When engineered to produce latent TGF-beta1, antigen specific T cells down regulate Th1 cell-mediated autoimmune and Th2 cell-mediated allergic inflammatory processes.

To determine whether T cells which produce large amounts of latent TGF-beta1 are capable of down-regulating autoimmune and allergic disease, myelin basic protein (MBP)-specific and ovalbumin (OVA)-specific BALB/c cloned Th1 cells were transduced with cDNA for murine TGF-beta1 by coculture with fibroblasts producing a genetically engineered retrovirus. The transduced MBP-specific Th1 cells were found to lose the capacity to provoke EAE in BALB/c mice, and to gain instead the ability to protect against EAE in (SJLxBALB/c) F1 mice immunized with proteolipid protein (PLP). This protective effect was not obtained with OVA-specific TGF-beta1 transduced Th1 cells. The transduced OVA-specific Th1 cells did protect against airway hyperreactivity induced by Th2-cell mediated responses to inhaled OVA. This effect was again antigen specific and it also could not be obtained with untransduced OVA-specific Th1 cells. In both cases these effects of antigen specific TGF-beta1 transduced T cells were nullified by administration of neutralizing anti-TGF-beta mAb. Thus, the antigen specificity of the cloned T cells allows the site-specific local delivery of therapeutic active TGF-beta1 to both Th1 and Th2 cell-mediated inflammatory infiltrates.

Gene therapy in allergic encephalomyelitis using myelin basic protein-specific T cells engineered to express latent transforming growth factor-beta1.

A myelin basic protein (MBP)-specific BALB/c T helper 1 (Th1) clone was transduced with cDNA for murine latent transforming growth factor-beta1 (TGF-beta1) by coculture with fibroblasts producing a genetically engineered retrovirus. When SJL x BALB/c F1 mice, immunized 12-15 days earlier with proteolipid protein in complete Freund's adjuvant, were injected with 3 x 10(6) cells from MBP-activated untransduced cloned Th1 cells, the severity of experimental allergic encephalomyelitis (EAE) was slightly increased. In contrast, MBP-activated (but not resting) latent TGF-beta1-transduced T cells significantly delayed and ameliorated EAE development. This protective effect was negated by simultaneously injected anti-TGF-beta1. The transduced cells secreted 2-4 ng/ml of latent TGF-beta1 into their culture medium, whereas control cells secreted barely detectable amounts. mRNA profiles for tumor necrosis factor, lymphotoxin, and interferon-gamma were similar before and after transduction; interleukin-4 and -10 were absent. TGF-beta1-transduced and antigen-activated BALB/c Th1 clones, specific for hemocyanin or ovalbumin, did not ameliorate EAE. Spinal cords from mice, taken 12 days after receiving TGF-beta1-transduced, antigen-activated cells, contained detectable amounts of TGF-beta1 cDNA. We conclude that latent TGF-beta1-transduced, self-reactive T cell clones may be useful in the therapy of autoimmune diseases.

Staphylococcal enterotoxin B and tumor-necrosis factor-alpha-induced relapses of experimental allergic encephalomyelitis: protection by transforming growth factor-beta and interleukin-10.

A study was made of the ability of the superantigen staphylococcal enterotoxin B (SEB) to induce relapses of experimental allergic encephalomyelitis (EAE) in SJL mice that had partially or completely recovered from acute EAE. We find that a single injection of 0.05 mg SEB i.v. induces mild relapses in 50% of such mice. In addition, tumor necrosis factor (TNF)-alpha (0.2 micrograms, i.p.) also induces EAE relapses in 43% of SJL mice when injected 1-2 months after recovery. SEB does not induce a second relapse if reinjected when V beta 17a+T cells are still partially deleted. In these mice, however, TNF-alpha is equally effective in inducing relapses as in mice that did not receive SEB previously. We showed earlier that transforming growth factor (TGF)-beta and TNF-alpha have antagonistic effects on experimental autoimmune diseases; e.g., in spontaneously relapsing EAE, TGF-beta and anti-TNF were protective, while anti-TGF-beta caused disease exacerbation. Interleukin (IL)-10 is also known to counteract certain TNF effects. We now find that both human IL-10 and TGF-beta 2 lower the incidence of EAE relapses when given simultaneously with SEB or TNF-alpha. The protective effect of TGF-beta is significant only against relapses induced by SEB (reduced to 9%), and that of IL-10 only against relapses induced by TNF (reduced to 0%) with the treatment regimens employed. Neutralizing anti-TGF-beta does not increase the incidence of SEB-induced EAE relapses. In contrast, anti-IL-10 increases both the incidence and the severity of such relapses. We conclude that TNF production is probably important in causing EAE relapses, but that other aspects of the SEB-induced reactivation of myelin-specific T cells also contribute. Furthermore, endogenous IL-10 rather than TGF-beta production appears to limit the susceptibility to induction of EAE relapses in this model.

