HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, L. Articles by Cloyd, M. W. Search for Related Content PubMed PubMed Citation Articles by Wang, L. Articles by Cloyd, M. W.

A Novel Mechanism of CD4 Lymphocyte Depletion Involves Effects of HIV on Resting Lymphocytes: Induction of Lymph Node Homing and Apoptosis Upon Secondary Signaling Through Homing Receptors¹

Liqiang Wang^*, Jenny J. Y. Chen, Benjamin B. Gelman, Rolf Konig^* and Miles W. Cloyd^2,*,

Departments of ^* Microbiology and Immunology and Pathology, University of Texas Medical Branch, Galveston, TX 77555

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, we reported that abortive HIV infection of resting human T lymphocytes up-regulated expression of CD62L, the receptor for homing to lymph nodes (LNs), and enhanced homing of these cells from the blood into the LNs (Wang et al., 1997, Virology 228:141). This suggested that HIV-induced homing of resting lymphocytes (which comprise >98% of all lymphocytes) may be a major mechanism for the reduction of CD4⁺ lymphocytes in the blood of infected individuals. This mechanism also could be partially responsible for the lymphadenopathy that often develops at the same time that CD4⁺ lymphocytes are disappearing from the blood. In this study, we show that secondary signaling through the homing receptors (CD62L, CD44, CD11a) of abortively infected resting CD4⁺ T lymphocytes induced apoptosis. These signals would occur as the cells home into the LNs. Apoptosis did not occur after secondary signaling through some other receptors (CD26, CD4, CD45, and HLA class I) or in HIV-exposed resting CD8⁺ lymphocytes signaled through the homing receptors. These findings indicate that HIV-induced homing of resting CD4⁺ lymphocytes to LNs results in death of many of these cells. This was confirmed in the LNs of SCID mice that were i.v. injected with HIV-exposed resting human lymphocytes. Thus, these effects of HIV upon binding to resting CD4⁺ T lymphocytes, which are not permissive for HIV replication, may significantly contribute to their depletion in vivo. These findings also offer an explanation for the bystander effect observed in the LNs of AIDS patients, whereby cells not making virus are dying.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Loss of CD4 T lymphocytes in the blood of HIV-infected subjects correlates with increasing levels of plasma virus and decreasing immunocompetence (1, 2, 3, 4). It has been thoroughly documented, however, that inversion of the CD4/CD8 ratio does not occur in lymph nodes (LNs)³ when it is occurring in the blood (5, 6). In fact, when blood levels of CD4⁺ lymphocytes begin to drop significantly, these cells often increase in number in the LNs (lymphadenopathy) (5, 6). This suggests that the loss of CD4⁺ cells in the blood does not necessarily reflect global depletion of these cells, but rather enhanced homing of CD4⁺ lymphocytes from the blood into the LNs. Recently, we reported that abortive HIV-1 infection of resting T lymphocytes up-regulated expression of cell surface CD62L (L-selectin), the receptor for homing to LNs, which resulted in a 12-fold increase in the number of these cells that bound to LN high endothelial venules in an ex vivo homing assay (7). When injected i.v. into SCID mice, HIV-exposed, resting human T lymphocytes rapidly homed from the blood into the LNs (60–70% reduction of cells in the blood by 2 h) (7). This action may help explain why most infected cells are found in the LNs and not in the blood, why CD4⁺ cells decline in the blood even though very few productively infected cells are found in the blood, and why lymphadenopathy occurs when the numbers of CD4⁺ cells are falling in the blood (8, 9, 10, 11, 12, 13, 14).

A large amount of evidence has indicated that HIV infection causes depletion of CD4⁺ lymphocytes by an indirect mechanism (15, 16, 17, 18, 19, 20, 21). The dying cells in the LNs of AIDS patients are bystander cells (i.e., cells not producing HIV mRNA) (21). This may explain why more CD4⁺ lymphocytes die than the few at any given time that produce HIV (13, 14). Apoptotic cells are generally not directly detected at higher than normal frequencies in freshly isolated PBLs from HIV-infected individuals (22), but infected subjects display significantly more apoptotic cells in their LNs (23). Lymphocytes, both CD4⁺ and CD8⁺, in HIV-infected subjects are in a general state of activation and prone to undergo spontaneous apoptosis when placed into culture or stimulated in vitro by Ags, mitogens, or superantigens (16, 17, 18, 19). It is possible that this state results in resting lymphocytes after abortive infection by HIV. The extent of CD4⁺ lymphocyte depletion via Ag activation is not clear, but any given Ag would only affect a very small percentage of the CD4⁺ lymphocyte population. Since our previous results demonstrated that resting CD4⁺PBLs are profoundly affected by HIV binding in the induction of homing properties, we wondered what happens to these cells as they home into LNs. We found that about one-half of the resting CD4⁺ lymphocytes that were preexposed to HIV were induced into apoptosis following signaling through their homing receptors (CD62L, CD44, CD11a).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation and infection of PBLs

Peripheral blood was obtained by venipuncture from healthy HIV-negative donors (low risk, HIV Ab negative) following informed consent. PBMCs were isolated by centrifugation through lymphocyte separation medium (Organon Teknika, Durham, NC) and washed twice with HBSS. Monocytes/macrophages were depleted by plastic adherence at 37°C for 2 h in medium. The nonadherent cells, PBLs, were collected and centrifuged. The cell pellet was resuspended in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 15% FBS, penicillin, streptomycin, L-glutamine (Sigma, St. Louis, MO), and IL-2 (10 U/ml) at 1 x 10⁶ cells/ml and seeded into 48-well tissue culture plates. HIV-1 stocks were prepared as described previously (7). HIV-1 at a multiplicity of infection (MOI) equal to 1 was added, and the PBLs were incubated for 12 h at 37°C. Control cells were mock treated with culture supernatant fluids from uninfected CEM cells, which is the cell line in which the HIV stocks were made.

