Cold exposure regulates the renin-angiotensin system.

The effect of cold exposure on the systemic renin-angiotensin system and on regulation of the angiotensin II (Ang II) receptor was examined in target organs for Ang II with cardiovascular relevance (left ventricle, kidney, lung) and metabolic relevance [interscapular brown adipose tissue (ISBAT), liver] to the functional consequences of cold exposure. In time course studies, the effects were examined of 4 hr or 1, 3 and 7 days of exposure to cold (4 degrees C) on plasma Ang II concentration and Ang II receptor binding characteristics in rat liver. Plasma Ang II concentration increased 10-fold after 4 hr of cold exposure, returned to control levels at days 1 and 3 of cold exposure, and was again increased (2-fold) at 7 days of cold exposure. The affinity of [125I]Sar1, Ile8-Ang II binding in membranes prepared from rat liver was not altered in cold-exposed rats. The density (Bmax) of binding sites in liver from cold-exposed rats was increased by day 1 and remained elevated over time-matched controls. Alterations in Ang II receptor density did not parallel plasma Ang II concentration in their time course, suggesting that cold-induced regulation of the Ang II receptor was not substrate mediated. In rats from the 7-day time point of cold exposure, Ang II receptor binding characteristics were examined in ISBAT and lung. Increases in Ang II receptor density were evident in ISBAT but not lung. To determine whether cold-induced increases in food intake contributed to elevations in plasma Ang II concentration and/or Ang II receptor density, a group of cold-exposed rats (7 days) were pair-fed to food intake levels of control rats. Pair-feeding of cold-exposed rats eliminated increases in plasma Ang II and norepinephrine concentration but did not prevent increases in Ang II receptor density in liver, ISBAT, kidney and left ventricle. Moreover, increases in Ang II receptor density were augmented in kidney and left ventricle from cold-exposed rats that were pair-fed. Results from these studies demonstrate that cold exposure resulted in an increase in plasma Ang II concentration through mechanisms related to increased food intake. Elevations in food intake in cold-exposed rats contributed to tissue-specific increases in Ang II receptor density. Moreover, cold-induced increases in Ang II receptor density were not related to plasma Ang II concentration.

Phenotypic and functional activation of monocytes in HIV-1 infection: interactions with neural cells.

To investigate mechanisms that facilitate transendothelial migration of HIV-infected leukocytes and their interactions with neural tissues early in the disease, we studied peripheral blood from Centers for Disease Control class A patients. Patients' monocytes displayed increased quantities of the adhesion molecules CD11a, CD11b, and very late antigen 4 (VLA-4). Expression of these correlated directly with the numbers of monocytes that migrated through confluent endothelium. These ligands also mediated leukocyte interactions with cultured human neural cell lines. Although patients' cells bound in greater numbers, there was no evidence of target cell injury. To evaluate the direct effect of HIV-1 on monocyte neuroadhesion, we compared infected with uninfected monocytoid (U-937,THP-1) and T lymphoblastoid (MT-4) cell lines. HIV infection increased the neuroadhesiveness of monocytoid lines only. By using lines with more than 95% HIV-infected cells, we demonstrated that HIV-1 gp120 participates with lymphocyte function-associated antigen 1 and VLA-4 to mediate monocyte-neural cell interactions.

Inhibition of transcription of HIV-1 in infected human cells by oligodeoxynucleotides designed to form DNA triple helices.