Tolerogenic forms of auto-antigens and cytokines in the induction of resistance to experimental allergic encephalomyelitis.

Resistance to experimental allergic encephalomyelitis (EAE) induction by homogenized myelin (MSCH) in complete Freund's adjuvant (CFA) and pertussigen (P) in SJL mice was seen 1 week after intravenous injection of PLP 139-151 coupled to spleen cells (PLP-ECDI-SP). Although this resistance could be transferred by spleen cells enriched for CD8+ T cells and thus had a component of immunoregulatory T cells, it was primarily due to anergy, as it was reversible by four daily injections of interleukin (II)-2 starting 3 days after the PLP-ECDI-SP. Earlier treatment with IL-2 did not reverse the tolerance. In view of the known higher sensitivity to anergy induction of Th1 than of Th2 cells, a change in the cytokine balance in the response to MSCH+CFA after anergy induction might be responsible for the resistance to EAE induction. The effect of treatment with cytokines alone on induction of EAE was therefore also determined. Short-term (1-2 weeks) daily pretreatment with IL-2 (4000 U) or TGF-beta 2 (1 micrograms) somewhat decreased the susceptibility to subsequent EAE induction, but IL-4 (5 ng), IL-10 (5 micrograms) or IL-12 (50-200 ng) had no effect under those conditions, even if low doses of PLP were injected simultaneously. Daily injections of IL-4 over an 8-week period prior to immunization, however, significantly lowered the incidence of EAE. Simultaneous injections of IFN-gamma (2000 U/day) completely abolished this effect of IL-4. The effect of these cytokines administered immediately after the immunization with MSCH + CFA + P was also examined. As shown earlier, TGF-beta 2 (100-1000 ng/day) caused a marked protection when it was given intraperitoneally on days 5-9 after injection of MSCH + CFA. IL-4 (5 ng/day), in contrast, was very protective when administered on days 0-4 and less so when given on days 5-9 or even on days 0-12. IL-10 (1 microgram/day) was not protective under these conditions and IL-12 (50 ng/day) significantly increased the severity and mortality of EAE when given on days 0-4 after MSCH + CFA.

Antagonistic effects of endogenous and exogenous TGF-beta and TNF on auto-immune diseases in mice.

Injection of transforming growth factor beta 1 (TGF-beta 1) for five days during the late phase of the immunization process leading either to collagen type II induced arthritis (CIA) or to experimental allergic encephalomyelitis (EAE) protects against the development of these auto-immune diseases. Tumor necrosis factor alpha (TNF-alpha) injected during this same interval aggrevates CIA. In addition, anti-TGF-beta exacerbates and anti-TNF protects against CIA, acute and relapsing EAE, suggesting an important regulatory role for the endogenous production of the two cytokines on the severity of these diseases. More detailed studies about the mechanism of action of TGF-beta in acute EAE show that there is no detectable effect of TGF-beta on the development of sensitized T cells in vivo, as assayed by the proliferative responses of T cells from lymph nodes and peripheral blood to myelin antigens. Nevertheless, the number of lymphoid cells infiltrating the central nervous tissue is much greater in untreated than in TGF-beta-treated, protected mice. We conclude that it is likely that TGF-beta protects against experimental auto-immune diseases by interfering with the entry of lymphoid cells into the target organs through inhibition of the upregulation of adhesion molecule expression on endothelial cells, and with subsequent inflammatory processes inside the target organs by antagonizing both the production and the effects of TNF.