Cross-linking of cell surface receptors

After 12 h of incubation, both HIV-exposed and mock-treated cells were centrifuged, washed, and incubated with one of the mAbs (2 µg/ml) against indicated surface molecules at 4°C for 30 min. Cross-linking of the surface molecules was then achieved by incubating the cells with goat anti-mouse IgG (1 µg/ml) at 37°C for 30 min. Subsequently, cell viability was monitored every day by trypan blue exclusion. The mAbs used were: anti-CD62L (PRREG-56), anti-CD44 (J.173), anti-CD11a (25.3), anti-CD103 (HML-1, 2G5), anti-CD26 (TaI), anti-CD8 (OKT8), and anti-CD4 (PRA-T4 or Sim 4). mAbs were purchased from PharMingen (San Diego, CA), ImmunoTech (Westbrook, ME), Coulter (Hialeah, FL), or American Type Culture Collection (Manassas, VA). Sim 4 was a gift from Dr. James Hildreth via the National Institutes of Health AIDS Repository. Each of these mAbs has been shown to be able to signal via cross-linking the receptor to which they bind (24, 25, 26, 27). Each experiment was performed in duplicate, and the results were calculated by subtracting the percentage of dead cells in untreated control cells.

Purification of CD4⁺ or CD8⁺ T lymphocytes and monitoring of cellular markers

CD8⁺ or CD4⁺ T lymphocytes were removed from PBLs by panning, as reported previously (28). Briefly, petri dishes (100 mm in diameter, 1 plate for 1–1.5 x 10⁷ cells) were treated with 10 ml of affinity-purified goat anti-mouse IgG (Sigma) in HBSS (5 µg/ml) overnight at 4°C. The dishes were washed with 10 ml of HBSS containing 2% FBS five times and incubated for 1 h at 4°C with 20 ml of the same solution. PBLs were incubated with OKT8 or Sim 4 hybridoma culture supernatant fluids for 1 h at 4°C with constant mixing, washed twice, resuspended in HBSS containing 2% FBS (10⁷ cells/10 ml), and placed onto the goat anti-mouse IgG (Sigma)-coated plates for 3 h at 4°C, with gentle tilting at 1.5 h. Nonadherent cells were then collected, washed with HBSS containing 2% FBS, and cultured. The percentages of CD4⁺ and CD8⁺ T lymphocytes following panning were determined by immunostaining the live cells and analyzing them by flow cytometry, and were always 95% enriched. The percentages and fluorescence intensity of cells expressing CD62L, CD44, and CD11a were monitored by flow cytometry of immunostained live cells.

Purification of memory and naive CD4⁺ T lymphocytes

Memory and naive CD4⁺ T lymphocytes were obtained by panning using anti-CD45RA or anti-CD45RO mAbs in addition to OKT8. The resulting CD4⁺ populations were >95% positive for CD45RO⁺ or CD45RA⁺, respectively.

Coating plates with various reagents

As reported by others (29, 30, 31), 48-well plates were coated with mannose-6-phosphate (M6P), hyaluronic acid (HA), mannose-1-phosphate (M1P), or chondroitin sulfate A (CHA) at concentrations of 5 mg/ml in PBS at 4°C overnight. The plates were washed six times with cold PBS to remove any reagents that did not attach to the wells.

In vivo assessment of the fate of HIV-1-exposed human PBLs that have homed in SCID mice

Fresh human PBLs were isolated and exposed to HIV-1 without stimulation, as described above. After 40 h of culture, the infected and mock-treated cells were centrifuged and then resuspended in culture medium at 2 or 3 x 10⁷ cells/300 µl for i.v. (retroorbital sinus) injection into SCID mice (Tac:Icr:Ha(ICR)-scidfDf; Taconic Farms, Germantown, NY). Before injection, SCID mice were transferred from University of Texas Medical Branch Animal Care Center (Galveston, TX) to the P3 lab and anesthetized with Nembutal (40 mg/kg of body weight). At 2, 4, and 6 days postinjection, the mice were anesthetized again and sacrificed; peripheral LNs were removed, frozen in liquid nitrogen, and subsequently stored at -70°C.

Detection of human T lymphocytes in SCID mice LNs

Frozen sections of the LNs were rehydrated in glass-distilled water and then PBS for 20 min each. For total human T lymphocytes, a polyclonal rabbit anti-human CD3 Ab (Biomed, Fullerton, CA) was used for 1 h at room temperature, followed by extensive washing in PBS. Sections were incubated with 1% normal goat serum (Vector, Burlingame, CA), 1% nonfat milk, and 0.2 M ammonium acetate to block nonspecific binding. The sections were then incubated with goat anti-rabbit horseradish peroxidase (Bio-Rad, Richmond, CA) at room temperature for 30 min. After extensive washing in PBS, sections were incubated in diaminobenzidine (Research Genetics, Huntsville, AL), according to the manufacturer’s instructions. After extensive rinsing in water, sections were counterstained for 2 min with Mayer’s hematoxylin solution, followed by 2 min in Blueing solution. After extensive washing in water, sections were mounted under coverslips. For human CD4⁺ or CD8⁺ T lymphocyte subsets, biotin-labeled anti-human CD4 or CD8 mAbs (Caltag, South San Francisco, CA) were used, followed by avidin-horseradish peroxidase (Vector) and diaminobenzidine as substrate.

