,13]. This early onset of LIF may suggest a role for LIF in the innate immune response against HIV-1. Moreover, it is evident that LIF is expressed concomitantly with other cytokines involved in the innate response, e.g. interferon-α/β, IL-12 and IL-18, which are also increased in LT during the early phase of acute SIV infection . It remains to be clarified if the CD4 and CD8 LIF expressing cells are HIV-1 specific or not. The early induction of LIF in PHI along with lower expression in LTNP and cHI argues for a role in the acute phase response to HIV-1 and less likely as an expansion of the adaptive HIV-1 immune response.
We have also shown that LIF receptors, gp130 and LIFR-α, were significantly upregulated in LT from PHI and cHI whereas they were significantly downregulated in the LTNP group versus HIV-1 seronegative controls. With the exception of the chronic phase, the expression of these receptors correlated with expression of LIF at various stages of HIV-1 infection. It should be noted that LIF is not the only cytokine that uses gp130 and LIFR-α . All cytokines of the IL-6 cytokine family; IL-6, IL-11, Oncostatin M (OSM), ciliary neurotropic factor (CNTF) and cardiotrophin-1 (CT-1) signal via the gp130 receptor . IL-6 and IL-11 signal through a gp130 homodimer while ligand binding of OSM, CNTF, CT-1 or LIF causes hetreodimerization of gp130/LIFR-α, and a third cytokine specific receptor subunit for CNTF and CT-1 . OSM signals via gp130/OSMR or via gp130/LIFR-α and displays some specific biological properties not shared by LIF . Therefore, persistent up-regulation of the LIF receptors, despite low LIF expression, may be a consequence of the total pro-inflammatory milieu present in LT throughout the course of HIV-1 infection . This may explain the up-regulation of LIF receptors in cHI in contrast with LTNP who are associated with reduced inflammation . The importance of LIF/gp130 mediated control of HIV-1 is further supported by the fact that significantly less viral replication (identified by HIV core antigen expression) could be demonstrated in CD4 cells expressing gp130 in LT. However it remains to be elucidated whether the virus down-regulates gp130 expression to counteract the anti-HIV effect of LIF.
Broor et al. have demonstrated that LIF activates HIV replication in the chronically HIV infected promonocytic cell line U1 . LIF may exert two different effects on HIV-1 replication; inhibitory activity in the early stage of HIV-1 replication and stimulatory activity in already chronically HIV-1 infected cells. It therefore remains to be determined in vivo if LIF inhibits de novo HIV infection in susceptible cells but not production in chronically infected cells.
The authors thank the patients involved and the recruiting sites for enrolling patients in the Quest PROB 3005 study and the assistance of those who provided additional biopsies.
Sponsorship: Supported by the Swedish Foundation for Strategic Research, Berth von Kantzow foundation, National Institutes of Health (grants no. AI 41536), Swedish Cancer Society (grant no.2490), Swedish Medical Research Council (grant no.10850), Swedish Physicians Against AIDS Research Foundation, Hedlunds Foundation and Swedish Society for Medical Research. This work was also supported by grants from the National Institute of Health to E. Connick (R29AI-42499 and UO1AI-41536).
1. Levy JA. The importance of the innate immune system in controlling HIV infection and disease. Trends Immunol
2. Patterson BK, Behbahani H, Kabat WJ, Sullivan Y, O'Gorman MR, Landay A, et al
. Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women. J Clin Invest
3. Andersson J, Abrams J, Bjork L, Funa K, Litton M, Agren K, et al
. Concomitant in vivo production of 19 different cytokines in human tonsils. Immunology
4. Andersson J, Fehniger TE, Patterson BK, Pottage J, Agnoli M, Jones P, et al
. Early reduction of immune activation in lymphoid tissue following highly active HIV therapy. AIDS
5. Andersson U, Litton MJ, Fehniger T, Ulgren A-K, Andersson J. Detection and quantification of cytokine-producing cells by immunostaining.
In Molecular Probes for Cytokine and Gene Detection.
Edited by Patterson B. Boston: Birkhauser CRC press; 1999:55–79.
6. Patterson BK, Landay A, Andersson J, Brown C, Behbahani H, Jiyamapa D, et al
. Repertoire of chemokine receptor expression in the female genital tract: implications for human immunodeficiency virus transmission. Am J Pathol
7. Lubbert M, Mantovani L, Lindemann A, Mertelsmann R, Herrmann F. Expression of leukemia inhibitory factor is regulated in human mesenchymal cells. Leukemia
8. Poli G. Laureate ESCI award for excellence in clinical science 1999. Cytokines and the human immunodeficiency virus: from bench to bedside. European Society for Clinical Investigation. Eur J Clin Invest
9. Patterson BK, Tjernlund A, Andersson J. Endogenous Inhibition of HIV: Potent anti-HIV Activity of Leukemia Inhibitor Factor. Curr Mol Med
10. Spina CA, Prince HE, Richman DD. Preferential replication of HIV-1 in the CD45RO memory cell subset of primary CD4 lymphocytes in vitro. J Clin Invest
11. Patterson BK, Czerniewski MA, Pottage J, Agnoli M, Kessler H, Landay A. Monitoring HIV-1 treatment in immune-cell subsets with ultrasensitive fluorescence-in-situ hybridisation. Lancet
12. Borrow P, Lewicki H, Hahn BH, Shaw GM, Oldstone MB. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol
13. Reimann KA, Tenner-Racz K, Racz P, Montefiori DC, Yasutomi Y, Lin W, et al
. Immunopathogenic events in acute infection of rhesus monkeys with simian immunodeficiency virus of macaques. J Virol
14. Giavedoni LD, Velasquillo MC, Parodi LM, Hubbard GB, Hodara VL. Cytokine expression, natural killer cell activation, and phenotypic changes in lymphoid cells from rhesus macaques during acute infection with pathogenic simian immunodeficiency virus. J Virol
15. Auernhammer CJ, Melmed S. Leukemia-inhibitory factor-neuroimmune modulator of endocrine function. Endocr Rev
16. Auguste P, Guillet C, Fourcin M, Olivier C, Veziers J, Pouplard-Barthelaix A, et al
. Signaling of type II oncostatin M receptor. J Biol Chem
17. Pantaleo G, Fauci AS. New concepts in the immunopathogenesis of HIV infection. Annu Rev Immunol
18. Broor S, Kusari AB, Zhang B, Seth P, Richman DD, Carson DA, et al
. Stimulation of HIV replication in mononuclear phagocytes by leukemia inhibitory factor. J Acquir Immune Defic Syndr