Studies on the mechanisms by which transforming growth factor-beta (TGF-beta) protects against allergic encephalomyelitis. Antagonism between TGF-beta and tumor necrosis factor.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease in which peripheral lymphoid cells are activated by immunization with myelin proteins and become effector cells that traverse the central nervous system (CNS) capillaries and initiate inflammatory demyelinating lesions. The administration of transforming growth factor-beta (TGF-beta) has been shown previously to decrease the incidence and severity of EAE. In our studies we have determined: 1) the effects of TGF-beta injected at different intervals after the EAE-inducing immunization; 2) the effect of TGF-beta on the development of sensitized T cells, as assayed by the proliferative responses of T cells from lymph nodes and peripheral blood; 3) the extent of lymphoid cell infiltration in CNS of TGF-beta-treated and control mice; and 4) the role of endogenous TGF-beta and TNF in determining the severity of both acute and relapsing EAE. The onset of acute-EAE in SJL mice, induced by immunization with spinal cord homogenate in CFA and pertussigen, is on days 10 to 15. Although daily i.p. injections of 0.2 to 2 micrograms TGF-beta 1 or TGF-beta 2 on days 5 to 9 after immunization are highly protective, injections on days 1 to 5 or 9 to 13 are not. Moreover, anti-TGF-beta accelerates and aggrevates EAE when given on days 5 and 9, but not on day 12. Anti-TNF, injected on days 5 and 9, provides a comparable degree of protection as does TGF-beta. Similarly, in relapsing EAE, anti-TGF-beta increases, whereas anti-TNF decreases the incidence and severity of relapses. TGF-beta treatment on days 5 to 9 does not influence the appearance of sensitized cells in peripheral blood and lymph nodes, but does prevent the accumulation of T cells in brain and spinal cord, as assayed on days 15 to 20. It is concluded that the protective effect of TGF-beta is exerted at the level of the target organ, CNS and/or its vascular endothelium, rather than through a direct effect on lymphoid cells, and that there is a small window of 4 days in which TGF-beta exerts its protective effect.

Isolation and amino terminal sequence of beta-trace, a novel protein from human cerebrospinal fluid.

beta-Trace, a 23.5 kDa glycoprotein of unknown biological functions, is present in all body fluids tested. It is found in higher concentration in human seminal fluid and cerebrospinal fluid (CSF) than in serum. A one-step procedure for the isolation of beta-trace from pooled CSF is described, by affinity chromatography using a specific antibody made against beta-trace. Amino terminal sequence analysis yields the sequence A P E A Q V S V Q P N F Q Q D K F L G with no homology to known proteins, indicating that beta-trace is a novel CSF protein.

Protective effect of transforming growth factor beta 1 on experimental autoimmune diseases in mice.

Interleukin 1 (IL-1) and tumor necrosis factor alpha are thought to contribute to the inflammatory response associated with autoimmune diseases. Transforming growth factor beta 1 (TGF-beta 1) counteracts many effects of these cytokines and has various immunosuppressive properties. In the present study, it is shown that microgram amounts of TGF-beta 1, injected daily for 1-2 weeks, protect against collagen-induced arthritis (CIA) and relapsing experimental allergic encephalomyelitis (REAE), the animal models for rheumatoid arthritis and multiple sclerosis, respectively. When administered during induction of the disease, TGF-beta 1 prevents CIA but only delays the onset of REAE by 2-3 days. However, when administered during a remission. TGF-beta 1 prevents the occurrence of relapses in REAE. The results suggest that TGF-beta 1 has powerful anti-inflammatory effects, mimicking in some respects the beneficial effects of immunosuppressive drugs in these experimental models of autoimmune disease, but without discernable adverse effects.

Immune response in draining lymph nodes and spleen after intraventricular injection of antigen.

Both Fischer 344 and Sprague Dawley rats showed significant numbers of antibody forming cells (PFC) in deep cervical lymph nodes after intraventricular injection of antigens, including trinitrophenylated (TNP)-hemocyanin, TNP-B. abortus and sheep erythrocytes. This indicated that particular as well as soluble antigens drained to these lymph nodes from the spinal fluid. Other lymph nodes examined did not show increased PFC over background, but levels of PFC in the spleen were significantly elevated after intraventricular injection of each of these antigens. Comparison with dose responses in the spleen after intravenous antigen injection suggested that approximately 20% of the intraventricularly injected immunogens drained to the peripheral blood. The relevance of these findings with respect to the brain as an immunologically privileged site is discussed.

Primary and secondary wattle swelling response to phytohemagglutinin as a measure of immunocompetence in chickens.