The effect on human immunodeficiency virus 1 (HIV-1) viral transcription and subsequent gene expression mediated by mixed purine-pyrimidine oligodeoxyribonucleotides (oligodeoxynucleotides) designed to form collinear DNA triplexes with purine-rich elements in the viral promoter was evaluated in intact mammalian cell lines (MT4 and U937). Oligonucleotides HIV31 (5'-GTTTTTGGGTGTTGTGGGTGTGTGTGGTTTG-3') and HIV38 (5'-TGGGTGGGGTGGGGTGGGGGGGTGTGGGGTGTGGGGTG-3') were designed to interact with the transcription initiation site (-16 to + 13) and nuclear factor Sp1 binding site (-81 to -44) of HIV-1, respectively. Oligonucleotides, synthesized with a 3' amine blocking group (5'-R-O-PO2-OCH (CHOH)-CH2-NH+3-3') to prevent degradation by cellular nucleases, were readily taken up by MT4 cells from the culture medium, achieving measured intranuclear concentrations higher than the medium in less than 2 h of incubation. The 3' amine modified oligonucleotides were recoverable from the cells after 24 h as greater than 90% intact material. Treatment of acutely infected MT4 cells with either HIV31 or HIV38 significantly inhibited viral-associated cytopathology and P24 antigen production (p less than 0.001). Additionally, inhibition of P24 antigen release, culture supernatant viral titer, and expression of the intact 9.2-kb HIV-1 mRNA was observed when the chronically infected promonocyte cell line, U937, was treated with 10 microM HIV38. Control oligonucleotides with similar base composition did not inhibit virus expression in either cell line. Furthermore, inhibition of viral expression was not due to alpha-interferon induction resulting from oligonucleotide treatment. Both HIV31 and HIV38 associate with their respective DNA target duplexes at micromolar concentrations, and a strong negative ellipticity near 210 nm, characteristic of DNA triplexes, was observed in the circular dichroism spectrum of either target-oligonucleotide complex. These observations suggest that oligonucleotides, designed to form nucleic acid triplexes with specific proviral target sequences, can selectively inhibit transcription of viral mRNA in intact cells and suppress accumulation of viral products.

HIV-1-stimulated expression of CD11/CD18 integrins and ICAM-1: a possible mechanism for extravascular dissemination of HIV-1-infected cells.

To identify mechanisms which might facilitate emigration of HIV-1-infected cells from the circulation, we studied the effect of HIV-1 infection on T lymphocyte and monocytoid cell expression of molecules involved in adherence and translocation of leukocytes across endothelial cell barriers. CD11a, CD18, and ICAM-1 were demonstrated on up to 80% of HIV-1-infected H9 T cells by flow cytometry; these molecules were not evident on uninfected H9. CD18 mRNA was detected in HIV-infected, but not in uninfected H9 T cells. Cell surface expression of CD11a and CD18, but not ICAM-1, was increased on HIV-infected, as compared to uninfected U937 and THP1 monocytoid cells. Increased cell surface expression of the leukocyte integrins was associated with a significantly increased tendency of HIV-infected monocytoid cells to adhere to human umbilical vein endothelial cell monolayers or aggregate homotypically. Preincubating the monocytoid cells with anti-CD18 or anti-CD11a or preincubating endothelial cells with anti-ICAM-1 suppressed these cell to cell interactions. These studies suggest that HIV-1 infection stimulates cell surface expression of molecules involved in leukocyte adherence and transendothelial migration in vitro. Similar mechanisms may influence leukocyte trafficking, in vivo, and may play a role in the localization of HIV-1 infected cells in the central nervous system and other tissues.

The activation of calcium/phospholipid dependent protein kinase and the association with interleukin-2 production.

The thymic leukemia cell line EL4 has been shown to produce the lymphokine Interleukin-2 (IL-2) following stimulation with phorbol ester (PMA). We investigated intracellular enzyme pathways triggered by phorbol stimulation using an EL4 cell line which responds to PMA with IL-2 synthesis (EL4r) and one which does not produce IL-2 following stimulation (EL4nr). By comparing these two cell lines we hoped to establish which enzyme activities were associated with IL-2 synthesis. The enzyme pathways studied included calcium/phospholipid dependent protein kinase (C-kinase) activity, the induction of polyamine synthesis, RNA, DNA and protein synthesis and finally IL-2 production. Our results indicate that both EL4 cell lines have a receptor for PMA, which can activate the C-kinase enzyme. Further, in both cell lines PMA activates the nuclear synthesis of polyamines as demonstrated by ornithine decarboxylase induction. Both RNA and protein synthesis measured by 3H-uridine and 3H-leucine uptake respectively appear comparable between EL4r and EL4nr. The only difference in cellular responsiveness between EL4r and EL4nr was in the 3H-thymidine uptake, and IL-2 production. IL-2 production or lack of production was established by 3H-uridine and 3H-thymidine incorporation as well as viable cell count using the IL-2 dependent cell line CTLL-2. We, therefore, conclude that EL4r and EL4nr cells show similar intracellular responses to phorbol ester except for 3H-thymidine uptake and detectable IL-2 production. Our results suggest that failure of PMA-stimulated EL4nr cells to produce IL-2 is either due to inability of this cell line to synthesize IL-2 or the production of defective IL-2. It is not due to failure of PMA to activate C-kinase or the subsequent nuclear events.