Immunocytochemical colocalization of DNA fragmentation (apoptosis) and surface markers of human T lymphocytes in SCID mice LNs

Frozen sections of tissues were prepared as described above and immunostained with either CD4 or CD8 Ab. Detection of DNA fragmentation was performed by the terminal deoxynucleotidyltransferase (TdT)-mediated 5'-uridine triphosphate (UTP) nick-end labeling (TUNEL) method (32) with some modifications. Briefly, sections were treated with Autozyme (Biomed) for 60 min at room temperature, according to the manufacturer’s instructions. After washing with PBS, sections were soaked in Tris-EDTA (pH 8) for 10 min and then in TdT buffer (25 mM Tris chloride, 200 mM sodium cacodylate, 5 mM cobalt chloride) for 10 min. TdT-mediated uptake of deoxyUTP-digoxigenin (Boehringer Mannheim, Indianapolis, IN) was performed at 37°C for 1 h, followed by washing in TBS (50 mM Tris HCl, 138 mM NaCl, 3 mM KCl (pH 8)) three times for 10 min each. Sections were blocked for 30 min at room temperature in TBS containing 2% BSA and 0.1% Triton, followed by incubation with alkaline phosphatase-conjugated anti-digoxigenin F(ab')₂ fragment (Boehringer Mannheim) diluted 1/500 in TBS plus BSA and Triton for 30 min at room temperature. After extensive washing with TBS, phosphatase activity was developed using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution (Sigma). After 5 min, the sections were washed with distilled water, covered with water-soluble media, and coverslipped. Sections of a previously tested LN with intense DNA fragmentation signal with or without TdT treatment were run in parallel as positive and negative controls, respectively. A total of 5–10 frozen sections of LNs from two to five mice sacrificed at 2, 4, and 6 days postinjection were photographed at x400 magnification, and the numbers of total human cells and TUNEL-positive human cells were visually counted.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
We first explored the fate of resting T lymphocytes abortively infected with HIV and induced to home into the LNs by signaling through their homing receptors. Cross-linking of the homing receptors CD62L, CD44, or CD11a with mAbs induced 25–38% of the HIV-exposed PBLs to die by 5 days, whereas mock-exposed resting PBLs did not significantly die when they were cross-linked (Fig. 1, A and B). Cross-linking of CD103 (a receptor involved in homing of a subset of T lymphocytes to mucosal lymphoid tissues), CD26, CD8, or CD4 with mAbs also did not affect viability of HIV-exposed PBLs (Fig. 1), demonstrating specificity of this effect following signaling through homing receptors. Since CD26 expression is low on resting lymphocytes, we examined cell death following cross-linking of some highly expressed receptors on resting lymphocytes, uninfected or abortively infected with HIV (Fig. 1C). Cross-linking of HLA class I or CD45RO and RA did not induce death of HIV-exposed PBLs. This is in contrast to cross-linking of CD62L on PBLs preexposed to HIV₂₁₃ or HIV_MCK (both T cell-tropic strains) or HIV_BAL (a macrophage-tropic HIV), which induced 30–50% of the PBLs to die by 4 days postexposure (Fig. 1C). CD62L cross-linking of mock-infected PBLs or PBLs pretreated with supernatant from ultracentrifuged HIV₂₁₃ stocks (to remove HIV particles (7)) did not induce death (Fig. 1C). Our previous study showed that supernatant from ultracentrifuged HIV stocks could not up-regulate homing receptors, but UV- or heat-inactivated HIV could induce CD62L expression (7). Similarly, heat-inactivated HIV₂₁₃-exposed PBLs still died following CD62L cross-linking (Fig. 1C). This effect followed a dose response, for if lower doses of infectious or inactivated HIV were used, reduced numbers of apoptotic cells following homing receptor cross-linking were observed (data not shown). Thus, binding of HIV particles to the cells appeared to induce a state in which second signals through homing receptors resulted in apoptosis.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Signaling through homing receptors CD62L, CD44, or CD11a induced death of HIV-exposed PBLs. Resting PBLs were exposed to HIV for 12 h in culture and then signaled through the indicated cell surface receptors by mAb cross-linking. Cell death was monitored by trypan blue exclusion. A, Kinetics of cell death following HIV exposure and mAb cross-linking. Data shown are the mean and SE of five separate experiments. B, Kinetics of cell death following mAb cross-linking of mock-treated PBLs. C, Summation of maximum cell death by 4 days for PBLs pretreated with CEM culture fluid (mock), supernatant from ultracentrifuged (30,000 rpm, 2 h) HIV213 stock (Sup), heat-inactivated (56°C, 1 h) HIV213 (HI-213), or stocks of HIV213, HIVMCK, or HIVBAL (at an MOI of 1) and cross-linked by anti-CD62L, anti-CD45, or anti-HLA class I mAbs.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. CD4+ but not CD8+ T lymphocytes died after HIV exposure and cross-linking of lymphocyte homing receptors. HIV exposure and receptor cross-linking were performed as in Fig. 1. Data shown are the average from duplicate samples minus the percentage of dead cells in untreated control cells. Representatives of four experiments for each are shown, with similar results in all four. A, CD4+ T lymphocytes. B, CD8+ T lymphocytes.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Secondary signaling through homing receptors on HIV-exposed resting CD4 PBLs did not induce the virus to replicate. HIV exposure and CD62L, CD44, and CD3 receptor cross-linking were performed as in Fig. 1. Culture supernatants were sampled and changed daily. HIV p24 was quantitated in these supernatants by Ag capture enzyme immunoassay (Coulter). Each point was performed in triplicate. Two separate experiments were performed, giving similar results (one is shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Cross-linking of L-selectin and CD44 with their natural ligands induced death of HIV-exposed CD4+, but not CD8+ T lymphocytes. Purified CD4+ and CD8+ T lymphocytes were prepared and treated as described in Fig. 1, and then seeded into 48-well plates (Costar) pretreated with M6P, HA, M1P, or CHA. Cell death was determined by trypan blue exclusion. The data shown are the average of duplicate samples minus the percentage of dead cells in untreated control cells. Representatives of three experiments for each are shown, with similar results in all three.