Wattle responses of 2-to-8-week-old chickens to phytohemagglutinins PHA-P and PHA-M were studied. Dilutions of PHA-M that did not induce wattle swelling after one injection did cause readily detectable swelling when injected a second time 1 week later. These responses were absent in birds subjected to thymectomy and gamma-irradiation at hatching and in birds treated with cyclosporin A during the week of sensitization, indicating that these responses are T-cell-dependent. Chickens bearing transplantable fibrosarcomas failed to show responses. It is suggested that reactivity to a second injection of PHA-M may be used as a measure of immunocompetence at the T-cell level in very young chickens.

Cerebrospinal fluid glucose: turnover and metabolism.

The turnover of cerebrospinal fluid (CSF) glucose was studied in cats during steady-state perfusion. In all experiments, the perfusion fluid contained either tracer [14C]glucose alone or tracer glucose along with 4.45 mM unlabeled glucose. In some studies, serum glucose was lowered with insulin. The concentration of glucose and [14C]glucose in the effluent fluid was measured, and the distribution of 14C between glucose and lactate was determined by chromatography. From these values, the extraction of glucose and the metabolism of glucose to lactate were calculated. From the decrease in the specific activity of glucose in the perfusion fluid, the influx of glucose from serum was also determined. During steady-state perfusion, 71% of the radioactivity was recovered in the effluent fluid: 50% in the form of glucose, 6% in the form of lactate, and 15% in forms that were not identified. Thus, 50% of the perfusion fluid glucose was cleared, of which 29% was extracted and 21% metabolized. The influx of glucose was proportional to the serum glucose when the latter was about 2.5 mM or 10.0 mM. During perfusion with tracer glucose only, the concentration of glucose in the effluent fluid was 25% that of serum. The transport of glucose from serum was independent of the glucose concentration gradient between serum and perfusion fluid. However, when perfusion fluid glucose concentration was greater than that of serum, transport was inhibited. These studies suggest that in maintaining CSF glucose at a lower concentration than serum glucose, with equal amounts of glucose entering and leaving the CSF, 50% of CSF glucose concentration cleared is replaced by 25% of serum glucose concentration.

Immunity to transplantable nitrosourea-induced neurogenic tumors. III. Systemic adoptive transfer of immunity.

The effect of intravenously injected tumor immune spleen cells on growth of 3 X 10(5) gliosarcoma T9 cells injected intradermally (ID) or intracerebrally (IC) into sublethally irradiated CDF rats was evaluated. Spleen cells from donor rats with sufficient immunity to reject 5 X 10(5) T9 cells inhibited the growth of T9 cells mixed with spleen cells in a ratio of 1:25 and injected ID, but could not act after intravenous transfer. However, donor rats which had rejected increasing T9 challenge doses up to 1 X 10(7) cells produced immune spleen cells which, upon IV transfer, could inhibit growth of ID T9 challenge but not of EB-679, an unrelated glioma, in recipient rats. Rejection of IC T9 challenge was also obtained after IV transfer, in recipients of such "hyperimmune" spleen cells, but was less (60% maximum) than that noted after ID T9 challenge (100% maximum). The removal of B cells from the transferred spleen cells did not affect the results, suggesting that the specific immunity was mediated by T cells. We conclude that the special immunological circumstances of tumors growing in the brain renders them less accessible to rejection by systemically transferred immune cells, but it is nevertheless possible to effect a significant incidence of rejection of syngeneic tumor growth in the brain by the intravenous transfer of hyperimmune spleen cells.

Cerebrospinal fluid glucose and leukocyte responses in experimental meningitis.

The fall in cerebrospinal fluid (CSF) glucose and CSF leukocyte response was studied in cats with experimental meningitis. Klebsiella pneumoniae or Streptococcus pneumoniae were injected intracisternally, and the latter organisms were incubated with CSF in vitro. When 10(6)-10(9)K. pneumoniae were incubated with 4 ml of CSF, the time time necessary for the glucose to decrease to less than 10 mg/dl ranged from 6.5 to 2.5 h, at a rate proportional to the size of the inoculum. When the same numbers of bacteria were injected intracisternally, the time ranged from 9 to 3 h, and the CSF leukocyte response did not exceed 1200 WBC/mm3. At this time, only minimal histological changes in brain and choroid plexus were seen. Twenty hours after intrathecal K. pneumoniae, large numbers of leukocytes (up to 4 X 10(4)/mm3) were recovered from the CSF. Regardless of the number of leukocytes, however, hypoglycorrhachia occurred when the CSF contained more than 10(7) bacteria/ml. At this interval, large numbers of leukocytes were seen invading the stroma of the choroid plexus, leptomeninges and perivascular spaces. When 10(8) S. pneumoniae were injected intracisternally, CSF glucose concentration decreased as rapidly as with K. pneumoniae. The spinal fluid leukocyte response to S. pneumoniae was, however, greater than that to K. pneumoniae. These results suggest that under the conditions of these studies, hypoglycorrhachia of bacterial meningitis is the result of metabolism of the bacteria with little contribution from the leukocytes.