Protein kinase activation and the immunosuppressant cyclosporine.

Cyclosporine (CsA), a potent immunosuppressant for the prevention of transplant rejection, modulates T lymphocyte activation by blocking antigen stimulation and the production of interleukin-2. The mode of action by which CsA generates this immunosuppressive effect is unknown. We have studied two early intracellular enzymes associated with mitogen activation. They include calcium/phospholipid-dependent protein kinase (C-kinase) and cAMP-dependent protein kinase (cAMPd PK). Changes in protein kinase activation were correlated with the immunosuppression of polyamine and DNA synthesis measured by ornithine decarboxylase (ODC) induction and 3H-thymidine incorporation, respectively. These studies utilized murine T cell tumor lines sensitive to the effects of CsA. Similar to the mitogen activation of human peripheral blood lymphocytes, CsA was capable of inhibiting the induction of ODC and 3H-thymidine uptake of T cell tumor lines cultured with either fresh serum or mitogen. In contrast, C-kinase and cAMPd PK activation stimulated by the addition of fresh serum was not affected by CsA. Further, CsA did not inhibit the direct activation of C-kinase with phorbol esters or the activation of cAMPd PK with exogenous cAMP. We conclude that CsA does not affect the activation of either C-kinase or cAMPd PK in T cell tumor lines when activated by either fresh serum or when stimulated with chemical agents. The suppression of ODC induction and 3H-thymidine incorporation associated with CsA treatment cannot be accounted for with changes in C-kinase and cAMPd PK activation.

Human peripheral blood monocytes release a 30,000 dalton factor (30 KD MF) that stimulates immunoglobulin production by activated B cells.

Supernatants (SN) of well-washed adherent human monocytes, obtained from T cell-depleted peripheral blood mononuclear cells, contain a 30,000 dalton protein (30 KD MF) that increases immunoglobulin (Ig) synthesis by EBV-activated B cells two- to fourfold. This factor is released spontaneously during the first 20 hr after monocytes are placed in culture. SN containing 30 KD MF are inactive in the thymocyte co-stimulator assay, under conditions that will detect as little as 0.5 U of purified IL 1. The addition of autologous T cells to isolated adherent monocytes, previously depleted of T cells, suppresses the release or activity of this B cell stimulator in a dose-dependent manner. In addition, 30 KD MF stimulates a two- to fourfold increase in IgA production by cells of an EBV-transformed B cell line (JB/FF line) without increasing incorporation of [3H]thymidine. In contrast, stimulation of this B cell line with up to 10 U of purified IL 1 increases IgA synthesis by less than 50%, and addition of up to 100 U of recombinant IL 2 causes no change whatsoever in IgA production. However, co-stimulation with 30 KD MF and recombinant IL 2 or recombinant gamma-interferon induces more Ig production than is caused by the monocyte factor alone. These observations suggest that the monocyte, in addition to acting as an antigen-presenting cell and source of IL 1, facilitates B cell differentiation by producing a factor which acts both independently and in synergy with cytokines produced by T cells to stimulate Ig production by B lymphocytes.

The effect of cyclosporine on ornithine decarboxylase induction with mitogens, antigens, and lymphokines.