The earliest detectable problem of the immune system in HIV-infected subjects is a poor Th response to recall Ags, which is known to be mediated by memory T cells (CD45RO⁺) (35, 36). One explanation may be that memory cells are eliminated earlier in the course of infection, perhaps because these cells express reduced amounts of Bcl-2 (37). We, therefore, asked whether CD4⁺ memory cells were more susceptible to induction of death than were naive cells (CD45RA⁺). We found both memory and naive CD4⁺ T lymphocytes preexposed to HIV were equally susceptible to induction of death by cross-linking of the homing receptors (Fig. 5). Thus, whether or not the cell dies following signaling through its homing receptors does not depend upon whether it is a naive or memory CD4⁺ T cell. Furthermore, the levels of expression of the homing receptors did not correlate to whether or not the cells died (data not shown).

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Memory (CD45RO+) and naive (CD45RA+) CD4 T lymphocytes were equally susceptible to induction of death after HIV exposure and cross-linking of the homing receptors. HIV exposure and cross-linking of the receptors were performed as described in Fig. 1. The data are averaged from duplicate samples minus the percentage of dead cells in untreated control cells. Representatives of two experiments for each are shown. Cross-linking of the homing receptors did not induce death of either memory or naive CD8+ T lymphocytes after HIV exposure (two experiments, data not shown).

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Cross-linking of the homing receptors of HIV-exposed CD4+ T lymphocytes induced apoptosis. Apoptosis was evaluated by the TUNEL (TdT-mediated biotin-deoxyUTP-end labeling) method (A) and electron microscopy (EM) (B), as described in Materials and Methods. A, At the indicated time points after mAb cross-linking of HIV-exposed or mock-treated resting CD4+ T lymphocytes, the cells were pelleted and washed twice in cold PBS, fixed in 2% paraformaldehyde, permeabilized in acetone, labeled by the TUNEL method (32), and analyzed by flow cytometry. B and C, At 3 days after HIV exposure and cross-linking with indicated mAbs, the CD4+PBLs were pelleted by centrifugation into Beam capsules (1000 x g), fixed immediately in a cacodylate-buffered 2% paraformaldehyde and 2% glutaraldehyde, and postfixed in 1% osmium tetroxide. After dehydration with a series of graded ethanol concentrations, the specimens were embedded in Polybed 812 resin. Thin sections were cut with a Reichert Ultracut ultramicrotome and poststained with uranyl acetate and lead citrate. The specimens were examined and photographed on a Philips 201 electron microscope.

View larger version (123K): [in this window] [in a new window]   FIGURE 7. Immunocytochemical staining of human T lymphocytes in SCID mice LNs. Frozen sections of LNs were obtained 4 days after i.v. injection of resting human PBLs. A and B were stained with anti-human CD3 Ab (brown) and counterstained with hematoxylin (blue). A is a LN section from a mouse i.v. injected with mock-treated resting human PBLs, and B is from a mouse given HIV-exposed PBLs. C, D, E, and F are immunocytochemical colocalization of DNA fragmentation and lymphocyte surface markers. DNA fragmentation (apoptotic cells) was labeled by the TUNEL method (dark blue/black). Human T lymphocytes were stained by anti-human CD8 (C, E) or CD4 (D, F) Abs (brown). Double-labeled cells are dark brown or with dark nuclei (arrows). A and B, x400 magnification; C and D, x650; and E and F, x1000.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Normally, T lymphocytes continuously circulate from the blood into lymphoid organs and then back to the blood (38). Exposure to HIV enhances homing of these cells from the blood into the LNs (7). These are resting lymphocytes that are not permissive for productive infection by HIV and comprise most (98–99%) of the total lymphocytes. Our results show that certain signals will induce apoptosis in the resting cells that were exposed previously to HIV, and that these dying cells are not producing virus. The high frequency of apoptosis following signaling through the homing receptors can help explain the bystander effect observed in the LNs of AIDS patients, in which cells not producing HIV are dying (21). Since most of the lymphocytes in vivo are resting and not permissive for productive HIV infection (33, 39, 40), any detrimental effect of HIV on these cells could have profound consequences.

Our findings that resting CD4⁺ lymphocytes preexposed to heat-inactivated HIV also underwent apoptosis certainly implicates that the virus is causing this cellular state via its binding to a receptor and inducing signals. It also could further implicate pathogenic roles for noninfectious virus. In the blood, most viral particles are noninfectious (41). However, most virus is produced from cells in the LNs, and by the time newly produced virus appears in the blood, it is most likely coated with neutralizing Abs. Thus, virus binding to resting lymphocytes is probably rare in the blood but frequent in the LNs. Many lymphocytes in the LNs recirculate back into the blood within a 24-h period, in numbers equal to those homing out of the blood into the LNs over the same period. There are data indicating that 1.5% of the blood lymphocytes home out per hour (42), and thus 35–40% of the blood lymphocytes (which in turn comprise 2% of the total lymphocytes in the body) circulate through the LNs and back into the blood per day (43). It has been shown that 7500 lymphocytes per million in the LNs at any given time are harboring unintegrated HIV DNA (14), and the vast majority of these are likely to be resting CD4⁺ lymphocytes abortively infected with HIV (33). Many of these cells will recirculate back into the blood, and since HIV-induced up-regulation of L-selectin reaches a maximum at 48 h, there is time for many of these cells to return to the blood before maximum expression of L-selectin. These cells, in turn, will possess an even greater propensity to home back into LNs, and this time, many of them will then be induced into apoptosis. These pronounced effects following exposure of resting lymphocytes to a high MOI (1) of HIV in vitro most likely also occur in the local environment of a productively infected cell in the LNs of HIV-infected patients. Our in vitro experiments exposed each lymphocyte to between 10–100 virions, resulting in the majority (70%) of the exposed cell population homing out of the blood within 2 h after i.v. injection into SCID mice (7). We suspect that CD4⁺ T cell numbers in the blood of patients would similarly drop very rapidly if 10–100 HIV particles per CD4⁺ lymphocyte could be achieved. More realistic is that only a limited number of resting CD4⁺ lymphocytes (those immediately surrounding a productively infected cell) in the LNs are signaled similarly to what we achieved in vitro, and that many of these cells that appear in the blood within the ensuing 24–72 h may be destined for homing-induced death.