Net transport of glucose from blood to cerebrospinal fluid in the cat.

The net transport of glucose from blood to the cerebrospinal fluid compartment of cats was measured by ventriculocisternal perfusion to determine over a large range of serum glucose concentrations the influence of serum glucose levels and their changes on the net transport rate. Changes in serum glucose levels were followed within minutes by corresponding changes in cerebroventricular effluent fluid glucose concentration. At mean values of serum glucose concentration of 6.2 mM and cerebrospinal fluid formation rate of 24.3 microliter/min, the net glucose influx rate was 1.6 mmol/min. The effluent fluid-to-serum glucose concentration ratio was 0.25 and decreased when serum glucose was greater than 11.1 mM. The rate of glucose transport from blood to effluent fluid during ventricular perfusion was saturable, and approached a maximum of 3.5 mumol/min at serum glucose levels above 22 mM. From the cerebrospinal fluid formation and net glucose influx rates the calculated glucose concentration of nascent cerebrospinal fluid was 6.5 mM and higher than the corresponding serum glucose of 5.6 mM. It is concluded that during perfusion over a wide range of serum glucose concentrations, a saturable mediated glucose transport mechanism can be demonstrated. Changes in serum glucose are rapidly reflected in corresponding effluent fluid glucose levels. From effluent fluid-to-serum glucose concentration ratios and calculations of the glucose in newly formed cerebrospinal fluid, the technique, however, overestimates the glucose influx rates at normal serum glucose levels.

Enkephalin-induced changes in ventilation and ventilatory pattern in adult dogs.

We studied the changes in ventilation induced by intracisternal administration of enkephalins in four unanesthetized adult dogs. Instantaneous minute ventilation (VT/TT) decreased markedly after D-Ala-Met-enkephalinamide (DAME). Mean VT/TT decreased maximally by 20-50 min after DAME and lasted an additional 15-60 min; by 2 h, VT/TT had returned to base line. Four doses (5, 25, 60, and 125 micrograms/kg) of DAME were used, and the ventilatory response depended on the dose. Mean inspiratory time decreased but mean expiratory time and mean TT showed a marked prolongation. Periodic breathing (2-3 breaths separated by long apneic pauses) occurred in every study and the frequency of sighs increased considerably. All these ventilatory changes were reversed by low doses of naloxone or naltrexone; in addition, VT/TT increased well above base line after the administration of these antagonists. However, naloxone did not increase VT/TT when injected without prior administration of DAME. We conclude that 1) the decrease in VT/TT is due to a decrease in respiratory duty cycle; 2) periodic breathing and increased frequency of sighs constitute part of the changes in the ventilatory pattern induced by DAME; 3) a ventilatory withdrawal reaction may occur after a receptor-agonist interaction of short duration; and 4) although enkephalins can modulate ventilation and the breathing pattern in a major way, these data provide no evidence suggesting that this modulation is tonic.

Spinal fluid formation and glucose influx in normal and experimental hydrocephalic rats.

Cerebrospinal fluid (CSF) turnover and glucose influx were measured in normal and kaolin-induced hydrocephalic rats. The CSF formation rate of normal rats was 2.8 microliter/min and after intracisternal kaolin it was reduced to 1.8 microliter/min. The CSF-serum glucose concentration ratio of normal rats was 0.57 and was reduced to 0.47 in hydrocephalic rats. The reduction was associated with a decrease in the net influx of glucose measured in hydrocephalic rats. The fraction of serum glucose transported from blood to CSF in normal and hydrocephalic rats decreased as serum glucose increased above 200 mg/dl. At all serum glucose concentrations studied, the influx of glucose in normal rats was always 2.6 times greater than that in hydrocephalic rats. These results suggest that because the glucose transport mechanism of both groups of rats is only quantitatively different, the number of sites available for glucose transport from blood to CSF is reduced in hydrocephalic rats.