Ornithine decarboxylase (ODC) is the initial enzyme in polyamine synthesis. An increase in ODC activity is associated with increased RNA, DNA, and protein synthesis. We have used the induction of ODC by mitogens and alloantigens in human peripheral blood lymphocytes as an intracellular marker of protein synthesis and lymphocyte activation. The immunosuppressive agent cyclosporine was found to inhibit both the mitogen and alloantigen stimulated induction of ODC in lymphocytes in a manner that parallels inhibition of subsequent 3H-thymidine incorporation. When purified T lymphocytes were stimulated with mitogen alone, minimal ODC activity was detected. The addition of 5% monocytes, human Interleukin-1 (IL-1), or T cell growth factor (IL-2) enhanced mitogen-induced ODC activity in T lymphocytes 4-10-fold. Cyclosporine inhibited the induction of ODC when T lymphocytes were combined with monocytes or growth factors. We conclude that (1) the induction of ODC in human lymphocytes by mitogen and alloantigen is inhibited in the presence of cyclosporine; (2) the induction of ODC activity in purified T lymphocytes requires the presence of both mitogen and monocytes or their products; (3) IL-1 and IL-2 can supplement for monocytes and augment the phytohemagglutinin induction of ODC in T lymphocytes; and (4) cyclosporine inhibits ODC induction in T lymphocytes stimulated with mitogen in the added presence of monocytes, IL-1, or IL-2. The inhibition of ODC induction and polyamine synthesis by cyclosporine adds insight into its mode of action on the mechanisms involved in early T cell activation.

The role of mitogens and lymphokines in the induction of ornithine decarboxylase (ODC) in T lymphocytes.

Polyamine synthesis occurs early in lymphocyte activation after stimulation with antigen or mitogen. Ornithine decarboxylase (ODC) is the primary enzyme in the polyamine cascade. We have examined the induction of ODC by mitogens and/or lymphokines in human peripheral blood T lymphocytes. When isolated populations of monocytes and T lymphocytes were stimulated with phytohemagglutinin (PHA) there was little or no change in ODC activity. The combination of T lymphocytes and monocytes enhanced mitogen-induced ODC activity 10-fold. Several interleukin 1 (IL 1)-containing supernatants and fractionated human IL 1 were capable of substituting for monocytes in supporting PHA induction of ODC in T lymphocytes. Interleukin 2 (IL 2) and IL 2-containing supernatants were also capable of increasing ODC activity in T lymphocytes in the absence of monocytes. Lymphokines alone in the absence of PHA could not induce ODC. We conclude that both mitogens and monocytes are required for the induction of polyamine synthesis in T lymphocytes, and that supernatants containing IL 1 or IL 1 and IL 2 can substitute for monocytes in the induction of ODC in mitogen-stimulated T lymphocytes.

A comparison of the regulatory effects of human monocytes, pulmonary alveolar macrophages (PAMs) and spleen macrophages upon lymphocyte responses.

Human monocytes, pulmonary alveolar macrophages (PAMs) and spleen macrophages were concentrated by immobilization on cold insoluble globulins. These cell preparations were 90 +/- 3%, 95 +/- 1% and 83 +/- 3% esterase rich, respectively, 87 +/- 4%, 95 +/- 3% and 66 +/- 11% phagocytic and 78 +/- 3%, 79 +/- 9% and 68 +/- 5% reactive with OKM1 monoclonal antibody. Spleen macrophages differed from the other two cell preparations in that significantly fewer reacted with 61D3 or 63D2 monoclonal antibodies. Monocytes and PAMs promoted the mixed leucocyte response by autologous lymphocytes when added at low concentrations, but suppressed this response at high concentrations. Spleen macrophages only promoted the mixed leucocyte reaction but were required in much higher numbers than either monocytes or PAMs for optimal promotion. Likewise, the added presence of monocytes or PAMs in high numbers suppressed Ig synthesis stimulated with pokeweed mitogen, while spleen macrophages were not suppressive in this system. This study shows that the distribution of macrophages that differ in their regulatory effects upon lymphocyte responses varies in different tissues. The human spleen is deficient in macrophage related suppression.

Suppression of lymphocyte responses by monocytes with untreated and treated multiple myeloma.