Our data also indicate that both macrophage-tropic and T cell-tropic HIV strains similarly induced this state in resting lymphocytes, whereby second signals through the homing receptors induced apoptosis. Studies using the SCID/hu model system have shown that the macrophage-tropic strains are more effective in depleting CD4 lymphocytes in comparison with T cell-tropic strains (44, 45). It is possible that these differential effects are not due to inherent differences in the abilities of the viruses to induce the state we are studying, but rather may be due to differences in the extent or degree of virus spread and replication in this mouse system (46). Studies have indicated that infected humans possessing either macrophage-tropic or T cell-tropic HIVs progress in disease (47).

We thus propose the following scheme as a major mechanism of HIV pathogenesis. HIV is produced in the LNs, as this is where most of the productively infected cells reside, and is binding to surrounding resting CD4⁺ lymphocytes. Many of these abortively infected bystander CD4⁺ cells will recirculate back into the blood within 1 or 2 days. These T lymphocytes then home back into the LNs at an enhanced rate, accounting for disappearance of the CD4⁺ lymphocytes in the blood. As they home in, signals are induced through the homing receptors that trigger induction of apoptosis in about one-half of the CD4⁺ T lymphocytes, but not in the CD8⁺ T lymphocytes. These dying CD4⁺ lymphocytes never make HIV (hence, a bystander effect). A few of the CD4⁺ T lymphocytes that survive may enter the cell cycle if signaled by Ag in the LNs, and then they will produce virus. Thus, as more of these cells home into the LNs, more productively infected cells result and HIV levels in the body should continuously rise. This in turn will induce more CD4⁺ lymphocytes to eventually home from the blood into the LNs and die. Only a small fraction of virus-producing cells is needed to account for the estimated amounts of HIV produced per day (10⁹–10¹⁰ virions) (48), since one productively infected cell produces 10²–10³ virions per 24-h period (49, 50). Thus, only 10⁷ productively infected cells of 10¹² total lymphocytes in the whole body would suffice to produce the observed amounts of virus, and most of these virions would be interacting with resting lymphocytes.

These effects of HIV (induction of LN homing and apoptosis after signaling through homing receptors) on resting T lymphocytes may explain the depletion of CD4⁺ T lymphocytes from the peripheral blood when very few blood CD4⁺ lymphocytes are productively infected, and the development of lymphadenopathy at a time when CD4⁺ T cell numbers are falling in the blood. It also can help explain some of the depletion of CD4⁺ T lymphocytes from the LN. Therapeutic approaches involving inhibition of viral-induced homing and/or homing-induced apoptosis, together with inhibition of viral replication, may prove beneficial for HIV-infected subjects.

	Acknowledgments

 
We thank Monica Wolf for valuable technical assistance in in situ staining and Jason Huang for blood collection. We gratefully acknowledge the National Institutes of Health AIDS Research and Reagents Program for mAbs.

	Footnotes

 
¹ This work was partially supported by a grant (AI38530) from the National Institutes of Health.

² Address correspondence and reprint requests to Dr. Miles W. Cloyd, Departments of Microbiology and Immunology and Pathology, University of Texas Medical Branch, Galveston, TX 77555-1019. E-mail address:

³ Abbreviations used in this paper: LN, lymph node; CHA, chondroitin sulfate A; HA, hyaluronic acid; M1P, mannose-1-phosphate; M6P, mannose-6-phosphate; MOI, multiplicity of infection; UTP, 5'-uridine triphosphate TdT, terminal deoxynucleotidyltransferase; TUNEL, TdT-mediated UTP nick-end labeling.