Studies were performed on 15 untreated and 14 treated patients with multiple myeloma. The monocyte content was normal in blood but elevated in mononuclear leukocytes (MNL) from treated but not untreated patients (p less than 0.001). This correlated with the severity of lymphopenia in blood (p less than 0.01). Three patterns of immunoglobulin(Ig) synthesis emerged. (1) Most untreated patients showed normal polyclonal responses to pokeweek mitogen. (2) Of 12 treated patients, the 8 whose MNL included greater than 30% monocytes had subnormal Ig responses to pokeweek mitogen. Ig synthesis increased when adherent cells that suppressed Ig synthesis were depleted. Suppression in vitro bore no relationship to polyclonal immunoglobulin levels in serum. (3) Three patients had early blood invasion by plasmacytoid cells. Their MNL spontaneously released large amounts of the Ig class of their serum gammopathies. Proliferative responses to phytohemagglutinin by MNL from all patients were reduced, in part due to monocytoid cell suppression and in part to intrinsic T-cell hyporesponsiveness. B- and T-cell responses in vitro are sometimes suppressed with myeloma. This is related to elevated monocyte percentages in MNL preparations. This excess of monocytes is a function of lymphopenia secondary to therapy, rather than the primary malignant process itself. No evidence was found that suppression by monocytes is qualitatively altered by myeloma or its treatment.

Angioimmunoblastic lymphadenopathy with hypogammaglobulinemia. Possible role of monocyte suppression.

A patient wit angioimmunoblastic lymphadenopathy had low serum immunoglobulin values and no antibodies to injected immunogens. This occurred despite the proliferation of polyclonal B cells. T cells were deficient in number and in lymphoproliferative responses, but their helper and suppressor functions were maintained. Ia-antigen bearing leukocytes from the patient stimulated poorly in mixed leukocyte culture. In vitro immunoglobulin synthesis by mononuclear leukocytes form the patient was severely impaired. These leukocytes actively suppressed immunoglobulin synthesis by normal cells from healthy subjects in co-culture. The responsible cell had characteristics of a monocyte. The suppression was selective for humoral immunity and was manifest despite normal numbers of monocytes. It appears that heterogeneous immunoregulatory abnormalities can underlie the syndrome of angioimmunoblastic lymphadenopathy. Furthermore, monocyte suppressor abnormalities may be implicated in clinical disease phenomena.

Spectrum of immunodeficiencies with Hodgkin's disease.

The role of six suppressor mechanisms upon T and B cell responses was studied on 17 untreated patients with Hodgkin's disease. Proliferative hyporesponsiveness to mitogen was greatly impaired in 8 of the 13 patients. 10 of these patients had an excessive degree of suppression by cells that adhered to foreign surfaces. Suppression by adherent cells correlated with impairment of proliferative responses and, in some instances, suppression was largely inhibited with indomethacin. Likewise, adherent cells suppressed immunoglobulin synthesis. A correlation was evident between suppression of T and B cell responses by adherent mononuclear leukocytes from individual patients. This suppression coincided with elevated percentages of monocytes in the patient mononuclear cell preparations. This excess of monocytes was not the result of a circulating monocytosis. The monocyte excess may have been acquired during isopyknic cell separation. A second form of suppression was observed in 5 of the 11 patients affected by a lymphocyte that neither adhered to glass wool nor required preactivation. It did not inhibit allogeneic lymphocytes, which contrasts with the suppressor abnormality of monocytoid cells.

Three suppressor systems in human blood that modulate lymphoproliferation.

Three suppressor system of T lymphocyte proliferation found in normal blood were characterized. The adherent cell suppressor system (ACSS) is effected by a steroid and radioresistant monocyte that survives well in culture. The prostaglandin-related suppressor system (PgSS) is effected by a similar cell but is distinct from the ACSS in terms of magnitude of suppression, effective monocyte concentration, and carrageenan or indomethacin sensitivity. The induced suppressor system (ISS) is effected by a T lymphocyte that is partially radiosensitive and loses activity after 24 hr in culture. Although the ISS is unaffected by age, ACSS and PgSS activity is, overall, higher among elderly than among young adult subjects. Activation of these three suppressor systems does not require cell replication. They are not restricted by histocompatibility barriers.