Received for publication June 4, 1998. Accepted for publication September 1, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Bagnarelli, P., S. Menzo, A. Valenza, A. Manzin, M. Giaca, F. Ancarani, G. Scalise, P. E. Varaldo, M. Clementi. 1992. Molecular profile of human immunodeficiency virus type 1 infection in symptomless patients and in patients with AIDS. J. Virol. 66:7328.[Abstract/Free Full Text]
2. Gupta, P., J. Kingsley, J. Armstrong, M. Ding, M. Cottrill, C. Rinaldo. 1993. Enhanced expression of human immunodeficiency virus type 1 correlates with development of AIDS. Virology 196:586.[Medline]
3. Furtado, M. R., R. Murphy, J. Armstrong, M. Ding, M. Cottrill, C. Rinaldo. 1993. Quantification of human immunodeficiency virus type 1 tat mRNA as a marker for assessing the efficacy of antiretroviral therapy. J. Infect. Dis. 167:213.[Medline]
4. Bagnarelli, P., A. Valenza, S. Menzo, A. Manzin, G. Scalise, P. E. Varaldo, M. Clementi. 1994. Dynamics of molecular parameters of human immunodeficiency virus type 1 activity in vivo. J. Virol. 68:2495.[Abstract/Free Full Text]
5. Janossy, G., A. J. Pinching, M. Bofill, J. Weber, J. E. McLaughlin, M. Ornstein, K. Ivory, J. R. Harris, M. Favrot, D. C. Macdonald-Burns. 1985. An immunohistological approach to persistent lymphadenopathy and its relevance to AIDS. Clin. Exp. Immunol. 59:257.[Medline]
6. Rosenberg, Y. J., P. M. Zack, B. D. White, S. F. Papermaster, W. R. Elkins, G. A. Eddy, M. G. Lewis. 1993. Decline in the CD4⁺ lymphocyte population in the blood of SIV-infected macaques is not reflected in lymph nodes. AIDS Res. Hum. Retroviruses 9:639.[Medline]
7. Wang, L., C. W. Robb, M. W. Cloyd. 1997. HIV induces homing of resting T lymphocytes to lymph nodes. Virology 228:141.[Medline]
8. Embertson, J., M. Zupancic, J. Beneke, M. Till, S. Wolinsky, J. L. Ribas, A. Burke, A. T. Haase. 1993. Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infection at single-cell resolution. Proc. Natl. Acad. Sci. USA 90:357.[Abstract/Free Full Text]
9. Pantaleo, G., C. Graziosi, J. F. Demarest, L. Butini, M. Montroni, C. H. Fox, J. M. Orenstein, D. P. Kotler, A. S. Fauci. 1993. HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature 362:355.[Medline]
10. Nuovo, G. J., J. Becker, M. W. Burk, M. Margiotta, J. Fuhrer, R. T. Steigbigel. 1994. In situ detection of PCR-amplified HIV-1 nucleic acids in lymph nodes and peripheral blood in patients with asymptomatic HIV-1 infection and advanced-stage AIDS. J. Acquired Immune Defic. Syndr. 7:916.
11. Schmitz, J., J. Van Lunzen, K. Tenner-Racz, G. Grossschupff, P. Racz, H. Schmitz, M. Dietrich, F. T. Hufert. 1994. Follicular dendritic cells retain HIV-1 particles on their plasma membrane but are not productively infected in asymptomatic patients with follicular hyperplasia. J. Immunol. 153:1352.[Abstract]
12. Harper, M. E., L. M. Marselle, R. C. Gallo, F. Wong-Staal. 1986. Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc. Natl. Acad. Sci. USA 83:772.[Abstract/Free Full Text]
13. Embertson, J., M. Zupancic, J. L. Ribas, A. Burke, P. Racz, K. Tenner-Racz, A. T. Haase. 1993. Massive covert infection of helper T lymphocytes and macrophages by HIV during incubation period of AIDS. Nature 362:359.[Medline]
14. Chun, T. W., L. Carruth, D. Finzi, X. Shen, J. A. DiGiuseppe, H. Taylor, M. Hermankova, J. Chadwick, J. Margolick, T. C. Quinn, Y. H. Kuo, R. Bookmeyer, et al 1997. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387:183.[Medline]
15. Gougeon, M. L., S. Garcia, J. Heeney, R. T. Schopp, H. Lecoeur, D. Guetard, V. Rame, C. Dauguet, L. Montagnier. 1993. Programmed cell death in AIDS-related HIV and SIV infections. AIDS Res. Hum. Retroviruses 9:553.[Medline]
16. Li, C. J., D. J. Friedman, C. Wang, V. Metelev, A. B. Pardee. 1995. Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein. Science 268:429.[Abstract/Free Full Text]
17. Groux, H., G. Torpier, D. Monte, Y. Mouton, A. Capron, J. C. Ameisen. 1992. Activation-induced death by apoptosis in CD4⁺ T cells from human immunodeficiency virus-infected asymptomatic individuals. J. Exp. Med. 175:331.[Abstract/Free Full Text]
18. Katsikis, P. D., E. S. Wunderlich, C. A. Smith, L. A. Herzenberg. 1995. Fas antigen stimulation induces marked apoptosis of T lymphocytes in human immunodeficiency virus-infected individuals. J. Exp. Med. 181:2029.[Abstract/Free Full Text]
19. Meyaard, L., S. A. Otto, R. R. Jonker, M. J. Mijnster, R. P. Keet, F. Miedema. 1992. Programmed death of T cells in HIV-1 infection. Science 257:217.[Abstract/Free Full Text]
20. Gougeon, M. L., H. Lecoeur, A. Dulioust, M. G. Enouf, M. Crouvoiser, C. Goujard, T. Debord, L. Montagnier. 1996. Programmed cell death in peripheral lymphocytes from HIV-infected persons: increased susceptibility to apoptosis of CD4 and CD8 T cells correlates with lymphocyte activation and with disease progression. J. Immunol. 156:3509.[Abstract]
21. Finkel, T. H., G. Tudor-Williams, N. K. Banda, M. F. Cotton, T. Curiel, C. Monks, T. W. Baba, R. M. Ruprecht, A. Kupfer. 1995. Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes. Nat. Med. 1:129.[Medline]
22. Gougeon, M. L., A. G. Laurent-Crawford, A. G. Hovanessian, L. Montagnier. 1993. Direct and indirect mechanisms mediating apoptosis during HIV infection: contribution to in vivo CD4 T cell depletion. Semin. Immunol. 5:187.[Medline]