A serum inhibitor of immune regulation in patients with systemic lupus erythematosus.

Normal mononuclear leukocytes were incubated with serum from patients with active systemic lupus erythematosus (SLE) and healthy subjects and then studied on lymphoproliferative tests. Serum from SLE patients that contained an autoantibody to a subpopulation of thymus-derived (T) lymphocytes inhibited suppressor T-cell activity induced with concanavalin A. These sera did not inhibit lymphoproliferative responses or suppression by monocytoid cells. Mitogen-activated suppressor cells were not inhibited with serum from SLE patients or healthy subjects lacking T-cell autoantibody. This abnormality may contribute to the altered immune response that occurs with SLE.

Suppression of lymphoproliferation by high concentrations of normal human mononuclear leukocytes.

Mononuclear leukocytes (MNL) include cells that suppress lymphoproliferation in unstimulated and antigen-stimulated cultures. Suppression is demonstrated by increasing the concentration of cells added to cultures and does not require preactivation of suppressor cells. Suprression of 3H-thymidine incorporation occurred if the high concentrations of MNL were added to cultures before the proliferative responses commenced. This suppressive effect of high cell concentration upon 3H-thymidine incorporation is removed by depleting MNL present in high numbers of cells that adhere to foreign surfaces or by preincubating these cells with cycloheximide, puromycin, or pactamycin. The suppressor cell, which only functions when present in a viable state, is radioresistant, adheres to foreign surfaces, remains active through 5 days in culture, and equates with the presence of a cell that is rich in cytoplasmic esterase. The suppressor cell may be of the monocyte series and did not appear to belong to either the T or the B lymphocyte series. This study provides additional evidence that normal immune reactivity in man is under regulatory control. The suppressor mechanism identified herein with normal human MNL is probably related to a similar type of suppression (but at a much lower cell concentration) that has been described with Hodgkin's disease and solid tumors.

Lymphoproliferative responses to antigens mediated by human pulmonary alveolar macrophages.

We evaluated the ability of human pulmonary alveolar macrophages (PAMs) to mediate (3H)-thymidine incorporation by blood lymphocytes severely depleted of monocytes when stimulated with soluble microbial and allogeneic lymphocyte antigens. Low (less than 2%) concentrations of PAM's from nonsmokers or blood monocytes did not support optimal responses. Over all, at greater than or equal to 10% concentrations, PAM's from nonsmokers supported higher responses than monocytes. At less than or equal to 10% concentrations, PAM's from heavy cigarette smokers mediated significantly less incorporation than did similar concentrations of PAM's from nonsmokers (p less than 0.05). The findings indicate that PAM's from healthy nonsmokers are functionally competent macrophages in terms of mediating lymphoproliferation in cultures stimulated with antigens. This classical macrophage function is impaired with cigarette smoking.

Reactivity of lymphocytes from primary neoplasms of lymphoid tissues.

Proliferative responses by blood and tumor lymphocytes to plant mitogens and allogeneic leukocyte antigens were tested concomitantly on 12 patients with Hodgkin's disease, 10 with chronic lymphocytic leukemia, and seven with non-Hodgkin's lymphomas. In 13 control studies, 3H-thymidine incorporation by blood and lymph node lymphocytes was brisk and, overall, comparable. With Hodgkin's disease, where extent of disease involvement and lymphocyte-depleted tumor histology were factors in the degree of responsiveness, incorporation was higher or at least comparable by tumor lymphocytes when compared with incorporation by autologous blood lymphocytes. Lymph node lymphocytes, especially with clinically stable disease, were more responsive than blood lymphocytes with chronic lymphocytic leukemia. Conversely, tumor lymphocytes were hyporesponsive compared with autologous blood lymphocytes with non-Hodgkin's lymphomas, where prognosis is usually less favorable than with chronic lymphocytic leukemia. Plasma from four out of 33 patients, although not lymphocytotoxic, inhibited lymphoproliferative responses.