23. Muro-Cacho, C. A., G. Pantaleo, A. S. Fauci. 1995. Analysis of apoptosis in lymph nodes of HIV-infected persons: intensity of apoptosis correlates with the general state of activation of the lymphoid tissue and not with stage of disease or viral burden. J. Immunol. 154:5555.[Abstract]
24. Wacholtz, M. C., S. S. Patel, P. E. Lipsky. 1989. Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules. J. Immunol. 142:4201.[Abstract]
25. DeMeester, I. A., L. L. Kestens, G. L. Vanham, G. C. Vanhoof, J. H. Vingerhoets, P. L. Gigase, S. L. Scharpe. 1995. Costimulation of CD4⁺ and CD8⁺ T cells through CD26: the ADA-binding epitope is not essential for complete signaling. J. Leukocyte Biol. 58:325.[Abstract]
26. Lorenz, H. M., T. Harrer, A. S. Lagoo, A. Baur, G. Eger, J. R. Kalden. 1993. CD45 mAb induces cell adhesion in peripheral blood mononuclear cells via lymphocyte function-associated antigen-1 (LFA-1) and intercellular cell adhesion molecule 1 (ICAM-1). Cell. Immunol. 147:110.[Medline]
27. McCallus, D. E., K. E. Ugen, A. I. Sato, W. V. Williams, D. B. Weiner. 1992. Construction of a recombinant bacterial human CD4 expression system producing a bioactive CD4 molecule. Viral Immunol. 5:163.[Medline]
28. Williams, L. M., M. W. Cloyd. 1991. Polymorphic human gene(s) determines differential susceptibility of CD4 lymphocytes to infection of certain HIV-1 isolates. Virology 184:723.[Medline]
29. Aruffo, A., I. Stamenkovic, M. Melnick, C. B. Underhill, B. Seed. 1990. CD44 is the principal cell surface receptor for hyaluronate. Cell 61:1303.[Medline]
30. Varki, A.. 1994. Selectin ligands. Proc. Natl. Acad. Sci. USA 91:7390.[Abstract/Free Full Text]
31. Oxley, S. M., R. Sackstein. 1994. Detection of an L-selectin ligand on a hematopoietic progenitor cell line. Blood 84:3299.[Abstract/Free Full Text]
32. Gavrieli, Y., Y. Sherman, S. A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119:494.
33. Zack, J. A., A. M. Haislip, P. Krogstad, I. S. Y. Chen. 1992. Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle. J. Virol. 66:1717.[Abstract/Free Full Text]
34. Harris, H., M. Miyasaka. 1995. Reversible stimulation of lymphocyte motility by cultured high endothelial cells: mediation by L-selectin. Immunology 84:47.[Medline]
35. Van Nosel, C. J., R. A. Gruters, F. G. Terpstra, P. T. Schellekens, R. A. Van Lier, F. Miedema. 1990. Functional and phenotypic evidence for a selective loss of memory T cells in asymptomatic human immunodeficiency virus-infected men. J. Clin. Invest. 86:293.
36. Shearer, G. M., M. Clerici. 1991. Early T-helper cell defects in HIV infection. AIDS 5:245.[Medline]
37. Akbar, A. N., N. Borthwick, M. Salmon, W. Gombert, M. Bofill, N. Shamsadeen, D. Pilling, S. Pet, J. E. Grundy, G. Janossy. 1993. The significance of low bcl-2 expression by CD45RO T cells in normal individuals and patients with acute viral infections: the role of apoptosis in T cell memory. J. Exp. Med. 178:427.[Abstract/Free Full Text]
38. Picker, L. J., E. C. Butcher. 1992. Physiological and molecular mechanisms of lymphocyte homing. Annu. Rev. Immunol. 10:561.[Medline]
39. Stevenson, M., T. L. Stanwick, M. P. Dempsey, C. A. Lamonica. 1990. HIV replication is controlled at the level of T-cell activation and proviral integration. EMBO J. 9:1551.[Medline]
40. Pomerantz, R. J., D. Trono, M. B. Feinberg, D. Baltimore. 1990. Cells nonproductively infected with HIV-1 exhibit an aberrant pattern of viral RNA expression: a molecular model for latency. Cell 61:1271.[Medline]
41. Layne, S. P., M. J. Merges, J. L. Spouge, M. Dembo, P. L. Nara. 1991. Blocking of human immunodeficiency virus infection depends on cell density and viral stock age. J. Virol. 65:3292.
42. Ford, W. L., J. L. Gowans. 1969. The traffic of lymphocytes. Semin. Hematol. 6:67.[Medline]
43. Smith, M. E., W. L. Ford. 1969. The recirculating lymphocyte pool of the rat: a systemic description of the migratory behavior of recirculating lymphocytes. Immunology 49:83.
44. Kaneshima, H., C. C. Shih, R. Namikawa, L. Rabin, H. Outzen, S. G. Machado, J. M. McCune. 1991. Human immunodeficiency virus infection of human lymph nodes in the SCID-hu mouse. Proc. Natl. Acad. Sci. USA 88:4523.[Abstract/Free Full Text]
45. Mosier, D. E., R. J. Gulizia, P. D. MacIsaac, B. E. Torbett, J. A. Levy. 1993. Rapid loss of CD4⁺ T cells in human-PBL-SCID mice by noncytopathic HIV isolates. Science 260:689.[Abstract/Free Full Text]
46. Picchio, G. R., R. J. Gulizia, K. Wehrly, B. Chesebro, D. E. Mosier. 1998. The cell tropism of human immunodeficiency virus type 1 determines the kinetics of plasma viremia in SCID mice reconstituted with human peripheral blood leukocytes. J. Virol. 72:2002.[Abstract/Free Full Text]
47. Mosier, D., H. Sieburg. 1994. Macrophage-tropic HIV: critical for AIDS pathogenesis?. Immunol. Today 17:332.
48. Perelson, A. S., A. U. Neumann, M. Markowitz, J. M. Leonard, D. D. Ho. 1996. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 271:1582.[Abstract]
49. Levy, J. A., B. Ramachandran, E. Barker, J. Guthrie, T. Elbeik. 1996. Plasma viral load, CD4⁺ cell counts, and HIV-1 production by cells. Science 271:670.[Medline]
50. Tsai, W. P., S. R. Conley, H. F. Kung, R. R. Garrity, P. L. Nara. 1996. Preliminary in vitro growth cycle and transmission studies of HIV-1 in an autologous primary cell assay of blood-derived macrophages and peripheral blood mononuclear cells. Virology 226:205.[Medline]

This article has been cited by other articles:

				J. Ji and M. W. Cloyd HIV-1 binding to CD4 on CD4+CD25+ regulatory T cells enhances their suppressive function and induces them to home to, and accumulate in, peripheral and mucosal lymphoid tissues: an additional mechanism of immunosuppression Int. Immunol., March 1, 2009; 21(3): 283 - 294. [Abstract] [Full Text] [PDF]

				J. Ji, J. J-Y. Chen, V. L. Braciale, and M. W. Cloyd Apoptosis induced in HIV-1-exposed, resting CD4+ T cells subsequent to signaling through homing receptors is Fas/Fas ligand-mediated J. Leukoc. Biol., January 1, 2007; 81(1): 297 - 305. [Abstract] [Full Text] [PDF]

				C. Debacq, N. Gillet, B. Asquith, M. T. Sanchez-Alcaraz, A. Florins, M. Boxus, I. Schwartz-Cornil, M. Bonneau, G. Jean, P. Kerkhofs, et al. Peripheral Blood B-Cell Death Compensates for Excessive Proliferation in Lymphoid Tissues and Maintains Homeostasis in Bovine Leukemia Virus-Infected Sheep J. Virol., October 1, 2006; 80(19): 9710 - 9719. [Abstract] [Full Text] [PDF]

				S. Kandula and C. Abraham LFA-1 on CD4+ T Cells Is Required for Optimal Antigen-Dependent Activation In Vivo J. Immunol., October 1, 2004; 173(7): 4443 - 4451. [Abstract] [Full Text] [PDF]

				V. Monceaux, R. H. T. Fang, M. C. Cumont, B. Hurtrel, and J. Estaquier Distinct Cycling CD4+- and CD8+-T-Cell Profiles during the Asymptomatic Phase of Simian Immunodeficiency Virus SIVmac251 Infection in Rhesus Macaques J. Virol., September 15, 2003; 77(18): 10047 - 10059. [Abstract] [Full Text] [PDF]

				J. J-Y. Chen, J. C. Huang, M. Shirtliff, E. Briscoe, S. Ali, F. Cesani, D. Paar, and M. W. Cloyd CD4 lymphocytes in the blood of HIV+ individuals migrate rapidly to lymph nodes and bone marrow: support for homing theory of CD4 cell depletion J. Leukoc. Biol., August 1, 2002; 72(2): 271 - 278. [Abstract] [Full Text] [PDF]

				D. C. Douek, M. R. Betts, B. J. Hill, S. J. Little, R. Lempicki, J. A. Metcalf, J. Casazza, C. Yoder, J. W. Adelsberger, R. A. Stevens, et al. Evidence for Increased T Cell Turnover and Decreased Thymic Output in HIV Infection J. Immunol., December 1, 2001; 167(11): 6663 - 6668. [Abstract] [Full Text] [PDF]

				S. M. Wahl, T. Greenwell-Wild, H. Hale-Donze, N. Moutsopoulos, and J. M. Orenstein Permissive factors for HIV-1 infection of macrophages J. Leukoc. Biol., September 1, 2000; 68(3): 303 - 310. [Abstract] [Full Text] [PDF]

				T. M. Tumpey, X. Lu, T. Morken, S. R. Zaki, and J. M. Katz Depletion of Lymphocytes and Diminished Cytokine Production in Mice Infected with a Highly Virulent Influenza A (H5N1) Virus Isolated from Humans J. Virol., July 1, 2000; 74(13): 6105 - 6116. [Abstract] [Full Text]

				T. A. Bennett, B. S. Edwards, L. A. Sklar, and S. Rogelj Sulfhydryl Regulation of L-Selectin Shedding: Phenylarsine Oxide Promotes Activation-Independent L-Selectin Shedding from Leukocytes J. Immunol., April 15, 2000; 164(8): 4120 - 4129. [Abstract] [Full Text] [PDF]

				M. D. Hazenberg, J. W. T. C. Stuart, S. A. Otto, J. C. C. Borleffs, C. A. B. Boucher, R. J. de Boer, F. Miedema, and D. Hamann T-cell division in human immunodeficiency virus (HIV)-1 infection is mainly due to immune activation: a longitudinal analysis in patients before and during highly active antiretroviral therapy (HAART) Blood, January 1, 2000; 95(1): 249 - 255. [Abstract] [Full Text] [PDF]

				J. J.-Y. Chen and M. W. Cloyd The potential importance of HIV-induction of lymphocyte homing to lymph nodes Int. Immunol., October 1, 1999; 11(10): 1591 - 1594. [Abstract] [Full Text] [PDF]

				R. L. Hengel, B. M. Jones, M. S. Kennedy, M. R. Hubbard, and J. S. McDougal Markers of Lymphocyte Homing Distinguish CD4 T Cell Subsets That Turn Over in Response to HIV-1 Infection in Humans J. Immunol., September 15, 1999; 163(6): 3539 - 3548. [Abstract] [Full Text] [PDF]

				R. Maroto, X. Shen, and R. Konig Requirement for Efficient Interactions Between CD4 and MHC Class II Molecules for Survival of Resting CD4+ T Lymphocytes In Vivo and for Activation-Induced Cell Death J. Immunol., May 15, 1999; 162(10): 5973 - 5980. [Abstract] [Full Text] [PDF]

				D. Shutt and D. Soll HIV-induced T-cell syncytia release a two component T-helper cell chemoattractant composed of Nef and Tat J. Cell Sci., January 11, 1999; 112(22): 3931 - 3941. [Abstract] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, L. Articles by Cloyd, M. W. Search for Related Content PubMed PubMed Citation Articles by Wang, L. Articles by Cloyd, M. W.